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Polymerix User Manual

1. Constant 1 Da W Peak detect and centroid Apply Proportional e00 ppr 0 4 Mey at nels 1200 Spectrum Select Peak Detect Mass Adiust Mass Mode Deisotope This displays the Peak Detect view shown above Merge to E peak width M Subtract baseline fram spectrum Baseline width 500 Da 1 ZAA TOF The scan law determines how the peak width changes as a function of m z The peak width value is measured using full width at 5 peak height or 10 valley Depending on your instrument type click the button to the left in the Peak Width Resolution section to select the appropriate type TOF spectra require two parameters the peak width in Da and the m z value at which the width was measured The peak width does not need to be precise within a factor of two is generally acceptable 36 DATA IMPORT AND PROCESSING 1 For very high resolution spectra such as produced by FT ICR or Orbitrap instruments Polymerix s peak detection algorithm works well with a very small peak width such as a constant width of 0 001 Da Polymerix however will not always detect peaks with a width of less than 4 data points 2 After choosing the peak width type and changing the width parameter also select the Peak detect and centroid check box in the Peak Detection Baseline Correction section and then click the Apply button This will process and redisplay the new processed target spectrum
2. Spectrum Select Peak Detect Mass Adiust Mass Mode Deisotope 1 The deisotoping algorithm operates by predicting an isotope cluster from a model chemical formula matching that to an experimental isotope cluster then moving the intensity found in the isotopic peaks back into the monoisotopic peak The model chemical formula is created by adding together an end group composition adduct and an appropriate multiple of the repeat formula to yield a model formula with mass close to the experimental mass The predicted peaks and their relative abundances are then matched to experimental data This process is repeated for each isotope cluster in the spectrum To enable deisotoping click the Deisotope spectrum checkbox 2 Next you must specify at least the repeat formula The end group and adduct entries are optional To specify a repeat formula click the button next to the Repeat Formula edit box 42 DATA IMPORT AND PROCESSING Select Formula ES none Acrylonitrile Cancel Butadiene Butylene Ethylene lsobutylene lsoprene xi New Edit Delete By Formula a 3 This displays the Select Formula dialog box In the top part of the dialog is a list of predefined monomers repeat units If your sample includes one of these you may select it and click the OK button 4 If your monomer is not on the list you may add a new definition which will be stored for use again in the future In this
3. 1 Click the button in the upper left corner of the Mass Ranges to Analyze table This adds a new row to the table Type the m z range start and end values Continue until all ranges have been defined and then click the Apply button 2 Alternatively you may right click and drag in the spectrum window Right click at the beginning of the range and then while holding the mouse button down drag to the end of the range Repeat this for each new range The ranges will automatically be added to the table If you wish you may fine tune the range setting by editing the table afterward Click the Apply button to calculate the percentages Relative Area results The relative areas and their percentages are displayed in the table in the middle pane Spectrum Mass Ranges Area Analysis Results Range From To Intensity Ranges Spectrum Total 264411 3 100 0 96 5 1 850 4 1164 1 66333 0 25 1 24 2 2 1162 6 1522 6 137556 4 52 0 50 2 3 1523 3 1987 4 60521 9 22 9 22 1 For each range Polymerix computes the total integrated intensity the percentage that intensity reflects of all of the defined ranges and of the spectrum as a whole which may include area outside of defined ranges 89 RELATIVE AREA In the next chapter In the next chapter we will describe the autocorrelation analysis process and how it can assist in determining the composition of an unknown polymer Autocorrelation analysis has been superseded by the functi
4. 5 However 43 946 Da does not correspond to any meaningful chemical formula for a monomeric repeat unit A better but still close value is 44 0262 Da corresponding to the mass of the ethylene oxide monomer with formula C H O Particularly in TOF spectra peak tailing due to detector dead time can result in intensity dependent variations in peak centroids This leads to broadening and shifts in the autocorrelation spectrum So let s make the assumption that the actual repeat mass is 44 0262 Da and recompute the end group spectrum accordingly 6 In the Repeat Mass Range section uncheck the Auto compute end groups checkbox In the End Group Mass section check the Use repeat mass checkbox and enter the value 44 0262 in the edit box Click the Apply button The end group spectrum will be recomputed this time using 44 0262 Da as the repeat mass f Repeat Mass Range r End Group Mass Minimum 10 drama 0 Maximum 300 MV Use repeat mass F fa HO T ompute end 44 0262 Apply Apply 7 Now as can be seen from the portion of the end group spectrum below it consists of just a single peak at 41 000 Da This implies a sodium adduct Subtracting the mass of Na 22 990 Da yields a 96 AUTOCORRELATION remainder of 18 010 Da very close to the exact mass for H O of 18 0105 Da 41 000 94076 What was covered in this chapter In this chapter we Showed how to access the autocorrelation analysis page Described
5. Next click in the Repeat box to display the Specify Monomer dialog box As with end groups there is a predefined list an option to create a new monomer definition and options to enter a temporary definition by either chemical formula or mass If you will be using Polymerix frequently on the same class of polymer it is very convenient to add new monomer definitions so they will be available for future use After defining the monomer click the OK button to close the dialog box and copy the monomer into the series definition For the Omega End Group follow the same procedure as for the alpha end group selection If you know only the total end group you may specify this as either an alpha or omega end group and leave the other entry empty 33 Next specify the charge state a signed quantity by typing into the Charge State box If more than one charge state is observed you must define additional homopolymer series to match each of those 34 Next specify an adduct if any Click in the Adduct box to show a button click the button to display the Specify Adduct dialog box As with other chemical entities there is a predefined list of common adducts an option 67 HOMOPOLYMER ANALYSIS 35 36 37 38 39 40 to define new ones and options to temporarily specify the adduct by formula or mass After choosing the adduct click the OK button to copy it into the definition Specify Adduct Charge the charge
6. Repeat A Statistics On this tab is a table of molecular weight distribution statistics for all A unit series by fixed B count That is keeping the B count fixed statistics are computed for every peak in the series with that B count and variable A count This is repeated for the next B count and so forth This is the equivalent of taking each column in the AB distribution table and computing statistics for it Repeat A Statistics For Constant Repeat B Counts RepeatB Mn Mw M PD DPn DPw Dz 8 3 000 3 000 3 000 614 404 614 404 614 404 1 000 9 672 627 672 627 672 628 1 000 3 004 3 004 3 004 10 730 645 730 646 730 646 1 000 3 003 3 003 3 003 11 766 114 769 114 7712768 1 004 2 450 200g 2 619 12 802 770 206 958 10 891 1 005 2 005 2 100 2 189 13 248 690 252 938 257 061 1 005 1 729 1 826 1 919 14 296 973 200 996 204 986 1 004 1 508 1 599 1 630 15 949 458 291 977 955 517 1 004 1 359 1 439 1 519 16 999 691 1002 342 1006 045 1 003 1 204 1 276 1 348 17 1050 523 1053 164 1055 880 1 003 1 040 1 100 1 162 18 1103 230 1111 758 1114 230 1 002 1 057 1 113 1 170 19 1165 434 1167 771 1170 110 1 002 1 015 1 067 1 120 20 1220 322 1222280 1224254 1 002 0 542 0 386 1 032 21 1277573 1279 383 1261247 1 001 0 524 0 365 1 008 22 1331 481 1333 054 133 678 1 001 0 830 0 366 0 503 23 1304301 1385 534 1386 810 1 001 0 712 0 740 0 763 24 1451 112 1452 491 1453 905 1 001 0 911 0 2 0 974 25 150349 1504873 1506 293 1 001 0 782 0 814 0 846
7. counts that will actually be considered are 2 1 4 2 6 3 100 50 since no others satisfy both the ratio and count range limits 79 COMPOLYMER ANALYSIS 5 After defining the necessary series click the Apply button to assign the spectrum Copolymer molecular weight statistics The table of copolymer molecular weight distribution statistics is identical to that created for homopolymers with the substitution of Repeat A and Repeat B columns for the single Repeat column i i e n ie ile at a a a Iada Ty cm tree ion tom mt oh mein een Me Cot NODC PI Bet the Iren Oe 1 Me CPt OM DLE PTB et oe Ings ru er ion w rr cM ip er Hi The statistics are computed in the same way as for homopolymers Visualizing copolymer series assignments Because copolymer assignments are a two dimensional result the results are presented in a two dimensional manner Since assignments are deconvolved into separate distributions by A and B count some additional tables and plots are provided to show the distribution of each repeat unit as a composite and separately Assignments Lapgyme Arugirenis De exe P3500 PET ren k ena TUA n gt oF Nee ee LAERE TE va 74 ape 0 91 1000 4 TnT 1700 8 308 0 tate 0 S00 8 iot o 17069 ao m Pitered Asuprerents Ramiai Det we AS Tate dopat A Statens Ragman A 5 spaar AP Rapant 2 Pit This is basically the same plot as for homopolyme
8. it will be created If you wish to install Polymerix in a different folder click the Browse button to choose an alternative Click Next gt to continue INSTALLATION LICENSING AND REGISTRATION Polymerix Setup Ioj x Choose a Start Menu folder for the Polymerix shortcuts J Select the Start Menu folder in which you would like to create the program s shortcuts You can also enter a name to create a new folder Accessories Administrative Tools Applied Biosystems MDS Sciex ComponentOne Games Microsoft Office Do not create shortcuts Mullsart Install system v2 45 4 Choose the name for the Start Menu folder If you do not wish to have an entry made on the Start Menu and a Polymertix icon placed on your desktop select the Do not create shortcuts option Click Install to begin copying files to the installation location INSTALLATION LICENSING AND REGISTRATION P Polymerix Setup Installing Please wait while Polymerix is being installed Waso Install system yi 4 5 As the installation program extracts and copies files to the destination folder the progress is displayed To see details of this process click the Show details button Two options for licensing INSTALLATION LICENSING AND REGISTRATION Polymerix Setup l G x Installation Complete Setup was completed successfully H Extract C Polymerix LCComponent dll 100 Execute regsyvr32 js
9. table by clicking on the in column one Remove an entry by clicking on the i column one of the entry s element table line 57 Repeat tolerance End group tolerance Maximum result count End group mass range Charge range DBE range Electron mode Element ratio restrictions Positive charge Mixed adducts Spectrum match tolerance FIND HOMOPOLYMER SERIES Repeat Tolerance is the maximum mass tolerance allowed when computing potential repeat formulas Can be specified in mDa milliDaltons or ppm End Group Tolerance is the maximum mass tolerance allowed in computing end group formulas Can be specified in mDa milliDaltons or ppm Maximum Result Count is the maximum number of repeat formulas or end group formulas that are displayed for user selection If more than this number of potential formulas satisfy the end group or repeat tolerance then only those formulas which best match the reference mass are shown The End Group Mass Minimum and Maximum are mass limits for the mass of the end group whose composition is being determined All end group formulas are computed using the minimum and maximum masses provided Charge Minimum and Maximum define the charge count range to be used for the computations To enable multiply charged ions Maximum Charge must be set higher than its default value of 1 DBE Minimum and Maximum is the allowed double bond equivalent range to be conside
10. 0 17 Only the processed target spectrum bottom pane is changed This is the convention used throughout the Data Processing page the middle pane shows the raw input spectrum the bottom panes shows the processed target spectrum that will be used for polymer series assignment 18 When you are satisfied with the mass adjustment click the Apply button to set the parameters 19 Often it is necessary to use the mass adjustment in concert with series assignment in an iterative process to ensure that all peaks are assigned For this purpose the Mass Adjust tab is duplicated on the Homopolymer Analysis and Copolymer Analysis pages Mass mode In Polymerix it is most common to do analysis on peak detected data with assignments to monoisotopic peaks For users of instruments with insufficient resolution to resolve isotopic peaks throughout the spectrum analysis is a bit more difficult when peak detection cannot be uniformly applied For this purpose Polymerix permits processing in monoisotopic chemical average or nominal mass modes and will apply a correction to the matched peaks to convert them to either mode as necessary For example if resolution is degraded above 2000 Da the detected peak m z values ate the chemical m z values of the whole cluster not of the individual isotopic peaks In this case you may choose to have molecular weight statistics reported as 40 DATA IMPORT AND PROCESSING monoisotopic or chemical mass
11. 0 04 0 19 Spectrum 1 5 111919 Points 1317 7 1287 1000 0 0 0 99 4202 6 419 8 620 7 220 226 219 213 236 192 1847 0 20628 2240 7 190 ro 1000 0 2000 0 Detect 0 4 Da 1000 500 6590 Peaks Mass Range 0 00 to 2402 00 1317 8 10854 10000 0 1449 8 6419 1561 9 TER 0 0 1846 1 ee 392 523 7025 568 266 222 z231 1000 0 2000 0 111 2310 6 137 RELEASE NOTES Polymenx Processing Report Homopolymer Analysis Results Printed Wed Mar 06 2013 15 42 07 Data file C Polymerixipeg1500_cal raw Monoisotopic mass mode Match tolerance 0 100 Da Homopolymer Assignments 51 R29 1148 3 10000 0 S1 Rae 1185 1103 91 R33 1493 9 2487 51 R35 1581 9 9595 g1 R34 1537 9 0266 5000 0 51 R44 5 R36 1097 6 1625 9 5724 5607 51 RJS 1053 6 Sl R37 4077q 1670 0 A432 sh R38 51 RAZ 1009 6 2519 51 R31 51 ae 924557 0 0 98 9 TERT ant 313 2 5273 702 5 gamed 268 222 23 429g l 1000 0 112 RELEASE NOTES Polymenx Processing Report Page 3 of 8 Homopolymer Analysis Results Printed Wed Mar 06 2013 15 42 07 Data file C Polymerixipeg1500_cal raw Monoisotopic mass mode Match tolerance 0 100 Da Filtered Homopolymer Assignments 51 R29 s1iRdg 3 12419 R R30 ha1 8 T2415 51 RA 122017 lea JER 13398 SUBST bese 10000 0 gA 51 RJS 1185 7 11098 cH R33 1493 9 2487 31 R45 31 R35 1741 1581 9 ToS 9595 g1 R44 4537 9 0268 5000 0 pb SH R36 ell 1625
12. 26 15 9473 1581214 1582 976 1 001 1 190 1 229 1 269 27 1621 900 1622 706 1623 519 1 000 0 835 0 853 0 872 28 1659 1650 448 1650 992 1 000 0 154 0 165 0 177 29 0 000 0 000 0 000 0 000 0 000 0 000 0 000 3 1807 244 1807 244 1807 24 1 000 1 030 1 030 1 050 Assignments Filtered Assignments Filtered Residuals Details Mass Ranges AB Distribution Repeat A Statistics Repeat B 84 COMPOLYMER ANALYSIS Repeat B Statistics Likewise by slicing the AB distribution table by rows the molecular weight distribution over B counts for each fixed A count can be computed RepeatA Mn Mw Me PD DPn DPw Dz 0 10 688 1025 574 106 607 1 021 16 999 17 359 17 722 1 1080 725 1080 050 1093 410 1 018 17 206 17 539 17 873 2 1099642 111 677 1135 749 1 016 17 118 17 429 17 740 3 106 184 1067 114 1088 205 1 020 15 439 15 799 16 163 Repeat A Plot More useful than tables of numbers even with color coding is to display spectra filtered by series and by A and B count In the Repeat A Plot tab the vertical slices taken through the AB Distribution table are displayed as spectra Wi 205 97 Ve 500 9 Ye O04 Gee FPO tod Oe 1 Soe OMe t OP Orr 1 oe Noun The screen shot above shows the A unit spectrum for a fixed B count of 14 The scroll bar at the bottom of the plot is used to step through each B unit count Clicking to the right increments the B count clicking to the left decrements it Each 85 COMPOLYMER ANALY
13. 3 p s i ya I _ os ne j x set 5 395 0 11086 12064 10048 14866 1800 4 1600 1700 100 0 13178 1361 8 17497 17217 1315 7 1340 013453 9 1353 2 100 47 46 55 1320 0 1330 0 1340 0 1350 0 1360 0 With one series defined our example looks like the screen shots above The series definition has been expanded into a list of targets appropriate for the 800 2000 Da mass range chosen and these have been matched against the spectral features Matched peaks have been labeled and colored The labels show the series name S1 repeat count Rn m z and intensity Homopolymer molecular weight statistics When homopolymer series are defined and the Apply button clicked Polymerix immediately calculates molecular weight statistics for the whole spectrum as well as for each series These are displayed in the middle pane of the Homopolymer Analysis page teresa ee Pent Sewer mm wm mw we em ee mm ee ee a R ee ae a ten _ i IMa E E Lowe i e Pit ile he Be HOMOPOLYMER ANALYSIS Series Total Average 1359 331 1396 488 1413 386 1 020 30466 31 083 31 694 51 1359 331 1386 488 1413 336 1 020 30 466 31 083 31 694 Percent Percent Alpha Omega Charge Series Series Spectrum End Group Repeat End Group State Adduct Loss Formula OH C2H40 n H Na 100 00 69 64 OH C2H40 H 1 Na OH C2H40 n H Na This table starts with a summary row for the entire sample followed by a row for each series If one or more of the o
14. 9 5725 5507 51 RB 1053 6 Sl R37 407 4670 0 B432 cH R38 51 Rae it 1009 6 pf Fos 2549 4758 0 poz 51 ROM s189 SIRA 324554 aeti S E P a jhi Ipi 0 0 SA tee 4 iB Sie TA SAT i jeier A is 12499 E a 1000 0 2000 0 113 RELEASE NOTES Polymenx Processing Report Page 4 of 8 Homopolymer Analysis Results Printed Wed Mar 06 2013 15 42 07 Data file C Polymerixipeg1500_cal raw Monoisotopic mass mode Match tolerance 0 100 Da Filtered Homopolymer Residuals 3000 0 2000 0 4000 0 1538 4 659 98 9 1 319 2 1407 4 1759 5 324 327 258 5973 702 5 659 6 aay 315 1 233 Tad 1a gai 2310 6 137 4000 0 2000 0 114 RELEASE NOTES Polymenx Processing Report Homopolymer Analysis Results Printed Wed Mar 06 2013 15 42 07 Data fille C Palymerixipeg1500_cal raw Monoisotopic mass mode Match tolerance 0 100 Da Assignments for Total Average Page 5o0f8 S Maximum 512839 1348284 R32 7 alt 50 4 100 0 11 FET 51 Raglang B 12742 1629 1601 1230 1339R g1 R33 1494 4 2487 51 RAS 1186 1109 i RH 1538 7 EpR35 1582 5 5595 51 RJS 1142 798 50 0 Si R36 1626 5 S1 Rad pee 1098 31 R37 572 1670 6 5432 ot R sh R38 1053 8 aa 407 B915 31 R39 51 Rap 758 7 1010 ita 25149 51 Rat eRe 966 Tete paz 51 A362 j 0 0 cff gS awT 17183 1000 0 2000 0 115 RELEASE NOTES Polymenx Processing Report Homopolymer Analysis Results Printed Wed Mar 06 2013 15 42 07 Data file C Polymeri
15. ANAIVSIS sccsicscedetecckeuedetiencduccedseedhieweerendeaesdadeies 60 Goals TOK VAIS Chaple heanas aa sent ewca tea eaetee eee eeaeee 60 Molecular weight distribution statistics cccccseeeeeeeseeeseeeeeeeens 60 The Homopolymer Analysis page user interface ccseceseees 62 Setting homopolymer processing paramMeters cccceseceeeeeeees 63 Defining homopolymer SELIES ccccccceeeeeeeeseeeceeeeeeeeneeeseeeeeeeens 65 Homopolymer molecular weight statistics ccccssceseeeseeeeeeeens 69 Visualizing homopolymer series assignments 6 ccceeeeeeeees 70 PS SIGNING aoire N er antes 70 Filtered Assignments seiman esi ai eaaa ai ariaa ai aia 70 Fitered FE SICU ANS sjeccete varia betoranscket n a iecacciara dealt 71 CTA NS aaan a O Winer ate 71 MASS Rangos career cecvauenat situa aa a ae siouimments 71 DIStiDUION PIOUS zoana ar a a 72 Assignment and matching details ccccccccceeecseeeseeeseeeeaeeeneees 73 The simple matching Algorithm ccccccccccsseeeeeseseeeeeeseeeeeeeeeeeneeeeesaees 73 Cister MAC MAG arcena a Meatde 73 GENIlOld CHS TEM MACANO vse itsecessted ince ese eet ratues E a 74 Centroid cluster Matching algorithm cccccceesseeeeeeeeeeeeeeeeeeseeeeeeeeeeas 74 What was covered in this Chapter cccccsececseeeceeeeseeeeeeeeeseeees 74 ITS OXU CM AOL CM sretatctcnctteanaetuttnshutedsnntetahtietwheltncedubiietwuididanetetes 15 Copolymer
16. ANGIVSIS cirein 76 Goals TOF TRIS CHAD esastan an Eer ED 76 GODOIVIMER deNIUON sasarina N E 76 The Copolymer Analysis page user interface ccccesseeeeeeees 77 Setting copolymer processing parameters ccccceeeeseeeeseeeeeees 78 Defining copolymer SELICS cccccccseeceeeceseecseeseeeeseeeseeseeeseeeens 79 Copolymer molecular weight statistics ccccccceseeecseeeeeeeseeeeees 80 Visualizing copolymer series ASSIQNMENHS ceeeeeeeeeeeeeeeeeees 80 PAS SIGN CIS Aca ticeutsccanse shen E E aye apdiant ciao 80 FINGKEO ASSIGNIMGINS ccies ceteris scnctancds ma aaa DA 81 Filtered Residual s iscnce tei oes a ee eet 81 Detalan E a uniictiaasmeslanan tctam dese untctan 82 WIASS IRAN S conyers aE ae yet codiaman tana N AES 82 PB DISUMOUN OM caseina ances ievibed eukweswadeiaas rpaude duels 83 Repeal A SiS CS eins tebe ce at aoe a a 84 Repeal B SQLS S assesses sandtcecd a E ENS 85 Repeat A Plotare E e nee 85 Ropod lO Geet icc tarnintheaciatantinmaniiuadanandtamethuasantanucinehcoumnsonandenscauansn aed 86 What was Covered in this Chapter ccccceececeeeeceeeeseeeeseeeeseeees 86 IA TNC NOX CMA DION cecs erotiniai adua aeaaea iian 87 The Relative Area TOOL ccccccccccceccseceeeeeeeeeeeeseeeeeseeeeeseeeaes 88 Goals TOF TRIS Chaple sew senarensseneconadenssndesi A 88 The Relative Area page cccccscccscecesecceeeceeeseeceueeseeeseeeeeaeenaees 88 SPECIE YING rang
17. Eas lie hia eed a did es 89 Relative Area TeSUNS asauicirinrini eka anaes 89 MNENE ENa sarigarri E 90 AUTOCONGIA HO Messrs ennnen enre 91 Goals tor tiS chaptar raien nena ain oe a T a 91 Accessing the Autocorrelation Dage ccccsscccssecseeeceeeeeeeeaeeeneees 91 The autocorrelation spectrum ccccecceececeeeceeceeeeceeesueeeeeenaees 91 neend group Spectrum sessie an Garnier eteunae bares 93 User interface and ParamMetelS ccccccceecceeeeseeeeseeeeseeesaeeesaeeeens 94 What was covered in this chapter cccccseccceeeeseeeeeeeeeeeeeeseeees 97 I TC OXE CHAD CN ensena n a REN 97 Creating and Customizing Output cccecceeeeeseeeeeeeeeeeeees 98 Goals Toris Cha PIET sea aaa enS 98 PONTE ODION Siiecauicstirabinsteunectraetieetruttianiatltand e 98 Copying to the clipboard cc cecccceececeeeeeceeeeeseeeeeceeeeesaeeeesaaeees 100 Saving and exporting results c ce ececseeeecseeeeeseeeeseeeeseeeeseeeees 101 PARUN atest tocianae acd Cia al a tol aget neat tngeen cet a aaa ein E anaes 103 What was covered in this Chapter cccccsseeceeeeeeeeeseeeeeaeeeens 106 AAEN CNA E arrmmrrpertrre meter emer rn nnn tert en errr ren cer rr rrr 106 Release NOLES szscsnausnacenanwimdendulsnaeamaiedadeindumocsvaciouidewdesnenseateteaes 107 NCES ION S Ohayeteesetus tage ida sonsehadenevagadasenedudesecetenutecsdasesensasausanses 107 Major new features cceeccccccceseeeeeeceeeeeceeeeeeeee
18. So 2 11415552 41142 Sei 51 S amp S 116126 17003004 5D A Seegrreerts Fipere degras Firet henhan etae pams targas Den Mote Distribution Plots This graphical view is linked to the Homopolymer Results Summary table Selecting a row in the table displays the peaks assigned to the series defined in that row In the first illustration below the first series S1 is selected in the second series S2 is selected 7 s lt 9 P om o owon ee ee OY om i faa l i wa neo O a WOOO p i e ee UMI ee ID Don Fin moi wed TEn JAIN 1EOON 1m I Lakad mum Ph TEO ae a 1H Oh EMIT H H aup A njstar REOT EE oom o w o am Se BE tt as ay b _ 1 Tae bowenge T Ie i ne m item 61k io uas UE I es wa ny te 8 s N 0H Sih ia Pasen it Iy 72 HOMOPOLYMER ANALYSIS Assignment and matching details The algorithms which Polymertx uses for quantitative analysis vary depending upon the data being analyzed and the settings being used for analysis When multiple series have been defined and there are series overlaps the different algorithms assign intensity values to series differently At its highest level mass matching can be divided into matching based upon single masses versus matching based upon computed isotope clusters When computed isotope clusters are being used for matching centroided data is matched diff
19. TET 3 C7H40 l 104 02621 36 42 36 421 349 955 4 C3H404 l 104 01056 51 677 51 677 496 594 Monoisotopic m z values are 1101 546513 and 1205 60877 repeat m z is 104 06254 tolerance is 0 1 Da electron mode is normal restricted element ratios DBE range is from 0 5 to 5 0 charge count is 1 maximum result count is 10 C count is from 0 to 10 H countis from 0 to 20 O count is from 0 to settings 10 N count is from 0 to 3 Choose chemical formulas for combined end group and adduct from the list provided by Polymerix and click the OK button 50 Automatic versus manual selection Excess Mass and errors FIND HOMOPOLYMER SERIES Select end group and adduct for series with monoisotopic peak at m z 1101 546135 Combined Computed Error Error Error C Se el ee ee lel 1 C4H10 0 0 Aq 1107 54620 0 064 O 064 0 058 2 C5H205 5 0 Na 1101 542655 3 267 3 287 2 984 3 C7H18N202 0 0 Ag 1101 542217 3 959 3 959 3 594 4 CBH1SN40 0 0 Ag 1101 5544 herd Ferd 5 604 5 H2N207 1 0 Na 1101 53882 r a09 7 309 6 636 6 C4HAN 204 5 0 Na 1101 554058 rot 54 rel 7 C8H1803 0 0 Ag 1101 523094 15 192 15 192 13 792 a HE05 4 0 H 1101 563770 17 563 17 563 15 944 9 C4H14N60 1 0 Ag 1101 52826 17 878 17 678 16 230 10 C5H18 6 0 0 Ag 1107 56464 15 508 15 508 16 502 EE Result End Groun ZEM 2 Monoisotopic m z is 1101 54513 repeat formula is C3H8 repeat mass is 104 06260 charge countis 1 tolerance is 0 1 Da electron mode is normal
20. The Find Homopolymer Series feature can help a user select chemical compositions for a homopolymer series from lists based upon pairs of reference peaks in the spectrum being analyzed Selecting compositions using Find Homopolymer Series is often easier and quicker than manually setting up polymer series definitions in Homopolymer Series Setup Find Homopolymer Series uses centroid cluster matching Updates bug fixes and new features e User interaction is essentially unchanged from Version 2 0 Some superfluous mouse clicks have been eliminated e Added support for multiply charged data with mixed adducts e Added a Filtered Residuals tab and plot to polymer analysis results e Added a Mass Ranges tab and table to homopolymer analysis results e Added error metrics to Homopolymer Details and Copolymer Details e Added percent of series molecular weight metric for series components e Changed the position of series annotations in plots so that their x positions always correspond to calculated monoisotopic peaks for the series e Depreciated the Autocorrelation tool but added a menu item to the View menu so that it is still accessible 107 RELEASE NOTES e Account for electron mass when computing mass ranges for use in matching Added a Positive charge checkbox to both Homopolymer Setup and Copolymer Setup e Fixed a problem with the calculations of intensity weighted mass averages Negative intensity values are now ignored i
21. analysis there is an additional parameter set used to define the type of copolymer and constrain the number of A and B combinations Match Tolerance E 01 song M Use clusters Positive ppm iv charge 05 Da at mrz 1000 Processing Maze Adjust Combine Within Series with Same Repeats Constraint Charge states 0 None Add Jl C Altemating ucts and losses A to E ratio Minimum fas End groups i B to A ratio Maximum 1 5 Proportional 100 TOF Constraint None Alternating C amp toB ratio Minimum 05 C B tod ratio Maximum f 5 1 To set a constraint on the relative counts of A and B units click the Processing tab On the right side is a new section Constraint There are four constraints None The A and B monomer units may occur in any combination Alternating The copolymer is strictly alternate and the A count B count 1 for all assignments This allows for one extra A or B unit at the end of the chain If the A and B counts must be strictly equal use the ratio constraint below instead Ato B ratio The ratio of A count to B count must fall within a specified range When either this or the next option is selected the Minimum and Maximum edit boxes are enabled Enter the minimum and maximum values of the A B ratio B to A ratio This is simply the inverse of the A to B ratio and is provided for convenience 41 After setting the desired matching combinatio
22. and Polymerix will smoothly adjust as the m z region changes The default mass mode is monoisotopic throughout the m z range To change the mass mode click on the Mass Mode tab in the Data Processing parameters pane Mass Mode Monoisotopic Convert Masses to Monoisotopic Above 1500 pa i i iy Bri C Chemical Convert Masses ta Chemical Below TU Da C Nominal Spectrum Select Peak Detect Mass Adjust Mass Mode 1 As can be seen above you may select one of three processing modes If Monoisotopic is selected all results will be reported based on assignments to monoisotopic peaks Likewise if Chemical or Nominal is selected results will be reported in that mode 2 For monoisotopic mode an additional option is to convert unresolved chemical mass peaks to the equivalent monoisotopic peak m z above the given m z value If this option is selected polymer formulas will be matched to the spectrum based on chemical mass and the result converted to the equivalent monoisotopic mass above the threshold 3 For chemical mass mode the additional option allows matches to monoisotopic peaks to be converted to the equivalent chemical m z below the given m z value If this option is selected formulas will be matched to monoisotopic peaks based on monoisotopic mass then converted to the equivalent chemical mass below the threshold 4 In either of the monoisotopic or chemical mode cases the optional processing ensur
23. and registration of Polymerix on up to 4 PCs using a combination of each PC s hardware information and a USB memory stick Once licensed the software may be run on any of the PCs but only one at a time The USB memory stick must be inserted at all times while A summary of the registration process Using the Registration Wizard to request a license key INSTALLATION LICENSING AND REGISTRATION the software is in use If the USB device is removed the software will cease working until the device is reinserted You must choose a license type when you first register Polymerix Once you have submitted a registration request and have installed a license key we may not permit you to change the license type until the license is renewed at the end of the license term We may issue a new license key under special circumstances such as a lost USB memory stick or an upgrade to a new PC but we may require that you first un register the software and send us proof via e mail These are the steps involved in registration 1 Run the Registration Wizard and enter information used to identify the type of license USB device if applicable and your contact details 2 E mail the registration information to us The Wizard will automatically create the e mail for you 3 Wait for our e mail in reply It will contain your license key When you start Polymerix for the first time it detects that the software is not registered and invokes the Re
24. as spectrum match tolerance and charge polarity are transferred from Homopolymer Analysis This transfers series definitions to the Homopolymer Analysis page for subsequent quantitative analysis In addition to the series definitions the spectrum match tolerance and polarity from the Find Series Settings dialog are transferred to Homopolymer Analysis Finally the Homopolymer Analysis mass mode is set to monoistopic and the use clusters option is enabled This shows the spectrum being analyzed Right clicking and dragging on the plot allows the user to select reference peaks for elemental composition determination This shows peaks from the spectrum being analyzed which match the currently selected series from the series list Peaks used as reference masses in the series are colored in red Right clicking and dragging on the plot allows the user to select a mass range for the series This shows the peaks which remain after peaks which match enabled series specifications in the series list have been removed Right clicking and dragging on the plot allows the user to select reference peaks for elemental composition determination Selecting the repeat unit This dialog is displayed when the user selects a new pair of reference masses It allows the user to select the polymer s repeat unit as well as charge state 54 FIND HOMOPOLYMER SERIES Select repeat and charge for mass difference 104 06264 from m z 11
25. can be computed The peaks in an autocorrelation spectrum indicate a frequently occurring m z difference where the mass spectral peaks have high relative abundance The autocorrelation spectrum can be computed by a brute force process For every point in a spectrum compute the m z difference between it and every other point in the spectrum and assign to each difference an intensity weighted by the intensity of the two points Create a difference spectrum by summing together the differences computed for every point For a large spectrum with many points this can be a very compute intensive process There is a simpler way based on the Fourier transform Without going into great detail the autocorrelation spectrum can be computed simply by taking the Fourier transform of the original mass spectrum convoluting it with itself and then transforming the result back What does the autocorrelation spectrum look like Take for example a singly charged ethylene oxide EO homopolymer spectrum Regardless of the polymer molecular weight there will be a series of peaks separated by the EO monomer mass or 44 Da There may be more than one EO series present possibly due to different end groups or adducts but each of these series will have a spacing of 44 Da as well The autocorrelation spectrum will therefore have a strong peak at 44 Da It will also have peaks of lower intensity at 88 Da 132 Da and higher multiples of 44 Da These overtones a
26. case click the New button and the Edit Chemical Entity dialog will be displayed Edit Chemical Entity ES Definition H me Ethylene Oxide Cancel Chemical Formula c2H 40 Ea Masses Monoisotopic Chemical Nominal 44 0262 44 0531 44 5 6 Enter a name for the monomer and supply the chemical formula in proper chemical nomenclature The formula will be validated and the masses automatically calculated Click the OK button close the dialog box The new entry will be added to the list in the Select Formula dialog To specify a repeat formula for a one time use type the formula in the By Formula edit box These formula are temporary and are not stored for re use Typing into this box will automatically select this option Click the OK button to validate the formula and close the Select Formula dialog box 43 DATA IMPORT AND PROCESSING 7 After closing the dialog box the selected entry or formula will be added to the parameters Isotope Correction iv Deisotope Repeat Formula Ethylene Oxide E spectrum End Group Ey Isotopic Mass Tolerance 100 ppm Adduct El Spectrum Select Peak Detect Mass Adjust Mass Mode Deisotope 8 You may optionally select an end group This may be important if the end group has a significant contribution to the observed isotope cluster for example if it contains chlorine bromine silicon or sulfur Click the button to the right of the End Group edit bo
27. may result in modification of this core structure including addition or one or more adducts and multiple charge states or a neutral loss For each combination of end group repeat unit adduct loss and charge state Polymerix builds a list of target species to be matched against the processed target spectrum In the nomenclature used by Polymertx this list of targets is called a serves Polymerix does not know in advance what target lists to build there is no built in database of polymers and their chemical and mass spectral properties You must specify everything Polymertx is to look for in the spectrum by defining homopolymer series The Find Homopolyer Series page introduced in version 3 0 of Polymerix provides an alternative means of defining homopolymer series in which the user can specify the details of a series definition by selecting from lists of chemical compositions generated by Polymerix based upon polymer spectral data Resultant series definitions can be transferred from the Find Homopolymer Series page Using the Find Homopolymer Series page it takes five mouse clicks and no keystrokes to set up the example which follows 65 HOMOPOLYMER ANALYSIS 1 Click on the Series Setup tab of the Homopolymer Analysis parameters pane With no series defined it should look as shown above 26 To start enable the first series by selecting the Enabled check box to the Zi 28 29 66 left of the first row Next i
28. on this page you will select the USB device used for the license key If the USB device is not inserted install it now and then click the Refresh button until it appears in the list of available devices Note If you are registering the software on more than one PC you must use the same USB device on each one We will not issue license keys based on different USB devices if you have only purchased a single license to the software Select the USB device from the list and click the Next gt button to continue 13 INSTALLATION LICENSING AND REGISTRATION Polymerix License Wizard 5 Please enter your contact details on this page We would appreciate complete details including mailing address We collect this information so that we can provide personalized support and updates for your software We do not share this information with anyone Click the Next gt button to continue 14 INSTALLATION LICENSING AND REGISTRATION Polymerix License Wizard 6 This final page shows the information that will be submitted as your license request Please review it carefully If you need to make changes click the lt Back button to go to the appropriate page If everything is correct click the Save button to create the registration request file 15 A summary of the license installation process Using the Registration Wizard to install a license key INSTALLATION LICENSING AND REGISTRATION Save License Reques
29. or panning in one by changes the other to the same axis range The Autocorrelation Analysis button is not displayed by default because Find Homopolymer Series see below supersedes the functionality of autocorrelation analysis in most cases To enable and show the button select View Autocorrelation from the Menu bar and then exit and restart Polymerix Clicking the Autocorrelation Analysis button on the control panel changes the right side to the Autocorrelation Page For an unknown polymer autocorrelation analysis may provide insights into the nature of the repeat units and total end group This page provides a way to review the autocorrelation and end group spectra and manipulate their settings 23 POLYMERIX WORKFLOW AND USER INTERFACE In the topmost pane the autocorrelation and end group parameters may be adjusted In the middle pane the autocorrelation spectrum is displayed In the bottommost pane the end group spectrum is shown Each spectrum is manipulated independently Find Homopolymer Clicking the Find Homopolymer Series button on the control panel changes the Series Page right side to the Find Homopolymer Series Page This page assists the user in determining the chemical formulas of a centroided spectrum s homopolymer repeat units end groups and adducts Once determined the chemical formulas can be transferred to the Homopolymer Analysis Page for quantitative analysis 24 Homopolymer Analysis Page POLYMERIX
30. pane is always the source for a copy operation The current spectrum pane is the one in which the last mouse click operation was performed To copy the pane do the following 1 Left click on the spectrum to select it Zoom in if desired the copied image will exactly as seen on screen 2 To copy the plot graphics select the Edit gt Copy Spectrum Plot menu command or press the Ctrl C keys to copy the spectrum image to the Clipboard The graphics can then be pasted into another application such as Microsoft Word or PowerPoint 100 Copying results COPYING EXPORTING AND PRINTING File Edit View Help bi Copy Spectrum Plot Ctrl C Copy Spectrum Data Ctrl D Copy Results Options tee 3 To copy the spectrum data as a peak list select the Edit gt Copy Spectrum Data menu command or press the Ctrl D keys This places a list of the spectrum peaks on the Clipboard These may be pasted into Microsoft Word or Excel as a table File Edit view Help Copy Spectrum Plot Ctrl C Copy Spectrum Data Ctri D Copy Results Options on The series definitions statistics and detailed assignments can be copied to the clipboard as a formatted table 1 Click on the Details tab to bring it to focus 2 Select the Edit gt Copy Results menu command to place a formatted table on the Clipboard This may be pasted into Microsoft Word or Excel as a table File Edit view Help D Copy Spectrum Plot Ct
31. peak width option helps when the spectra are very noisy and peak detection results in a cluster of smaller peaks around the true centroid Selecting this option will merge together each of these peak clusters to yield a single peak using an intensity weighting algorithm The peak width mode and peak width parameter selected for peak detection will be used to set the width of the merge window 8 The MS Threshold option is used to filter the smallest peaks out of a peak detected spectrum based upon a relative abundance threshold value 37 DATA IMPORT AND PROCESSING 9 Once all parameters have been chosen click the Apply button to set them Mass adjustment Overview of mass Occasionally the m z assignment read from the data file is incorrect due to inadequate adjustment calibration in the instrument software Polymerix offers a simple way to adjust the mass axis by applying a zero order mass offset linear slope or both If you suspect a problem in mass assignment click on the Mass Adjust tab of the Data Processing parameters pane m Mass Adjustment Offset 0 Da a Slope 0 ppm et a Adjust spectrum masses Spectrum Select Peak Detect Mass Adjust Deisotope 14 Click the Adjust spectrum masses checkbox to enable mass adjustment mode 15 To apply a mass offset click the Offset checkbox to enable offset adjustment You may either type an mass adjustment value in Da into the edit box or use the two set
32. peak within an isotope cluster is the monoisotopic peak This accounting is important for higher mass polymers when silver Ag is used as an adduct Automatic reference peak finding ignores peaks in the spectrum being analyzed which match any existing polymer series specifications Because the automatic reference peak finding algorithm is based on a heuristic it may not always correctly identify monoisotopic reference peaks from the same series and it may select reference peaks which seem less than ideal If the wrong peaks are identified the repeat unit selection dialog and the end group selection dialog will often show no potential chemical compositions or only chemical compositions which don t make sense to the user In such cases manual selection by right clicking and dragging on spectrum plots may provide a workable alternative 52 Series list Find button Clear button FIND HOMOPOLYMER SERIES Find Homopolymer Series page In the topmost panel series specifications and reference masses are displayed The top spectrum plot shows the complete spectrum being analyzed The middle spectrum plot shows the currently selected series with its reference peaks highlighted in red The bottom plot displays the residual spectrum in other words all peaks still unassigned In the above example the residual spectrum mass peaks are all less than 2 RA The Series list shows a list of polymer series specifications which
33. restricted element ratios DBE range is from 0 5 to 5 0 minimum end group m z is 0 maximum end group m z is 200 maximum result count is 10 C countis from 0 to 20 H countis from 0 to 40 O count is from 0 to 20 N countis from 0 to 6 ox cancel In this case Polymerix creates a new polymer series definition using C8H8 styrene for its repeat formula a combined end group of C4H10 and Ag silver as the adduct Peaks from the spectrum being analyzed which match the series definition are extracted and displayed Understanding reference peaks The Find Homopolymer Series tool uses pairs of adjacent monoistopic peaks in a polymer mass spectrum to determine potential repeat compositions and combinations of end group and adduct Pairs of reference masses are assumed to differ only by the monoisotopic mass of the repeat unit corresponding to molecular ions whose formulas differ in composition only by a single repeat unit Monoisotopic reference masses can be selected either automatically or manually Automatic reference mass selection is accomplished using the Find button Manual reference mass selection is accomplished by right clicking and dragging on spectrum plots Whenever Polymerix has both a reference mass and a hypothetical polymer formula the amount that the reference mass is in excess of the m z of its closest corresponding molecular ion in the polymer series is calculated and displayed either as Excess Mass in mDa mill
34. statistics are commonly used in polymer chemistry to describe a polymer The statistics calculated by Polymerix are M Number average molecular weight M gt Mind gt mi m M Weight average molecular weight My X MP m gt Mind l l 60 HOMOPOLYMER ANALYSIS m M Third order molecular weight M gt ui m Mi ni Polydispersity PD Polydispersity PD M M Degrees of DP Number average degree of polymerization polymerization DP M R DP Weight average degree of polymerization DP M R DP Third order average degree of polymerization DP M R Relative s Percentage of series of i th entry abundances S 100 nil Y n l S Percentage of spectrum of i th series entry S 100 nil OD ni J l w Weight percentage of series of i th entry Y w 100 n i M Y Cu Mi l W Weight percentage of spectrum of i th series entry W 100 n M X CY u MD j l Miscellaneous Formula and mass of i th series entry 61 HOMOPOLYMER ANALYSIS Monoisotopic peak error for i th series entry the difference between the m z value where the most abundant matched peak occurs and its calculated value Cluster error for i th series entry the difference between the intensity weighted mass average for a matched entry and its calculated value Totals by series and by spectrum Where M mass of i th series member n moles relative abundan
35. the two dimensional nature of copolymer distributions Clicking the Relative Area button on the workspace control panel switches the right hand side to the Relative Area Page This page is used to subdivide the spectrum into two or more regions to measure relative percentages of spectral intensity in each region This is useful if the polymer has a multimodal distribution or if there are multiple charge states and you are interested in the relative amount of polymer in each distribution 26 POLYMERIX WORKFLOW AND USER INTERFACE 19283 2 The top panel allows creation of areas by m z range The middle panel displays a tabulation of the total relative abundance in each range and the bottom panel shows a graphical view of each range What was covered in this chapter In this chapter we Described the analytical workflow modeled by Polymerix Explained how that workflow is mapped to the user interface and Introduced the major components and views in the user interface In the next chapter We will discuss the first part of the workflow data import peak detection and internal recalibration 27 DATA IMPORT AND PROCESSING Data Import Spectrum Selection and Processing How to import and reduce raw input spectra to a spectrum appropriate for polymer series assignment he most critical initial steps in assigning polymer compositions to mass spectral components are selecting spectra to be co a
36. 0 0 100 0 120 0 140 0 94 AUTOCORRELATION 43 409 17217 37 561 39 339 41 195 193 215 340 340 360 380 400 420 440 There are two sets of parameters used in the autocorrelation analysis The first controls the computation of the autocorrelation spectrum itself and whether the repeat mass determined automatically from it is used to compute the end group spectrum Repeat Mass Range Minimum 10 300 Maximum iv Auto compute end groups Apply The default repeat mass range for the autocorrelation spectrum is from 10 to 300 Da This will cover a large number of common monomers If observation of the experimental spectrum gives evidence for a larger repeat mass adjust these parameters and then click the Apply button The second set of parameters controls how the end group spectrum is computed By default the mass of the maximum intensity peak in the autocorrelation spectrum is chosen as the repeat mass In this case the only end group parameter available is the minimum end group mass which is usually set to zero To set a different end group minimum mass change the value then click the Apply button 95 AUTOCORRELATION End Group Mass So Minimum 4 In the illustration above the repeat mass value 43 946 Da was chosen using the mass of the maximum abundance point in the autocorrelation spectrum Applying this to compute the end group spectrum leads to a cluster of peaks near 43 Da
37. 01 54613 to m z 1205 60877 Repeat Error Errar Erro a n E E Long 0 035 0 035 i STE i 10404734 15 291 15 291 146 944 3 C7H40 j 104 02621 36 421 36 421 343 955 4 C3H404 I 104 01056 51 677 51 677 496 594 Monoisotopic m z values are 1101 546513 and 1205 60877 repeat m z is 104 06254 tolerance is 0 1 Da electron mode is normal restricted element ratios DBE range is from 0 5 to 5 0 charge count is D Se SS C count is from 0 to 10 H countis from 0 to 20 O count is from 0 to settings 10 N count is from 0 to 3 The currently selected row represents the users selection of repeat unit composition In the bottom panel all parameters that were in effect are shown These parameters can be adjusted using the Settings button which brings up the Find Series Settings dialog Selecting end groups and adducts End group and Polymertx displays this dialog when reference peaks are selected or when the Excess adduct selection Mass column for an entry in the Series list is clicked and either the dialog Apha End Group or Omega End Group column for the entry is empty 55 Find Series Settings dialog FIND HOMOPOLYMER SERIES Select end group and adduct for series with monoisotopic peak at m z 1101 546135 Combined Computed Error Error Error 1 C4H10 0 0 Aq 1107 54620 0 064 O 064 0 058 2 C5H205 5 0 Na 1101 542655 3 267 3 287 2 984 3 C7H18N202 0 0 Ag 1101 542217 3 959 3 959 3 594 4 CBH1SN40 0 0 A
38. 266 206 618 180 330 491 404 107 008 836 846 436 807 1289 486 1508 680 Page 8of8 79383 43 11038 52 13392 53 16017 27 17349 32 17214 57 16291 07 16357 00 12487 15 10269 04 2595 10 6606 95 5431 56 3914 74 2942 41 1427 70 1389 92 1013 69 600 23 585 97 205 46 234 87 131 16 10 70
39. 3 u L wi EL 4 i T s a h moe meee r b a yb s t ts gt A oe Hil 1800 3 aod 1700 5 t300 i4002 t5000 teoa 17000 T ea Amge Farag amyoera Fire Hessuns tetai sae Barges Deraan Het Filtered Residuals The Filtered Residuals tab shows only the peaks in the spectrum which have not been assigned to a series In the current example since the peaks corresponding to the polymer have been matched all that remains in the spectrum is low intensity noise Pere Mermoochy er Heo rdials 1303 990 8 10 108 0 1200 0 moo wht 10 9 ewes rea 8 1ene amp sen 8 gt Tiwas ampret riwer Peetak Ontee am Ortar Pet Details This presents a table containing quantitative details of every peak assignment for all series The left side of the table summarizes assignments for all series followed by additional groups of columns giving assignment details for each series by repeat unit count Depending upon the data being analyzed and the parameters being used for quantitative analysis columns for Homopolymer m z and Cluster m z may or may not be shown Vemneciew i wyan Daaa Twa i n H Beee i Desa fami A feared thew fean Peres DSim fect Mrargs s Cee eer fort 8 feet See feces 1 PET Piet os 1005 one w gs ooM se sss sez T E 1199 po ins IES jiin Cie a oom s jan g as mi a o a mti wii pH pii e i Ma iN a1 134 aa o S Sn ta Aiit OU SS me 22t teda bows ue ints US Ca SiL TA
40. 4 321 572 48 418 6393 41 21 0 73 0 50 0 25 965 550 965 561 10 767 1361 97 22 1 34 0 97 0 47 1009 577 1005 608 31 269 2518 73 23 2 17 1 64 0 76 1053 603 1053 608 5 570 4076 74 24 3 05 2 40 1 06 1037 629 1097 625 4 315 5723 39 25 4 25 3 45 1 48 1141 655 1141 654 1 438 7383 43 26 5 91 5 04 2 06 1185 681 1185 684 2 684 11058 _52 27 7 13 6 32 2 48 1225 708 1229 710 2 653 13332 53 28 8 53 7 83 2 97 1273 734 1273 741 6 965 16017 27 29 3 24 8 78 3 22 1317 760 1317 761 0 552 17349 32 30 3 17 3 01 3 19 1361 786 1361 795 9 234 17214 57 31 8 67 8 81 3 02 1405 812 1405 817 4 322 16251 07 32 9 03 9 46 3 14 1449 839 1449 844 5 413 16357 00 33 6 65 7 18 2 32 1453 865 1493 867 2 578 12487 15 34 5 47 6 08 1 50 1537 891 1537 893 1 436 10269 04 35 5 11 5 85 1 78 1581 917 1581 914 3 718 3535 10 36 3 52 4 14 1 23 1625 944 1625 981 37 130 6606 95 37 2 85 3 50 1 01 1669 970 1663 976 5 817 5431 56 38 2 08 2 59 0 73 1713 996 1713 995 0 870 3914 74 a9 1 57 2 00 0 55 1758 022 1758 043 21 251 2942 41 40 0 76 0 33 0 26 1802 048 1802 108 593 407 1427 70 41 0 74 0 33 0 26 1846 075 1846 059 16 036 1389 92 42 0 54 0 74 0 19 1890 101 1890 118 17 454 1013 63 43 0 32 0 45 0 11 1534 127 1534 105 22 173 600 23 44 0 31 0 45 D_11 1978 153 1978 118 34 847 585 97 45 0 11 0 16 0 04 20223 179 2022 100 79 001 205 46 46 0 13 0 153 0 04 2066 206 2066 187 18 865 234 87 47 0 07 0 11 0 02 2110 232 0 000 0 000 131 16 45 0 01 0 01 0 00 2154 258 0 000 0 000 10 70 Repeat Percent S
41. C Polymerix LCComponent dll Extract C Polymerix SAShimadzuProvider dll 100 Extract C Polymerix SADEProvider dll 100 Created uninstaller C Polymerix Uninstall exe Create folder D ProgramData Microsoft Windows Start Menu Programs Polymerix Create shortcut D ProgramData Microsoft Windows Start Menu Programs Polymeri Create shortcut D Users Public Desktop Polymerix Ink Create shortcut D ProgramData Microsoft Windows Start Menu Programs Polymeri Completed a Mullsart Install System v2 45 cae 6 Once installation has completed click the Close button to end the setup program This screenshot shows the installation wizard after the Show details button was clicked The details shown for your installation might be different Registration After installation the next step is to register the software and submit a request for a license key Registration is required The software will not function until registration has been completed and a valid license key has been entered You may license your installation of Polymerix using either a fixed license or a floating license A fixed license locks your copy of Polymerix to a specific PC The registration procedure reads information from the PC hardware creates a license key request Using this request and the term of your license we create a license key that permits Polymertx to run only on the chosen PC A floating license allows installation
42. CESSING Polymerix File Edit View Help New Ctrl N Open Ctrl O Save Ctrl S Save As Open Template Save Template Ctrl V Import Chromatogram Import Spectrum Export Results Export Spectrum Print Ctrl P Print Setup Recent File Exit 2 To load a previously saved template use the File gt Open Template menu command This will display a file selection dialog from which you can choose the desired template file When you close the file dialog by clicking its OK button the template file will be read in and all of the current parameters replaced with the new ones read from the file Parameter template files do not contain any experiment information only parameters Therefore parameter templates can be applied to experimental data of any sample of a similar type They can be transferred from PC to PC since they also do not contain any absolute folder or file path information What was covered in this chapter In this chapter We presented the Data Import browser and described how to select and import a data file We also described how in the case of a multiple scan dataset you can select a single spectrum or a range of spectra to be co added prior to impott We described how to select spectral sub ranges for processing We discussed the peak detection process and how to set peak detection parameters 47 DATA IMPORT AND PROCESSING We presented ways to overcome inade
43. Homopolymer analysis parameters are spread among three tabs at the top of the Homopolymer Analysis page Match Tolerance Combine Within Seres With Same Repeat Constant 0 1 Da 100 M Use clusters M Charge states Positive iv charge 0 9 Da at mz 1000 Processing Mass Adjust 1 The first tab Processing contains parameters to control the tolerance for matching m z values predicted from the homopolymer series definition with experimental peaks in the target spectrum Proportional ppm Adducts and losses End groups TOF 2 First the Match Tolerance parameter should be set to correspond to the instrument scan function type As in peak detection these choices are Constant used for quadrupole and ion trap instruments where the peak width is constant or nearly so over the mass range of interest This choice also works well for very high resolution data such as is produced by FT ICR or Orbitrap instruments Proportional used for magnetic sector instruments and TOF used for time of flight instruments 20 Select the appropriate scan type and then set the matching tolerance appropriately In general for well resolved peak detected spectra where there are now very closely spaced peaks the match tolerance can be set to any reasonable value that would not result in a tolerance window so wide as to cause mismatches In the example spectrum peaks are separated by 44 Da s
44. Note that the plots will be displayed in the report at their current on screen zoom state Homopolymer spectrum Produces a page containing the plot shown on the Assignments tab of the Homopolymer Analysis page 104 COPYING EXPORTING AND PRINTING Homopolymer filtered spectrum Produces a page containing the plot shown on the Filtered Assignments tab of the Homopolymer Analysis page Homopolymer residuals spectrum Produces a page containing the plot shown on the Filtered Residuals tab of the Homopolymer Analysis page Homopolymer distribution plots Produces multiple pages one containing a plot of the total series spectrum and followed by additional pages each containing an individual series spectrum plot Homopolymer summary Produces a page containing the Homopolymer Results Summarty as seen in the middle pane of the Homopolymer Analysis page Homopolymer details Produces one or mages of the tabulated homopolymer assignment details as seen on the Details tab Copolymer spectrum Produces a one page plot of the copolymer Assignments spectrum Copolymer filtered spectrum Produces a one page plot containing the copolymer Filtered Assignments spectrum Copolymer residuals spectrum Produces a one page plot containing the copolymer Filtered Residuals spectrum Copolymer summary Produces a page containing the Copolymer Results Summary as seen in the middle pane of the Copolymer Analysis p
45. Polymerix s peak detection parameters allow for three different instrument scan function types e Constant or near constant width peaks such as produced by quadrupole or high resolution FT ICR or orbitrap instruments e Proportional width peaks such as produced by magnetic sector instruments and e TOF mode peak widths as produced by time of flight instruments Select the mode appropriate for your data type then click the Apply button Evaluate the results of peak detection by zooming in on a small section of the bottom target spectrum and compare the middle profile spectrum with the bottom peak detected target spectrum 35 Setting peak detection parameters DATA IMPORT AND PROCESSING To zoom out one step simply click the left mouse button without dragging Are there detected peaks in the bottom spectrum where you would expect them to be based on the profile peaks in the upper spectrum In this case the peak detection parameters are probably OK and do not need to be changed Are closely spaced profile peaks merged into a single centroid peak Are profile peaks split into two centroid peaks Either of these situations indicates that the peak detection parameters are not set correctly To set peak detection parameters click the Peak Detect tab on the top pane of the Data Processing page Peak Width Resolution 102 Valley Peak Detection Baseline Subtraction Merge Spectrum MS Threshold
46. RT AND PROCESSING Restricting the processing mass range Occasionally a spectrum will contain contaminants or other non sample peaks such as MALDI matrix ions or chemical noise If these are in a region of the spectrum that can be isolated from the region containing sample peaks then you may use the Processing Mass Ranges parameters to define one or more spectral ranges to be included for peak assignment 8 On the Spectrum Select tab there is a table containing a list of mass ranges that will be included for processing By default then entire spectral range is included a PALU a wen ae EL Ip ane ah mie 9 You chose a restricted mass range in one of two ways First by directly editing the mass values in the table or second by a right click and drag operation in the spectrum window If you edit the mass values manually click the Apply button after setting them If you right click and drag the table and spectrum will be updated automatically In the spectrum above there are no useful peaks outside of the 800 2000 Da range so a right click and drag was used to restrict the processing range as shown below Processing Mass Ranges Include From To a a 10 The processed target spectrum in the bottom pane is also updated to eliminate peaks outside of the chosen range 33 DATA IMPORT AND PROCESSING 11 You may select additional ranges if needed for example if a major imp
47. SIERRA ANALYTICS INC Version 3 0 0 M Polymerix Software User Manual VERSION 3 0 0 MARCH 2013 Polymenx Software User Manual ANALYTICS Copyright 2010 to 2013 Sierra Analytics Inc 5815 Stoddard Road Suite 601 Modesto CA 95356 USA 1 209 545 8506 Polymerix MassSpec com http www MassSpec com Table of Contents MEO AUC UO osise ian ceessnaaaieuteohadoiet een Siheceeenemiecceets 1 How this Manual is organized cccecccsececeeeceeeeeeeeseeeseeeeaeeeseeeeaes 1 Some conventions used in this MANUAl ceccecseeeeceeeeeeeeeeeeeees 1 Senra SW k 6c gt eee eee ee E E eee ae ee ae ee 2 CE NNO te Oh PAE E E A TE TE 3 Goals TON IMIS CHADICM sereen aa 3 INSLANING FONMETX cessere eeue TETSU 3 Rog raUo E E 9 Installing the license key cccccccececseeceeeeceeeceeeceeeecseesseeseeeensees 16 What was covered in this Chapter ccccseecceeeeceeeeceeeeseeeesaees 19 NANE AEX CHADICN ea E ES 19 Polymerix Workflow Model and User Interface Overview 20 Goals Tor This CHAD R oi scse acta ip acess a eee eee 20 The workflow model cccceeccececeeeceeeeeeeeseeeceeeeseeeseesueeseeesaess 20 Introducing the Polymerix user interface ccccceeecseeeeeeeeeeeeees 22 What was Covered in this Chapter cccccececseeeceeeeseeeeeeeeeseeees 27 In the next chapter ccasnsaideasarntandonirivacunddie tends ireranduliudantadiadaauddetonas 27 Data Import Spe
48. SIS plot is initially scaled to actual relative abundance on a 0 100 scale left click with the mouse in the plot window to autoscale it to the full height of the plot Repeat B Plot Likewise horizontal slices through the AB Distribution table can create plots of the B unit spectrum for fixed A counts Similarly the scroll bar is used to step through the A count increments Two steps in A count are shown in the screen shots below The first is the B spectrum for AO the next is the B spectrum for A1 Aasegremernts bor A Wn 1094 636 Ww 1028574 Ne VE 20 PD tO DAs 06959 Dew 17 0E Os 17T Uatun ov erm 285 223 ew 1h8e 6 eo seen 3 an PLPR ay t213 azz PL 7 7 7377 82 N i 17782 2 ar ace n3 a a 8 Joha d ised a seca o rete s NN ee a ee ee ee ee ee et ie Ae a8 1000 wen toos 13850 1400 2 1232 0 1000 e tase aoto ana e 2000 0 2j Fitnes rst Getai Mons dh Utter A Mipoees Repor E Sete Giai he Anuga far As Wn 1080 729 biw 1080 020 Me SOON 410 PO 10 DPn 11 56 Ow t7 420 Dre Ther A Uot me cs as i7 i aw b aft p Bu nua g Lo F ste Bie 11 ooo 4y m wee GI it r on 8M 43 5 84 oe ee 2 ax ji ta7 WS j S52 1485 8 vaya 4 vaata 1688 1 129 2 en PO Pr cee eee re pene Ge o ay me mot teat lt t0000 oon tiee 0 1xH 8 14901 t00 gt gt f t700 ta p 1888 0 DIA d sj iter Peret Demians Detsis Aang DOrttuan Zopest A Sistitzs A Stutates Gepmet A Per ort Here it can be s
49. Sut ay i das Mib sii roS teat base ism on Ai gt ae i 0i ae 177 Jiss I7 OC h2 ihote wie 51 SAL 7 tab LEA oa j la 5 i enn i i 17 thi i th fe toy 1 a aad TUS E ame FEAL tw om 4 E gt the hw 17A pile as mya sie iha re wr Maen ts D s 2 1 1412 gt ay B75 aiy 0077 U EE oe tit oa E E 2 it Fa 735 Oa Sat wh c was iD tgal ies Lets Ko AIT Sel TES J 207 lay sues mog 7 Th TIm D ti 27 eeece TEN EMS IS 7 EXE Soom ISR on7N 200 WEIR uas IBIU J UII OOD 4 now LSJ 7a CON tena 771m cono Wits UTIN cs UNAS UN Ga cix gons pous maos Smear Sco CSD Sime Tir SSI IIN UNNI osu mese m mmh ian a a m CSET rR inse 641 IT S 320 DEW Ixi 2ST iA Eni oem poti 3i WDY tux sn CHa 2 954 E an ci Jaang 4 reat 430 1c wer O06 eS se tim bot j l Anget fim igeret Piers n p l A TA Rro Mass Ranges The Mass Ranges table displays a list of the calculated m z ranges used for determining series assignments ordered by m z value The table lists the m z window 71 HOMOPOLYMER ANALYSIS as defined by the matching constraints series name repeat unit count and the number of isobaric overlaps collisions if any ae gas Wass re Ae Moss ias S Sh ee a i F nins paii te s DIIT TKSD 5i i 5455 SSENT S 1 ms moms 13 899467 02 450 E 2 ia erosi x Mie pim zi siasio sit s n Wiis WHI a 1084S tie S1 a WIE SA TMII GZ a WES Sa Tri S41 a uT ISTE Tee ST 7 1087 5235 1900 7975 Si x muso 1 TE
50. WORKFLOW AND USER INTERFACE oe Ose SAAT al Tees bass ats tss 1180 8 nesa 13 0 wisa iia In the topmost pane series parameters and setup results are displayed The top spectrum plot shows the spectrum being analyzed The middle spectrum plot shows a selected series The bottom plot displays the residual spectrum in other words all peaks still not assigned to a series Clicking the Homopolymer Analysis button on the workspace control panel switches the right hand side to the Homopolymer Analysis page WORE SEE 1257 158 i EN 1 eT AA Sr MRA TS ee k mera TAT a 55 IRAI RA duki Aes oy ae OSB ae oes ee Ser ayd veh FUME tta FAAA eps Sess ASS aoe INAS IIVO RIFTA 39 IET S eP aL SL E E I T E a Hemi peer Foon Aesha j Der Manes Ringes Darian Pt 25 Copolymer Analysis Page Relative Area Page POLYMERIX WORKFLOW AND USER INTERFACE The top pane of this page contains tabbed views for defining homopolymer series and editing peak matching parameters The middle pane contains a table of the polymer series molecular weight distribution statistics and relative amounts and the bottom pane contains a series of tabbed views that display the assignment results in graphical or tabular form Clicking the Copolymer Analysis button on the workspace control panel switches the right hand side to the Copolymer Analysis Page It is very similar to the Homopolymer Analysis page but with additional features specific to
51. a number of choices for the items to include in the report Polymerix File Edit View Help New Ctrl N Open Ctrl O Save Ctrl 5 Save As Open Template Ctrl T Save Template Ctrl Import Chromatogram Import Spectrum Export Results Export Spectrum Print Setup Recent File Exit 1 Select the File gt Print menu command An enhanced Print dialog will be displayed 103 COPYING EXPORTING AND PRINTING Printer Mame he Lasenlet 1320 PCL 5 Setup Status Offline 0 documents waiting Type hp Lasenet 1320 PCL 5 Where 10 210 0 50 Comment Print range C All fe Fages from li bor i Selection Copies Number of copies 3 Reports to print Data processing Copolymer spectrum i Homopolymer spectrum Copolymer filtered spectrum i Homopolymer filtered spectrum Copolymer residuals spectrum Copolymer summary i Homopolymer distribution plots Copolymer details i Homopolymer summary lf Homopolymer details Preview Cancel 2 In addition to the normal printing options at the top of the dialog the lower part of the dialog contains Polymerix specific printing options Selecting an option will create one or more pages in the output containing the chosen results These are Data Processing Produces a page containing data file header information the raw input spectrum plot and the processed target spectrum plot
52. age Copolymer details Produces one or more pages tabulating the copolymer assignment details sorted by series and by increasing B count within A count Autocorrelation results This check box is only shown if autocorrelation has been enabled by use of the View menu s Autocorrelation item Produces a page with the autocorrelation and end group distribution plots at their current on screen zoom level An example homopolymer analysis report is included in the Appendix 105 COPYING EXPORTING AND PRINTING What was covered in this chapter In this chapter we Described how to customize plot display options Showed how to copy plots spectral data and results to the Clipboard Explained how to save and export Polymerix results In the next chapter There is no next chapter you ve reached the end of the manual It s time to go to work and analyze some polymers 106 RELEASE NOTES Release Notes Version 3 0 Major new features The major new features improve polymer analysis of centroided data with high mass accuracy The new centroid cluster matching feature matches individual peaks in each isotope cluster for a polymer series This allows matching of centroided data without deisotoping For data with high mass accuracy the new matching feature allows use of much smaller match tolerance values Use of smaller match tolerance values can reduce or eliminate mass overlaps between different series
53. at were in effect are shown These parameters can be adjusted using the Settings button which brings up the Find Series Settings dialog Setting constraints on chemical composition The Find Series Settings dialog is used to set constraints on the chemical compositions considered when attempting to match reference peaks The available chemical elements used for constructing repeat units end groups and adducts are specified here 56 Element tables FIND HOMOPOLYMER SERIES The Find Series Settings are made persistent on the user s local computer so that when Polymerix is next started the same settings will be in effect None of the Find Series Settings are saved with Polymerix document files The screen shot below shows the default Find Series Settings for Polymerix Find Series Settings Atomic Min Ma Atomic Min Max ewe S es I Element Ratio Restrictions M Positive Charge Mixed adducts When considering possible chemical formulas Polymerix restricts the formulas under consideration using element tables There are three independent tables which correspond to repeat units end groups and adducts These tables are shown on the right side of the Find Series Settings dialog For repeat units and end groups the number of atoms of a particular element can be numerically constrained for the repeat unit or end group Change an entry by selecting and modifying its text Add a new entry to an element
54. ce of i th series peak R mass of repeat unit The Homopolymer Analysis page user interface Switch to the Homopolymer Analysis page by clicking the Homopolymer Analysis button on the Workspace Control Panel The Homopolymer Analysis page is shown below after the data processing steps have been completed For the PEG example spectrum shown the data processing options included limiting the mass range to between 800 and 2000 Da and applying peak detection and baseline correction arames ie or j pe twas TT arna Febe i pigi C Agoes T g P ip P Miis ori Mio M Secs l tipo At the top of the page are the parameter setting tabs There are three of these a Processing tab for setting peak matching tolerances and options for combining related homopolymer series a Series Setup tab used to define the homopolymer homologous series that will be matched as part on the analysis and a copy of the Mass Adjust tab described previously in the Data Processing chapter 62 Match tolerance HOMOPOLYMER ANALYSIS In the middle of the page is a table of the homopolymer molecular weight distribution statistics These are calculated for the sample as a whole and for each series individually At the bottom of the page there is a series of tabbed panes showing the assignment results in various graphical and tabular formats Each of these will be described in more detail in the sections below Setting homopolymer processing parameters
55. ceessseaaeeeesseaaeeeeessaaeeeees 107 Updates bug fixes and NeW features ccccceeccccceeeceeceeeeeeseeeeeeeaees 107 WEES ION Osona aint laiaiataldnansdutucansdutueaaucntuntauutunde 108 Updates bug fixes and new features ccccceeccccceeeceeceeeeeeeeeeeeeeaees 108 EXA MPIC COO MM orar 110 INTRODUCTION Introduction Some tips on using this manual effectively elcome to Polymerix We re looking forward to helping you get the most out of your data with a minimum of headache But before getting started we would like to explain some of the conventions we use in this manual which we hope will make it easier for you to understand how to use the manual and software to maximum effectiveness How this manual is organized Our goal in writing this manual is to guide you from initial installation and registration to expert use of Polymerix by presenting the material in a way that models your workflow Instead of a programmer s point of view Here s the File menu It has five submenus we try to take a task oriented approach in which each major task is broken into a series of smaller subtasks We use a minimum of explanation and a maximum of illustration to help make the tasks as clear and easy to follow as possible So the first part of this manual is a description of the tasks to get you started in using Polymertx In many cases the steps will be sufficient to solve most problems However Polymerix i
56. ctrum Selection and Processing 28 Goals for tiS Chaple nA 28 IMPONG AA Aea E E E A EE 29 Restricting the processing mass range ccceccceeeeseeeseeeseeeeeeeens 33 DETECIING DEAKS raa ee 35 Mass adjustment aaas hart ene ee eee 38 MaS 90 0 ener er meee ener cee eee eee ee eee ene ee eee 40 DCIS O10 DING canoa 41 Saving and loading parameter template files ccceeceeeeeee ees 46 What was Covered in this Chapter cccccsececeeeeceeeeceeeeseeeeeeeees 47 I TNE MEXL CNADLSN seit aes a tin ee ee 48 Find Homopolymer SEVICS cccceeecceeeceeeeceeeeeeeeeeeeseeesaeees 49 Goals TfortNi S ONapte en E EA E E 49 OW CIV EW reaR E eo ane aes 49 Simplest USE CASE example cccccccsseeeeceeeeeceeeeeeeeeeeseeeeesaeeeeeas 50 Understanding reference peaks cccccccseeeceeeecseeesseeesseeeseeeeees 51 How Polymerix handles reference peak Selection ccssee 52 Automatic reference peak selection cccccceeeeeeeneeeeeeeeaeeeneees 52 Find Homopolymer Series PaQe ccccceececseeecseeeeseeseeseeeeesaeees 53 Selecting the repeat UNIt csacsi 54 Selecting End groups and ACCUCTS cecceececeeeeceeeseeeeeeeeeeees 55 Setting constraints on Chemical COMPOSITION cccceeeeeeeeeee es 56 TFOUBICSHOOUAG TI DS arsina aa 59 What was Covered in this Chapter cccccseccceeeeeeeeeseeeeseeeeeeeees 59 IMC OX UCM AlCl een 59 Homopolymer
57. dded from a multi scan data set accurately processing the input spectrum to create an optional centroid peak list correcting the mass axis deisotoping and applying corrections to accommodate loss of resolution at high m z Goals for this chapter In this chapter we will explain how to Use the data browser to select and import raw data from vendor format data files Select a range of spectra to be co added from a multi scan data set m Select an m z sub range to exclude non polymer peaks Set peak detection parameters Apply a mass axis adjustment to reduce mass calibration errors Adjust spectral assignment to accommodate resolution loss at high m z m Use deisotoping to merge higher isotopomer peaks into a single monoisotopic peak in each isotope cluster 28 DATA IMPORT AND PROCESSING Importing data When you installed the Polymertx software you selected one or more data format options This then installed specialized data import software that recognizes files in those vendors formats and a browser that can display information contained in those files when navigating through your hard disk folder hierarchy In addition Polymerix supports import of peak list data contained in ASCII mass intensity peak list files Ei a Polymerix The data import 1 Start the software as usual by double clicking the Polymerix icon Click browser the Data Processing button in the Workspace Control panel 2 To impo
58. determining series assignments ordered by m z value The table lists the m z window as defined by the matching constraints series name A and B unit counts and the number of isobaric overlaps collisions if any 82 COMPOLYMER ANALYSIS EFE 639 4593 51 3 t 691 3606 653 4754 51 2 9 665 4039 667 4916 51 1 10 679 4191 681 5077 51 0 11 695 4135 697 5030 51 3 9 409 4287 711 5191 51 2 10 7734440 725 5352 51 1 11 73 4592 740 5540 51 0 12 793 4537 756 494 51 3 10 76 4689 770 5655 51 2 11 781 4842 784 5815 51 1 12 795 4994 796 5976 51 0 13 611 4939 814 5929 51 3 11 625 5007 828 6090 51 2 12 639 5244 942 6251 51 1 13 Goj0d0 896 6411 317 0 14 669 5347 872 6364 51 3 12 663 5495 286 6525 51 2 13 297 2647 900 6606 51 1 14 911 5800 914 6846 31 0 15 9775 45 930 6799 31 3 13 941 5898 544 6959 1 2 14 955 6051 958 7120 1 1 15 AB Distribution This interesting table is a top down view of the A vs B distribution A repeat unit counts increase downward on the Y axis while B counts increase along the X axis For every AB combination where a matching peak was found the table cell contains the relative abundance on the peak In addition the cells are color coded the more intense the color the higher the relative abundance i ist EN an lo 28 2 Ve AO 8 HE nH 7 Sw aa a 26 tij 20 6 n amp WH Bo wn 2 Si ti tS 20O ib i Gu MS JATSEN Say Be MT Di nin s dt e 83 COMPOLYMER ANALYSIS
59. e to easily set up homopolymer series definitions by selecting from candidate lists of chemical compositions generated from polymer spectral data Described reference masses and how they are used by the Find Homopolymer Series page Showed how to manipulate the various settings which are used when constructing candidate chemical compositions In the next chapter In the next chapter we will present the procedure for homopolymer analysis and describe the tables and graphics produced as a result of homopolymer series assignment 59 HOMOPOLYMER ANALYSIS Cl Molecular weight averages Homopolymer Analysis Defining and renewing homopolymer seres assignments produce a processed target spectrum the time has come in the case of a homopolymer sample to set up the definitions of the homopolymer series and assign compositions to spectral peaks N fter Polymerix has appled all of the data processing parameters to Goals for this chapter In this chapter we will Describe the molecular weight distribution statistics calculated by Polymerix Illustrate in detail the user interface for creating homopolymer series definitions Explain how to browse the series assignment results in tables and graphics Molecular weight distnbution statistics One of the primary goals of the Polymerix software is to produce molecular weight distribution statistics on the total sample as well as each of the polymer series within it These
60. een that the B distribution is smooth for each of the A counts What was covered in this chapter In this chapter we Described the definition of a copolymer used by Polymertx Explained how to set processing parameters and define copolymer series 86 COMPOLYMER ANALYSIS Presented the various result plots and tables and illustrated the utility of each one In the next chapter In the next chapter we will describe the relative area tool 87 RELATIVE AREA The Relative Area Tool Using the relative area tool the total relative abundance due to spectral features in selected parts of the spectrum T he Relative Area tool is just that a simple tool that gives an estimate of Goals for this chapter In this chapter we will Describe the Relative Area page Explain how to select spectral regions for relative area measurement The Relative Area page Like other Polymertx pages the Relative Area page consists of three panels RELATIVE AREA At the top of the page are parameters for specifying the relative area regions In the middle is a pane containing a table of the relative area ranges and relative abundance percentages Finally the bottom pane displays the processed target spectrum with each area highlighted in color Specifying ranges There are two ways to specify relative area ranges Mass Ranges to Analyze Range From To 1 850 4 1164 1 11626 15226 1523 3 1987 4
61. eing able to find any results Overview The spectrum being analyzed must have been peak detected and should not have been deisotoped The masses of selected monoisotopic peaks from the spectrum are analyzed and candidate lists of the potential chemical components which correspond to those masses are generated The user chooses from these lists of candidate compositions generating polymer series definitions which can be used for quantitative analysis When multiple components are present the process can be repeated 49 FIND HOMOPOLYMER SERIES Spectral data with high mass accuracy will work best with the Find Homopolymer Series page Correct selection of neighboring monoisotopic peaks from the same polymer series is crucial for the correct determination of chemical compositions Finally the tools are constrained to determining composition of homopolymer series only as opposed to copolymer series Simplest use case example The simplest example of using the Find Homopolymer Series tool to determine the chemical composition of a homopolymer is as follows Load a dataset and select a peak detected polymer spectrum for analysis Navigate to the Find Homopolymer Series page m Click the Find button Choose a chemical formula for repeat unit from the list provided by Polymertx and click the OK button Select repeat and charge for mass difference 104 06264 from m z 1101 54613 to m z 1205 60877 x zE l 104 04734 15291 15251
62. erently than continuum data Matching based upon single masses and matching of continuum data based upon isotope clusters share the same algorithm for making intensity assignments and resolving intensity assignments in overlapping series Isotope cluster based matching of centroid data uses a different algorithm for resolving intensity assignments in overlapping series The simple matching algorithm This algorithm is used when the Use clusters option is not selected or when Use clusters is selected and the data being analyzed is continuum data The steps in the algorithm are as follows 1 Use polymer series definitions and the match tolerance to generate mass ranges corresponding to each series Each mass range corresponds to a distinct repeat count or for copolymers a combination of repeat A and repeat B counts 2 Partition the mass ranges into those without m z collisions and those with m z collisions 3 Assign intensity values from the spectrum being analyzed to the collision less mass ranges 4 Construct absolute intensity estimates for the mass ranges which have not yet been assigned using the assignments determined in the previous and linear interpolation and extrapolation 5 Assign intensity values from the spectrum being analyzed to the collided mass ranges The intensity estimates from the previous step are used to determine a proportional to the estimates intensity distribution to each mass range for a spectrum da
63. erface is similar to that used in many popular software applications There are menu and tool bats a set of main workspace pages and a control panel for rapid switching between these pages These are indicated in the screenshot below p y be MONU Sig DJE Aa et toolbars Workspace page divided ipto panels as ta Ia ow an 1 Posce Mant borage 0 09 te Of The buttons in the workspace control panel are used to switch between the top level pages on the right In almost all cases one of the first steps in your workflow will be to import raw data perform peak detection and other processing and review the results to ensure that the processing is correct The Processing page shown below is used for that purpose 22 Autocorrelation Page POLYMERIX WORKFLOW AND USER INTERFACE DEU Au PF LLI Late 7 soem Pp On teatar mer H Oo C29 x oe osu polystyrene 1000 bt HT 200 Spectrum 1 717055 Ports ore aa S 100000 3 M7055 Ports Mass Range 195 20 to 3561 00 Ria 1BECCO 3 4i The topmost pane uses tabs to choose between different data import and processing parameter specification editors In the second pane is the TIC or equivalent for multi scan infusions or other sampling The third pane shows the raw data as imported prior to any processing In the bottom pane shows the final processed spectrum after all processing parameters have been applied The m z axes of the spectrum plots are linked and zooming
64. eries Percent Computed Found Error Found Count Series Weight Spectrum Cluster m2 Cluster m z na Intensity 18 0 07 Oo 04 o o02 233 938 535 106 1167 264 124 29 19 D_ 17 0 07 0 04 877 989 877 713 276 026 139 43 20 0 34 0 22 0 12 322 039 321 867 172 183 6393 41 21 0 73 0 50 0 25 966 089 966 063 26 874 1361 397 22 1 34 0 97 0 47 1010 139 1010 052 87 239 2518 73 23 2 17 1 64 0 76 1054 189 1053 946 243 443 4076 74 24 3 05 2 40 1 06 1098 239 1098 085 154 035 5723 39 117 RELEASE NOTES Polymenx Processing Report Homopolymer Analysis Results Printed Wed Mar 06 2013 15 42 07 25 26 27 28 Dua be aR SB 39 40 41 43 44 46 47 46 CO0O0O FC FF O O e B A w OO g Wo oo i ODOH W W i i O a oe b PSbpeERZRAJBBEL AR H S E E oer Fo tb i i i l p r r REP POR A OOP OB ON eR BOB Ho 1142 288 1186 337 1230 386 1274 435 1318 518 1362 569 1406 620 1450 671 1494 722 1538 773 1582 823 1626 873 1670 924 1714 974 1759 024 1803 073 1847 123 18931 203 1935 254 1979 305 2023 357 2067 408 2111 459 2155 510 118 1142 052 1186 023 1230 018 1274 151 1318 206 1362 279 1406 317 1450 418 1494 443 1535 653 1582 490 1626 529 1670 589 1714 741 1758 697 18503 07 1846 917 1891022 1334 763 1379 412 2024 193 2066 971 2112 748 2157 319 236 025 313 878 367 631 284 208 312 340 290 354 303 756 253 130 278 712 119 716 332 725 344 434 334 780 232 861 327 147 5
65. es that mass errors due to lack of resolution are minimized and that mass values are consistently reported across the entire spectral range Deisotoping The final topic in this data processing chapter is deisotoping This is the process of applying a model chemical formula to each isotope cluster in a centroided spectrum and merging all higher isotopomeric peaks into a single monoisotopic peak 41 DATA IMPORT AND PROCESSING The new centroid cluster matching feature which was added to Polymerix in version 3 0 should be used instead of deisotoping Tools on the Find Homopolymer Series page will only work with non deisotoped spectra In monoisotopic mass mode Polymerix will match predicted polymer m z values only to monoisotopic peaks and use the resulting matched intensity in the molecular weight statistics calculations For polymers with a wide polydispersity and thus wide mass range this will result in bias since higher m z isotope cluster have more abundance in higher isotopes To eliminate this bias the deisotoping option 1s provided Note Deisotoping is only available when the processed spectrum is peak detected If peak detection is not selected the deisotoping parameters will be disabled To set deisotoping parameters click the Deisotope tab on the Data Processing parameters pane m lsotope Correction r Deisotope Repeat Formula End Group Isotopic Mass Tolerance IUU ppm Adduct
66. ever removes more of an isotopic peak than the model predicts If there are overlapping isotope clusters where a higher isotopomer of one cluster is isobaric with the monoisotopic peak of another only as much of the higher isotopomeric peak is removed as is predicted from the formula leaving a residual peak This could happen for a polymer 45 DATA IMPORT AND PROCESSING mixture with two different end groups one containing a C C double bond and the other a C C saturated bond This is a 2 Da difference and could result in an isobaric overlap between the monoisotopic peak of the saturated species with the M 2 isotope of the unsaturated one Saving and loading parameter template files After all that work of creating appropriate processing parameters it would be a shame if you had to re enter all that information every time you used Polymerix on a similar sample To avoid this Polymerix allows storing parameters in files as named templates Polymerix parameter template files use the pmt file extension Edit view Help New Ctrl N Open Ctrl O Save Ctrl S Save As Open Template Ctrl T Save Template Ctrl Import Chromatogram Import Spectrum Export Results Export Spectrum Print Ctrl P Print Setup Recent Fle Exit 1 To save parameters to a template file use the File gt Save Template menu command This allows you to choose a file name and save the parameters 46 DATA IMPORT AND PRO
67. f desired change the name of the new series by clicking in the Label box and typing a new name By default Polymertx entitles each series as S1 S2 S3 and so forth You may change these to more descriptive names Specify End Group xi By End Group Name x New Edit Delete By Formula OH By Mass EE Next click in the Alpha End Group box to display the Specify End Group dialog box shown above If the desired alpha end group appears in the list you may select it and then click the OK button Otherwise you may define a new end group by clicking the New button This will display the Edit End Group dialog box which was described in an earlier chapter End groups defined in this way will be added to the list for future use Alternatively you may temporarily specify the end group either by chemical formula or by mass Using a chemical formula is strongly preferred because then Polymerix can calculate monoisotopic chemical HOMOPOLYMER ANALYSIS 30 31 32 or nominal mass to high precision If you do not know a chemical formula and must enter a mass do so to the highest precision possible as many decimal places as can be experimentally determined Click the OK button to close the dialog box and place the selected end group information in the series definition Specify Monomer x By Monomer Name none Acrylonitrile Cancel Butadiene By Formula AA a By Mass a
68. font is used to present items from Polymerix s parameter and results formats or to indicate text you would write in scripting files used to extract and process these formats Sierra s Web site Visit our Web site at http www MassSpec com for more Polymertx information application notes publications and other useful information We plan to update the site frequently with new content INSTALLATION Starting the installation program LICENSING AND REGISTRATION Cl Getting Started How to install Polymerix submit registration details and install a license Rey yout putchase you may have a limited or a perpetual term license In either case we will issue a license key valid for the term of your license which must be installed on your PC before the software will function At the end of a limited license term the software will cease to run until you renew yout license and obtain a new key P olymerix is a registered and licensed product Depending on the details of Goals for this chapter In this chapter we will explain how to Install Polymerix from the distribution media or through a Web download Register the software and obtain a license key Install the license key and begin using the software Installing Polymernx We deliver Polymerix software in one of two ways physically on a CD ROM or electronically via an Internet download from Sierra s Web site If you received Polymerix on a CD ROM i
69. g 1101 5544 herd Ferd 5 604 5 H2N207 1 0 Na 1101 53882 r a09 7 309 6 636 6 C4HAN 204 5 0 Na 1101 554058 rot 54 rel 7 C8H1803 0 0 Ag 1101 523094 15 192 15 192 13 792 a HE05 4 0 H 1101 563770 17 563 17 563 15 944 9 C4H14N60 1 0 Ag 1101 52826 17 878 17 678 16 230 10 C5H18 6 0 0 Ag 1107 56464 15 508 15 508 16 502 CEE Result End Groun ZEM 2 Monoisotopic m z is 1101 54513 repeat formula is C3H8 repeat mass is 104 06260 charge countis 1 tolerance is 0 1 Da electron mode is normal restricted element ratios DBE range is from 0 5 to 5 0 minimum end group m z is 0 maximum end group m z is 200 maximum result count is 10 C count is from 0 to 20 H countis from 0 to 40 O countis from 0 to 20 N countis from 0 to 6 ox cancel This is actually two lists each of which has a highlighted selection In the top list chemical compositions which differ only by an integral number of repeat units are eliminated Additionally the columns which ate presented in the top list will vary slightly depending upon whether the end group being determined is an Alpha or Omega endgroup versus determining a combined end group The bottom list corresponds to the composition which is currently selected in the top list When the End group maximum mass from Find Series Settings is large enough then the bottom list will present multiple choices of end group which differ by integral numbers of repeat units In the bottom panel all parameters th
70. gistration Wizard The wizard is a dialog based procedure that guides you stepwise through the registration process Polymerix 1 Double click the Polymerix icon on your desktop or select Programs gt Polymerix gt Polymerix from the Start menu to start the Polymerix application The Polymerix splash screen will appear for a few seconds and then the first page of the Registration Wizard will be shown 10 INSTALLATION LICENSING AND REGISTRATION Polymerix License Wizard 2 Since you do not yet have a license key click the Next gt button to continue 11 INSTALLATION LICENSING AND REGISTRATION Po lymerix License Wizard 3 On this second page you choose the type of license fixed or floating If you wish to lock the software to a single PC choose the Fixed option otherwise choose the Floating option A floating license permits installation on up to 4 PCs but requires use of a USB device that you can transfer from PC to PC when you wish to run Polymerix The USB device must be installed in the PC during the entire duration of your Polymerix session Usually a USB memory stick is the most convenient way to do this Any kind of USB device may be used as long as it has a unique name and serial number that can be detected by the software Click the Next gt button to continue 12 INSTALLATION LICENSING AND REGISTRATION Polymerix License Wizard 4 If you chose the floating license option
71. h Fount 3 rt Pt iiam feet Meier trl mr Mrt l el Some mept i Spo emputed Fs Fon Erot balla ETS 40 y UY ow maL ann Gm Cne OII 573 44 Des 7358 nen o wi niw ons ews aR eC ann W 7 s Wi M ZEIN an H wW uem 2wa 152 2am Z I6 2m e MA pZ i MT Ws Baw 0 Hi T o 35 3445 g77 ayy 3460 aon rs Se me tim m We Jman 39800 Sisu aSr Sawo Si SUITS Eds pis jiz J ee oe Oo w 440600 essa ESE CEO cset ESS Eme SEM REM Smo ON 1 381 ad 80 J 17 409600 7a Fisel FOD atte TEN NTT Tn AWI 1028327 iaee 15302 ED 5 A 30 Gasa EAA 5S5 Sae GASS SIS ESTED 10357 be IOa WREST 8620888 SN oi 3 297100 aan Som aai am So ann aT M PMS Has aea man n 0 wi awo aw eT Om I Sor Ra at a 25 WR Wet IM Ms 0 i nsw eS eT pyr tame N 6M 100 l 23 YO 11m Tena tnai TS 140 rr 1h nite Hi Ha Ihana toita MPD 3 200 Oan aee Oan Q7 Oene OAND SESE FS 267 iein 1M tans B60 l 34 80 ana easi CAm enno O sOi SCS 436883 Se 1681 89 135 00 l zs aso as eio O73 amm OnO oGs Ito eae S527 igas MSS IO CoD 0 Ej w oez ems Omio omz 0m omw 1mos 51042 WSIS lasm leo 0 z 7 on ewa oom oon Ooa doe TEND temu WT zem 20 i 38 no ons mn oam ee Ooi Gos lisin iona J r e TS DN tT iN Ty 0 apit CON Ob 7AA me et Sesh ae Aa i ayer m red Ane T Phm paana Dotais Ma San AS Lar eater seat b arad Mass Ranges The Mass Ranges table displays a list of the calculated m z ranges used for
72. iDaltons or as Error in both mDa and ppm Result lists of chemical 51 FIND HOMOPOLYMER SERIES compositions corresponding to a reference peak are sorted in order of increasing magnitude of calculated errors How Polymerix handles reference peak selection When the user selects a pair of reference peaks either automatically with the Find button or manually by right clicking and dragging on a spectrum plot Polymerix responds as follows The reference peaks are highlighted in red where they are shown in spectrum plots The mass difference between the peaks is computed for use in analyzing the repeat unit s chemical composition If no repeat unit is currently specified or if the mass difference is incompatible with the currently specified repeat unit then the user is presented with a list of repeat unit chemical formulas to choose from The user is presented with a list of end group and adduct formulas to choose from Polymerix creates a new polymer series definition from the users choices Reference peak highlighting is removed where it is shown in spectrum plots Automatic reference peak selection Polymertx uses a heuristic algorithm when attempting to automatically select reference peaks from a spectrum The algorithm attempts to identify large monoisotopic peaks corresponding to a homopolymer series It takes account of the range of possibilities for adduct chemical composition when attempting to determine which
73. in the bottom pane 3 If you observe peak splitting in the peak detected spectrum profile peaks are split into two or more centroid peaks this indicates that the peak width parameter is too small Missed peaks or in TOF mode too many noise peaks or baseline anomalies indicate a peak width that is too large Change the value click Apply and evaluate the result Repeat if necessary until the centroid spectrum is satisfactory 4 Polymerix s peak detection algorithm always assigns intensities to the centroided peaks based upon mathematical areas calculated from the spectrum being peak detected 5 There are three additional sets of parameters on this tab Baseline correction peak merging and peak thresholding 6 The Subtract baseline from spectrum option is used to flatten a sloping baseline to eliminate systematic errors in peak intensity due to a non zero baseline It is automatically selected when Peak detect and centroid is chosen If you choose to process in profile mode i e no peak detection the Subtract baseline option is available separately Using this option is generally recommended The Baseline width parameter determines the width of the spectrum used when averaging to compute the baseline offset at each point The default value of 500 Da is usually adequate to compensate for broad baseline drifts If the baseline has more narrow artifacts using a smaller parameter might provide a better fit 7 The Merge to
74. includes a mass range for each series and chemical formulae for repeat unit end groups and adduct When a series is selected as the current series its row is highlighted and the current series spectrum plot will show peaks which match the series The Series list also displays the two reference masses used to determine the chemical composition of the polymer and the excess mass at the first reference mass which indicates the difference between the reference mass and its closest calculated mass from the polymer series which is being specified Clicking on an excess mass field when either end group is empty will bring up the End group and adduct selection dialog Clicking on most of the other columns in the series list will allow individual components of the polymer series definition to be entered or edited The reference masses cannot be entered or edited This invokes the automatic reference peak finder which attempts to find a pair of reference peaks from the residual spectrum which correspond to a polymer series This clears all polymer definitions in the series list 53 Settings button From Series Setup button To Series Setup button Source spectrum plot Current series spectrum plot Residual spectrum plot Repeat unit selection dialog FIND HOMOPOLYMER SERIES This brings up the Find Series Settings dialog This transfers series definitions from the Homopolyer Analysis page Additionally some settings such
75. ion The Polymertx splash screen will appear for a few seconds and then the first page of the Registration Wizard will be shown ps Polymerix License Wizard 2 On the first page of the wizard click the Register button This will open a file selection dialog Choose the registration key file you saved from online registration process 17 INSTALLATION LICENSING AND REGISTRATION Select Polymerix License Key File Look in E Polymerix f e Es My Recent Documents E Desktop 9 My Documents _ MassHunter EJ INSTALLATION txt 5 InstallationLog txt lt 3 Peg2000 txt Polymerix_License_Key FRACTALZ txt Polymerix_License_Request_FRACTAL2 txt PolymerixUninstall txt E ReadMe txt E SRM2888 txt S My Computer File name Polymerix_License_Key_FRACTAL2 trt La ETE Files of type Text files txt Cancel Places Open as read only 3 Select the license key file and click the Open button The Registration Wizard will read the file contents and verify them LT x e i J Thank you For registering You may now use the software E 4 If everything is OK the Thank you dialog will be displayed Click the OK button to close this dialog then the Finish button on the Registration Wizard 5 The main Polymertx page will then be displayed If the registration failed for any reason a message will be displayed with that informat
76. ion and the registration wizard will remain open If you used a USB memory device for the registration request be sure it is inserted and recognized by your PC If everything seems OK but the licensing still fails please contact us at Polymerix MassSpec com and we will try to work through the problem with you 6 Please note that registration keys are specific to the PC that was used to generate the license request You cannot use the same file to license Polymerix on a different PC If you are using a floating registration you 18 INSTALLATION LICENSING AND REGISTRATION must create a registration request and receive a license key file for each PC using the same USB device each time et ar ey Be ET Bee eS Sr SI SE FEST TI ST FTE SE Re Si SE SF SF SE SE SI Sr EI SE SI SE SE 3 to 7 eee 7 Time to get to some real work What was covered in this chapter In this chapter we described how to Install the Polymertx software from the distribution Create a registration request and submit it by e mail to obtain a license key Install the license key in the software In the next chapter We will describe the workflow model used in the Polymertx software and show how that workflow is implemented in the main parts of the Polymerix user interface 19 POLYMERIX WORKFLOW AND USER INTERFACE Cl l Goals of polymer analysis Steps in the workflow Polymerix Workflow Model and User Interface Overview How P
77. m Match Tolerance values are the same as in Homopolyer Analysis 58 Reset button FIND HOMOPOLYMER SERIES The To Series Setup button transfers this setting to homopolymer analysis and the From Series Setup button sets this setting using the corresponding value from Homopolymer Analysis This restores all the parameters in the Find Series Settings dialog to default values Troubleshooting Tips With high mass accuracy data and chemical composition constraints which have been properly set up Polymerix should be able to correctly determine chemical compositions If Polymerix is showing no results or only results which don t make chemical sense the problem is usually that Polymerix has done a poor job of automatically selecting reference peaks for use in determining chemical compositions Restricting mass ranges using the Data Processing page may help automatic reference peak selection work Once a series is found the mass range restriction may be removed Double check the results of automatic reference peak selection Manual reference peak selection even of a different pair of reference peaks from the same series may solve the problem Double check the chemical constraints being used If multiply charged ion series aren t being recognized make sure the Charge Maximum setting is set higher than its default value of one What was covered in this chapter In this chapter we Showed how to use Find Homopolyer Series pag
78. n and constraint parameters click the Apply button 78 COMPOLYMER ANALYSIS Defining copolymer series As was mentioned above the major difference between a homopolymer and a copolymer series definition is the presence of a second repeat unit 10 osat 1 Click the Series Setup tab to view the copolymer series definition table Copolymer Seres Definition Alpha Repeat Min L 51 0 none apes Min Max Omega Charge Adduct none 100 1 2 Most of the editable components in this table are the same as for homopolymer series setup and behave in the same way click in a box click the button and then select or define a chemical entity from the dialog 3 As can be seen from the screen shots above there are a few new items as well First instead of a single repeat unit there are now separate selections for A and B repeats Next there are lower and upper limits on the A repeat count and B repeat count set by typing into the Min A Max A Min B and Max B edit boxes These repeat count constraints are used in conjunction with the ratio constraints on the Processing tab to limit the number of combinations of A and B count generated for matching to the spectrum 4 For example suppose the A to B ratio constraint was chosen with minimum and maximum ratio both set to 2 This means that for every B there must be two A units So if the A and B count limits are each set from 1 100 then the only A B
79. n of the sample In this case Polymerix provides options to combine the results for these different series into a composite result In all cases though the series to be combined must have the same repeat unit The choices are Charge states By selecting this option results for series with the same repeat unit end group and adduct that differ only by charge state will be combined into a single composite series result Adducts and losses By selecting this option results for series with the same end group repeat unit and charge state that differ only by an adduct or a neutral loss will be combined into a single series results End groups Finally by selecting this option results for series that differ only by end group are combined into a single composite result 64 HOMOPOLYMER ANALYSIS 24 Any combination of these options can be chosen depending on the nature of the sample and ionization artifacts With nothing selected all series will be assigned and reported separately 25 Once the match tolerance and series combination options have been chosen click the Apply button to set them Defining homopolymer series A homopolymer series consists of three patts a Repeat w Adduct n e An alpha end group which starts the polymer chain e Some number of repeat units or monomers and e An omega end group that terminates the chain In addition the process of sample introduction and ionization
80. n these computations e New data file format Data files created by earlier versions of Polymertx can be loaded by Polymertx version 3 0 but data files created by Polymerix version 3 0 cannot be used with earlier versions of Polymerix e Fixed problem with multiple charge states combined with a loss e Added RA threshold to MS peak processing e Added the Reset Mass Ranges checkbox to the Data Processing page e Improved peak detection Version 2 0 Updates bug fixes and new features User interaction is essentially unchanged from Version 1 0 Moved copolymer processing trom Polymerix V2 beta to the 2 0 Release version including full copolymer reporting Implemented improved mass spectral peak detection Fixed known printing and report generation problems including missing lines blank pages incorrect page count when switching between landscape portrait incorrect sizing of collate icons blank copolymer assignments spectrum Fixed bug in copolymer details display which used to manifest when copolymer series other than the last were disabled Result summaries now include entities specified by mass and consolidate shared formulas Polymer statistics now have zero values when intensities don t match Fixed heap corruption bug in Homopolymer spectrum view when attempting to color unassigned masses Fixed homopolymer assignment bug where assignments were generated for masses of Zero Added Filtered Assignments
81. nal grid Keeping the count of one repeat unit fixed the second repeat unit typically has a repeat distribution similar to a homopolymer Increment the first repeat unit count there is another distribution for the second repeat unit and so forth T he definition and analysis of copolymer series is very similar to Goals for this chapter In this chapter we will Describe Polymerix s definition of copolymers Present the Copolymer Analysis page and how to use it Show how to define copolymer series Explain the tables and plots produced by Polymertx during copolymer series assignment Copolymer definition B xw Adduct n mo 76 COMPOLYMER ANALYSIS In the Polymerix world a copolymer series is a polymer containing two different repeat units which we label as A and B in the schematic above Like a homopolymer a copolymer series also has alpha and omega end groups an adduct and or neutral loss and a charge state In a mass spectrometric determination of a copolymer molecular weight distribution there is information only about the total m z value of a given peak Nothing can be inferred about the copolymer sequence responsible for the observed m z value It could be a block alternate or random copolymer or something else All that is known is that after subtracting out the end group and adduct masses there are 7 counts of repeat unit A and counts of repeat unit B However as will be shown in the descrip
82. nsert the disk into your CD ROM drive The installation program should start automatically If it does not start browse to the CD ROM folder and double click the Polymerix_3 0_FullSetup EXE file to start the installation program If you received an e mail with a Web link double click the link in the e mail or copy the link to your Web browser if your e mail program does not permit opening links and follow the instructions on the Web page to download an appropriate setup program which differs depending upon the data importers which are provided Save The installation program is a dialog based wizard that guides you through the setup steps INSTALLATION LICENSING AND REGISTRATION the downloaded file to your disk unzip the distribution and then double click the Polymerix_3 0_FullSetup exe file or the Polymerix_3 0_ Setup exe file where represents a vendor name to start the installation program Polymerix Setup O x Choose Components Gn Choose which features of Polymerix you want to install F Check the components you want to install and uncheck the components you don t want to install Click Next to continue Select components to install Pas Agilent file support Ne ANDI netCDF file support F Agilent MassHunter file support F Analyst file support I al m I i ao space required 78 5MB Position Your mouse over a component to see its description Mullsart Install s
83. o any window narrower than that is acceptable Typically a value of 0 5 Da or less or the equivalent in ppm is acceptable 63 HOMOPOLYMER ANALYSIS 21 The Use clusters option should be selected when the target spectrum has not been deisotoped or has not been peak detected and it is desirable to match all peaks belonging to the target isotope cluster With this option selected and continuum data the monoisotopic peak will be matched then the window will be broadened to match the additional peaks in the isotope cluster With this option selected and centroided data individual peaks within computed isotope clusters will be matched using the tolerance In the example below an isotope cluster is shown The match tolerance is set to 0 1 Da which matches the isotope cluster peaks but not the peaks which lie between isotope cluster peaks 22 The Positive charge option should be selected to match the polarity of the spectrum being analyzed It controls whether Polymerix adds or subtracts electron masses when computing the m z values for a polymer series Combining series 23 Combine Within Series With Same Repeat Next are optional choices for combining homopolymer series In many cases multiple series are found in the spectrum simply due to sampling or ionization artifacts For example the appearance of multiple charge states in ESI or of both proton and sodium adducts is simply a result of the ionization process and not a true reflectio
84. olymerix models the data reduction analysis and reporting workflow and the principal elements of the user interface steps you would use if you were to perform this data analysis and reduction manually The user interface implements this workflow model in an intuitive and graphical fashion olymerix s workflow model is designed to simulate the same processes and Goals for this chapter In this chapter we will cover the following material We will describe the model used in Polymerix for the data reduction analysis and reporting workflow We will present the major parts of the Polymerix user interface and how they fit the workflow model The workflow model A typical polymeric or related sample consists of one or more homologous series each consisting of one for a homopolymer or two for a copolymer repeating units the monomers With alpha and omega ot tota end groups When analyzed by mass spectrometry the resulting spectrum may likewise show spectral features for each of these series and possibly more dependent on the sampling and ionization method and resolving power of the instrument The goals of polymer analysis are to assign a unique chemical composition and abundance to as many of the detected features as possible and to compute the molecular weight distribution statistics for each series The workflow for this analytical process can be broken down into a series of dependent steps 20 Mapping workflow
85. onality provided by the Find Homopolymer Series page so the chapter can be skipped 90 COPYING Summary of the algorithm EXPORTING AND PRINTING Autocorrelation How to use autocorrelation to analyze unknown pobsers molecular weight distributions for materials of known composition What can you do when you have an unknown polymer or a polymer where only partial information is known pP olymerix is normally used to deconvolve polymer series and compute Goals for this chapter In this chapter we will Describe how to enable and access the Autocorrelation page Describe the autocorrelation algorithms and what the resulting spectra mean and Show how to use the information in the autocorrelation and end group spectra to assist in determining repeat unit or end group composition Accessing the Autocorrelation page The Autocorrelation Analysis page and its button are not displayed by default because Find Homopolymer Series supersedes the functionality of autocorrelation analysis in most cases To enable and show the button select the View menu s Autocorrelation item and then exit and restart Polymerix Once Polymerix has been restarted there will be a new Autocorrelation icon not previously shown Clicking on the icon will access the Autocorrelation Analysis page The autocorrelation spectrum 91 AUTOCORRELATION Autocorrelation is a method by which all m z differences between peaks in a mass spectrum
86. peak and assign it as the mass of the repeat unit monomer Then for each peak in the experimental spectrum subtract from it the highest integer multiple possible of the repeat mass Insert these remainder mass values as peaks in the end group spectrum If the repeat mass was correctly chosen there will be clusters of peaks in the end group spectrum at positions corresponding to each of the total end group plus adduct masses To give a concrete example suppose the experimental spectrum has peaks at 63 85 107 129 151 173 195 and 217 Da The autocorrelation spectrum shows among other differences a strong peak at 44 Da If we use 44 as a repeat unit then we compute an end group spectrum according to the results shown in the table below Expt Max of 44 m us Z repeats Max x 44 Remainder s pe 9 a o o oa pa oe oa m a As should be clear from examining the table the end group spectrum will have only two peaks at 19 Da and 41 Da The difference between these two is 22 Da leading to the inference that there are two homologous series in the sample one with proton adduct and the other with sodium adduct So the peaks at 19 Da must be due to the total of the end groups plus a proton and likewise the peaks at 41 Da are due to the total of the end groups plus a sodium ion Subtracting 1 Da from the 19Da peak and 23 Da from the 41 Da peak leaves 18 Da in both cases This leads to the conclusion that the total end group is wate
87. processing page which displays the total ion chromatogram TIC if present the input profile spectrum and the fully processed spectrum that will be used for series assignment 2 A page with graphical tools for helping to determine from spectral data the chemical compositions of the units which construct a homopolymer The resultant chemical compositions can be transferred to the homopolymer analysis page 3 A homopolymer analysis page used to view the quantitative results of performing homopolymer series assignments Homopolymer series definitions can be specified here 4 A copolymer analysis page used to setup copolymer series definitions and to view the assignments and decomposition into spectral subsets by repeat unit count 5 A relative area page where relative areas of multimodal distributions can be selected and measured 6 An optional autocorrelation page which can assist in determining repeat units and total end groups for an unknown polymer The functionality of 21 Processing Page POLYMERIX WORKFLOW AND USER INTERFACE this page has largely been superseded by the Find Homopolymer Series page in item two above In support of these pages are many dialogs and other user interface items for data import and export setting parameters displaying tables and graphics and switching between the various pages and data views Introducing the Polymernx user interface The general layout of the Polymertx user int
88. ptions to combine series was selected during setup the combined series will be shown instead of individual series e g S1 S2 The left side of the table presents the statistics and the right side summarizes the series definition Visualizing homopolymer series assignments There are four tabs in the bottom pane of the Homopolymer Analysis page each presenting the assignment details in a different way Assignments The Assignments tab presents the target spectrum with all matched series peaks labeled This can be a cluttered plot if there are many series with many assigned peaks However it shows at a glance if there are major unassigned peaks in the spectrum Here we have defined an additional series with a proton adduct i ms 4 SIR e rasss foes ites eee pape e s ota EL i TESS ET tA Sine fiT A gaj Ore ees TTEN Filtered Assignments The Filtered Assignments tab shows only the assigned peaks in the spectrum 70 HOMOPOLYMER ANALYSIS P ered hornopi Actaprirents a am remap wT tI pry ei xis IRL iem ees hs 1449 xet Ia Vr 1720 ier ia hale 1492 3 nee 5B ag TE ENDR tet zt meer moet ie er AL ll bad azr 1887 ere aere ss te idd ALS es tliy ETE E bane s Ate Meat meee RST STATS yy Ga FO raes uted xe ade n aaz 4 de gt code Rida AS RI aes merges be yy Ea joe leaner ies Isr resis 122 8 DES Myer avers t2 ait rid Artia se E N ap eee eth ai T ae
89. quate resolution and mass assignment by changing the mass mode and by adjusting the mass axis We discussed deisotoping and how this important algorithm can overcome bias in computing molecular weight distribution statistics And finally we described how to save and load parameters using Polymerix template files In the next chapter In the next chapter we will describe the Find Homopolymer Series page and how it can be used to assist in determining and specifying the composition of an unknown polymer 48 FIND HOMOPOLYMER SERIES Cl l Find Homopolymer Series How to use the find homopolymer senes page to analyze unknown or partially known polymers molecular weight distributions for materials of known chemical composition The Find Homopolymer Series page can help a user determine and specify the chemical composition of polymers in a given spectrum pP olymerix is normally used to deconvolve polymer series and compute Goals for this chapter In this chapter we will Provide a brief high level overview of the polymer series finding process and its requirements Show how the tools are used with a very simple example Introduce the concept of reference masses and describe how they are used Show details about how to use the tools on the Find Homopolymer Series page to construct repeat unit formula end group formula and adduct formula specifications Provide tips for solving common problems such as not b
90. r H O With this information and some knowledge of polymer chemistry it seems likely that the sample is an ethylene oxide polymer with H and OH end groups A real example would undoubtedly be more complex and subject to errors in calculation of the repeat mass and remainder masses In particular if the total end group plus adduct mass is larger than the repeat mass then the algorithm described above will subtract too many repeat units leading to masses in the end group spectrum that are too small by one or more repeat masses This will lead to nonsensical 93 AUTOCORRELATION compositions In such cases you may need to add one or more repeat masses into the peak values to see if that makes more sense as a total end group mass Nevertheless autocorrelation analysis can often give valuable insights into composition of an unknown material User interface and parameters The autocorrelation and end group spectra are computed automatically from the process target spectrum The see these spectra click the Autocorrelation Analysis button on the Workspace Control Panel to switch to the Autocorrelation Analysis page Dm Te fe few ee t Ai i A magnified view of portions of these two spectra is shown below Autocorrelation Spectrum base peak mass 43 946 43 946 67 966 100 96 131 955 Response 89 80 0 60 0 40 0 20 0 15 967 27 906 59 987 71 925 104 006 115 945 148 02 1 1 1 1 20 0 40 0 60 0 8
91. r analysis showing the entire target spectrum with all assigned peaks labeled Instead of an R repeat count label there is an AB label A detail view of the screen shot above illustrates this 80 COMPOLYMER ANALYSIS Coputyrer Assupiniens Filtered Assignments This plot is also the same as for homopolymer analysis only the assigned peaks are shown in the plot Filesi Copetyrret Assignments sane dist on te see 0 ur aide moa OFE LS 2730 To et O 2n Jp ao ets Te Fh 1000 paun 28 pin Na ere 30S oa R ae war mr 1ee 0 di p the rg i TE a 42011 oe Y pre a1 O al 41 pis i 109 mre CA me le 17 tas i paza G Bfp ie a ree ete eee 43 nas om t J1 111 t 3 Tiee 1 i us y Mnrusn t f ens 1 Bi To tid 4 1866 0 iio tyme x09 1 0 1400 6 oo 6 Ts 1699 0 Agmar Peed Ameren Piteret Pentas ete Mem Tarpe AS Devt ter Fepeat Saneso Ta narte Hapast A Pet Tapaat A Pit Filtered Residuals This plot is also the same as for homopolymer analysis only peaks which have not been assigned to any series are shown in the plot 81 COMPOLYMER ANALYSIS gt E BALT mmo 12060 i testtuter Ameni Siatates Repent g Sianas Repeat A tot Resesi Srat Details The Details table groups the assignments by B count within A count That is the first group contains the range of B counts matched for the minimum A count the next group contain the B counts for the next A count and so fort
92. re In this case the MassLynx file contains a single scan function in positive ion mode produced with MALDI LD ionization Click the OK button to import the selected 30 DATA IMPORT AND PROCESSING data Click the Cancel button to close the browser without importing data Some datasets may contain multiple spectra for example where a sample was infused over time or multiple MALDI laser shots were acquired These are automatically recognized by the data import browser After selecting the dataset and clicking OK the total ion chromatogram TIC or its equivalent for infusion or other multi scan acquisitions will be displayed in the TIC pane of the Data Processing page To select a single spectrum simply right click once in the Total Ion Chromatogram pane at the appropriate scan time or number To select and co add a range of spectra right click and while still holding the mouse button down drag across the desired time range in either direction then release the mouse button In each case the selected scan or scan range will be extracted from the dataset co added and displayed in the middle and bottom panes To zoom in on a region of the TIC or spectrum left click drag and release with the mouse To zoom out simply left click once Each time you right click and drag to select a spectrum range from the TIC the input middle pane and target lower pane spectra will be replaced with the new selection When you a
93. re satisfied with the chosen spectrum it is time to move onto peak detection and processing To import a single spectrum from an ASCII peak list file from the File menu click on the Import Spectrum command 31 DATA IMPORT AND PROCESSING Polymerix File Edit view Help New Ctrl N Open Ctrl 0 Save Ctrl 5 Save As Open Template Ctrl T Save Template Ctrl V Import Chromatogram Import Spectrum Export Results Export Spectrum Print Ctrl P Print Setup 10 A standard file browser will be displayed Select the desired file then click OK If the file contains valid spectral data it will be imported and displayed in the middle and bottom spectrum panes The TIC pane will be empty 11 By default Polymerix assumes that data read from peak list files is in centroid mode If this is not the case change to profile mode by clicking the Continuum button in the Raw Spectrum Format section of the Spectrum Select page Raw Spectrum Format Continuum C Centroid Alternatively you may edit the ASCII file to add the word continuum as the first line in the file to permit the file reader to automatically identify the mode 12 ASCII peak list files must have the following format Each line contains a single m z intensity pair separated by a comma space or tab delimiter Most mass spectral data systems will automatically create this format when exporting a peak list 32 DATA IMPO
94. red for both repeat formulas and end group formulas In the case of a polystyrene polymer a maximum of at least 4 must be used since the styrene repeat formula C8H8 has a DBE value of 4 This sets another restriction on DBE values limiting whether integral and or non integral values are allowed For repeat units selecting an Electron Mode of Normal will limit result chemical compositions to those with integral DBE values and selecting Reversed will limit result compositions to those with non integral DBE values For end groups composition results are limited in such a way that the sum of end group DBE values is integral when Normal is selected and non integral when Reversed is selected Selecting None disables electron mode restrictions Enabling Element Ratio Restrictions requires that the formulas for repeat and end group ate reasonable for example H14 won t be allowed Specifies ion polarity Polymertx uses this to account for electron masses when determining elemental composition The To Series Setup button transfers this setting to homopolymer analysis and the From Series Setup button sets this setting using the corresponding value from Homopolymer Analysis Mixed Adducts is only used for multiply charged data It specifies that different adducts may be combined with each other when forming multiply charged ions For instance a doubly charge species with H and NH4 would be allowed The Spectru
95. rl C Copy Spectrum Data Ctrl D Copy Results Saving and exporting results Polymertx results and the input unprocessed spectrum can also be saved as ASCII files for import into Microsoft Excel or other software 101 COPYING EXPORTING AND PRINTING Polymerix File Edit view Help New Ctrl N Open Ctrl O Save Ctrl S Save S Open Template Ctrl T Save Template Ctrl Import Chromatogram Import Spectrum Export Results Export Spectrum Print Ctrl P Print Setup Recent File Exit Exporting results 1 To save Polymerix results select the File gt Export Results menu command A file dialog will appear to allow the choice of file name and location t Polymerix File Edit View Help New Ctrl N Open Ctrl O Save Ctrl 5 Save As Open Template Ctrl T Save Template Ctrl Import Chromatogram Import Spectrum Export Results Export Spectrum Print Ctrl P Print Setup Recent File Exit Exporting the input 42 To export the input spectrum as an ASCII peak list select the File gt spectrum Export Spectrum menu command A file dialog will be displayed to allow file name and location selection The spectrum will be saved as a tab delimited file with one m z intensity pair per line 102 COPYING EXPORTING AND PRINTING Printing Polymerix produces a preformatted printed report as output There are
96. rt vendor format MS data from the File menu click on the Import Chromatogram command t Polymerix File Edit View Help New Ctrl N Open Ctrl O Save Ctrl 5 Save As Open Template Ctrl T Save Template Ctrl V Import Chromatogram Import Spectrum Export Results Export Spectrum Print Ctrl P Print Setup Alternatively click the Chromatogram button in the Import section of the Spectrum Select tab pane from the Data Processing page 29 DATA IMPORT AND PROCESSING Import Chromatogram Spectrum IW Reset Mass Ranges Spectrum Select Peak Detect 3 The data import browser appears In the figure below there are two MS data sets in Waters MassLynx data format shown Both have been recognized by the browser as raw MS data formats so they are displayed with a special spectrum icon If you selected additional formats when you installed Polymertx and those datasets were also in this folder they would also be shown with spectrum icons Select Dataset ri J xj a A MS C LD 3 D Gp E ep F ee G ce Files S E Polymerix E i peg1500_cal raw i PPG_LIQUID raw H RECYCLER E J System Volume Information ee J Se k L H P HE Ww Fg Network Neighborhood ERE Functions OK Cancel 4 Selecting one of the datasets will display a list of the scan functions it holds using the vendor s nomenclatu
97. s complex software with many parameters and options to handle a wide range of sample types and analytical approaches The second part of the manual goes into much greater detail to describe this complexity explaining each setting and its effect on the analysis Finally because no single software product can accomplish all things for all people the last part of the manual details the export format used to store all of Polymerix s parameters and results and gives some examples of how to extract information for use in other software Some conventions used in this manual We have standardized on certain typographical conventions to help distinguish normal text from labels prompts or other text you see onscreen to highlight important items and to point out areas where you must pay attention to avoid mistakes that could affect the quality or accuracy of your results ICON KEY amp Valuable information Warning Z Lengthy process INTRODUCTION Text that appears in boldface normal font is used to display menu items labels or other prompts that appear onscreen Text that appears in boldface Arial font is used to emphasize some point in this manual An icon is displayed to indicate an important point or warning An arrow sets off an item of importance An exclamation point indicates a warning Z An hourglass indicates a step that might require lengthy processing In the later parts of the manual text in computer
98. s of arrow buttons to interactively change it The left hand set of arrow buttons adjusts by 1 Da while the right hand set adjusts by 0 1 Da As the buttons are clicked the spectrum will shift left or right accordingly as shown in the pictures below Mass Adjustment Moist oE Slope 0 ppm Set iv Adjust spectrum Masses Spectrum Select Mass Adjust Deisotope Peak Detect 38 DATA IMPORT AND PROCESSING 1317761 1361 795 10244 10262 1320 0 1340 0 1360 0 Mass Adjustment Iv Offset iv Adjust spectrum masses IV Slope Mass Adjust Deisotope Spectrum Select Peak Detect 1318 761 1362 795 10244 10262 1320 0 1340 0 1360 0 16 To apply a linear slope adjustment click the Slope checkbox As with offset adjustment the left hand arrow buttons adjust the slope in coarse increments of 10 ppm while the right hand set adjusts in increments of 1 ppm Note that in the plot above the two monoisotopic peaks differ by 44 034 Da while in the plot below the difference is 44 065 Da The 10 ppm positive slope results in an increasing mass difference with m z whereas an offset alone simply shifts the spectrum while maintaining the same mass differences 39 DATA IMPORT AND PROCESSING Mass Adjustment M Offset 1 Da a Freee ee neee iv Adjust spectrum masses eee ee een e nee Spectrum Select Peak Detect Mass Adjust Deisotope 1318 774 1362 809 10244 10262 1320 0 1340 0 1360
99. spectrum view Added homopolymer distribution plots Added intensity weighted mass metrics Changed MS data access to use ordinary DLL instead of COM DLL If low level data access tools supplied by the vendor use COM this is not affected by this change Only Sierra s high level data access is changed Updated installation script to use new DLLs 108 RELEASE NOTES e Changed licensing to use a new scheme that does not depend on MAC address Two methods are provided o fixed license licensed to a single PC or o floating license using a designated portable USB device licensed up to 4 PCs Copies using a floating license must have the USB device installed at all times when the software is in use The software will exit if the USB device is removed 109 EXAMPLE REPORT Example Report An example of a homopolymer analysis report On the following pages is an example of a full report for a PEG homopolymer analysis On the Print dialog the Data Processing and all Homopolymer report options were selected RELEASE NOTES Polymenx Processing Report Data Processing Results Printed Wed Mar 06 2013 15 42 07 Data file C Polymerixipeqi500 cal raw Processing mass range 0 0 to 2402 0 Peak width TOF 0 400 Da at 1000 000 m z Peak detected area intensity Baseline subtraction width 500 0 Da C 2 de pierix pegi500 cal raw Scans 1to5 Page 10f3 2 5 0 0e 0 a OS i 2 4 6 Scan Number pegi500 cal raw RT
100. state corresponding to the adduct Polymerix uses Adduct Charge and Charge State to compute the number of adducts used to create a molecular ion A few more options remain in the series definition If a loss occurs in sampling or ionization specify that by formula or mass by typing in the Loss box If you wish to have the molecular weight distribution statistics adjusted for the mass difference of the adduct or loss click the Adjust For Adduct Loss check box Finally to restrict the mass range searched for the newly defined series edit the range by typing into the Low Mass or High Mass boxes This completes the definition of the first series Click the Apply button To define additional series click the button next to the Enabled box This will make a copy of the last series onto a new line Make whatever changes are necessary then click the Apply button again Continue until all needed series are defined To remove a series definition click the button in the column to the left of the Enabled box At this point it might be a good idea to save the definitions into a template file for later use 68 HOMOPOLYMER ANALYSIS Hamepolvenw Assignee St az oy zY syer 27 431 inet 4 m a7 tEn ene pocos panes 9 lay gt a wen RIT I 13 AR IETT tet 6600 5 wT 1S arz Cie mt RJE ID i0 z mn atot 3 oa ny fasg Akis i i ON RIRE DIRA 5 lt x tR p33 F Ae aia t Raj 2400 3 Srats 3183 gera MSE OSTE oe dake
101. t File xj Save in gt Polymerix e Fe _ MassHunter e E INSTALLATION txt My Recent InstallationLog txt Documents at Peg2000 txt E 5 Polymerix_License_Key_FRACTALZ txt sa Polymerix_License_Request_FRACTAL2 txt Desktop SS PolymerixUninstall txt ReadMe txt so j SRM2888 txt My Documents My Computer 5 My Network File name Polymers Lic Request FRACTALZ Places Save as type License request files txt Cancel 7 Once the license request file is saved continue with the Polymerix redeem download instructions They will allow you to retrieve a license key file which you will use in the nest section Installing the license key If registration was successful we will send you a license key by reply to your e mail You will use the Registration Wizard again this time to copy the license key into the software and license the copy These are the steps involved in installing your license key 1 Open the saved file with the license key 2 Run the Registration Wizard and load the license key file into the wizard 3 Start using Polymertx Until you have installed a valid license key each time you start the Polymerix software it will run the Registration Wizard 16 INSTALLATION LICENSING AND REGISTRATION Z Polymerix 1 Double click the Polymerix icon on your desktop or select Programs gt Polymerix gt Polymerix from the Start menu to start the Polymerix applicat
102. ta point which matches multiple mass ranges Cluster matching When the Use clusters option is enabled Polymerix will calculate theoretical isotope clusters corresponding to a series and use these theoretical clusters together with the match tolerance to construct the mass ranges described above The low m z value for 73 HOMOPOLYMER ANALYSIS a mass range corresponds to the monoisotopic peak of its isotope cluster The high m z value for a mass range corresponds to the highest mass peak in the isotope cluster for which the peak s intensity is greater than or equal to 2 relative abundance Centroid cluster matching When the Use clusters option is enabled and centroided data is being analyzed the assignment process is similar to that used for simple matching but with an important difference the individual peaks in theoretical isotope clusters are used for matching in addition to the mass ranges described above With small match tolerance values this excludes some intensity values from being assigned to a series The example below shows an isotope cluster Because the match tolerance is set to 0 1 Da only the isotope cluster peaks are matched Match contributions from data between the isotope cluster peaks are filtered out Centroid cluster matching algorithm The steps used for centroid cluster matching are as follows 1 Determine mass ranges as described above 2 Assign intensity values from the spectrum being anal
103. te due to the fact that for every group of peaks with a difference of 44 Da the first and third peaks second and fourth peaks and so on ate separated by 88 Da while the first and fourth second and fifth etc has a 132 Da spacing etc However there are fewer pairs of peaks with each higher multiple of the base separation so these overtones form a progression of decreasing abundance in the autocorrelation spectrum In addition if there is more than one homologous series there will be autocorrelation peaks corresponding to the mass shifts between the series For example if a sample contains both protonated and sodiated series there will be an autocorrelation peak at 22 Da the difference in m z between H 1 Da and Na 23 Da Likewise if there are two or more end groups there will be autocorrelation peaks corresponding to the mass differences between them Thus the peaks in the autocorrelation spectrum give clues to the nature of the repeating unit chemical composition If the original spectrum is singly charged then the peaks in the autocorrelation spectrum that are due to real chemical differences can be assigned a chemical composition 92 Computing the end group spectrum AUTOCORRELATION The end group spectrum Once an autocorrelation spectrum has been computed it is possible to get an estimate of the end groups by computing an end group spectrum The process is as follows From the autocorrelation spectrum pick the base
104. the process for creating autocorrelation and end group spectra Demonstrated how these spectra might be used to help identify an unknown polymer In the next chapter the next chapter we will describe how to get all results out of Polymerix and into other documents files or printed reports and how to set some plot customization options 97 HOMOPOLYMER ANALYSIS Plot labeling options Creating and Customizing Output How to do get creative with Pobmenx results A Polymerix and into your other documents reports and files Goals for this chapter In this chapter we will Describe how to customize the appearance of Polymertix s plots pp y p Explain how to copy plots spectral data and tabular results to the Windows Clipboard Show how to save and export Polymerix results Polymernx options There are few options that can be set to customize plot colors and labeling Untitled Polymerix File Edit view Help Copy Spectrum Plot Ctrl C Copy Spectrum Data Ctrl D Copy Results Options i prion 1 To set options select the Edit gt Options menu command This will display the Polymerix Options dialog box 98 fter spending a lot of time and thought setting up polymer analysis parameters series and analyzing data it would be nice to be able to do something with it besides look at it on screen In this chapter we will present all of the various options for getting information o
105. tion of copolymer series setup below you can add your knowledge of the chemistry to help guide the assignment process by putting constraints on the type of copolymer and ranges of A and B counts This may setve to eliminate any possibility of isobaric overlaps or at least minimize them to compositions that are chemically meanineful The Copolymer Analysis page user interface Switch to the Copolymer Analysis page by clicking the Copolymer Analysis button on the Workspace Control Panel The Copolymer Analysis page is shown below after the data processing steps have been completed ontew utan Sener ee ere ore Coreto M Di RNT D Veschann I Cheng titer Nene Penny C staneg z am r adina i Promentonat to iotas NS mi 7 ie oaa 7 tan F poga M Sedo vesan ahd In il Uda 22 As with the Homopolymer Analysis page there are three panes the upper pane contains several tabs of copolymer analysis parameters the middle pane displays molecular weight distribution statistics and the bottom pane is a tabbed display of plots and tables with different views onto the assignment results 77 COMPOLYMER ANALYSIS The analysis parameters pane contains a copy of the Mass Adjust tab This is a copy of the same tab in the Data Processing page and will not be described further here Setting copolymer processing parameters In addition to the same match tolerance and series combination options as are available for homopolymer
106. to the user interface POLYMERIX WORKFLOW AND USER INTERFACE 1 Data import and reduction of the profile continuum mode spectrum to an m z and abundance peak list followed by optional mass adjustment of the peak list to assign highly accurate m z values and or deisotoping to merge higher isotopomers into the monoisotopic peak 2 Creation of one or more homologous series definitions to permit assignment of the sets of peaks in the spectrum 3 Visualization of sample composition and series assignments through the use of graphs and tables 4 Creation of a report of the results of the analysis Each of these steps depends on successful completion of the previous step Failure to accurately peak detect the profile spectrum can lead to missed peaks or false additional peaks which will result in the failure of subsequent steps to produce correct results Likewise bad mass assignment will result in inaccurate correction of peak m z values which in turn leads to incorrect or missed series assignments and so forth Mapping this workflow onto an intuitive user interface is a major goal of the software The user interface must permit specification of the relevant parameters for each processing stage and then allow browsing the processing results to validate their correctness Corresponding to the workflow described above the user interface is organized into pages each of which corresponds to a step in the workflow 1 A data reduction and
107. urity peak occurs in the midst of sample peaks and you wish to exclude this peak from processing In this case click the button at the upper left of the Processing Mass Ranges table to add another row Select the new sub range by editing or a right click and drag in the spectrum Both the table and processed spectrum will be updated with the new range In this case an arbitrary new range was selected for illustration even though it obvious contains only noise Processing Mass Ranges Include From To 801 2001 592 759 12 To remove a sub range click the button at the left edge of the row You may also temporarily remove a mass range by unchecking the checkbox on that row 13 When all mass ranges are specified click the Apply button to set them in the parameters list 34 Evaluating peak detection parameters DATA IMPORT AND PROCESSING Detecting peaks After importing data and selecting a spectrum range the Data Processing page should look similar to the figure below ao ee r m I tr Fwar Mess Amor 1 wT For most modern instruments the resolution is high enough to resolve individual isotopic peaks in isotope clusters over most if not all of the m z range In this case it is generally desirable to process data in peak detected or centroid mode For data where peaks are not fully resolved it is possible to process without centroiding Le in profile mode with often very satisfactory results
108. ut of COPYING EXPORTING AND PRINTING Polymerix Options 2 The Display tab allows you to specify the X axis labeling preference for the chromatogram TIC plot as either scan number or retention time and to set options for labeling of peaks in mass spectra To include a label on the spectra click the checkbox to enable it Optionally change the number of decimals of precision in the label iPolymerix Options Plot color options 3 The Colors tab allows you to specify the colors used for the TIC spectrum plot titles and assigned series The colored rectangles to the right of each item are buttons Clicking the button displays a Color 99 Copying a spectrum COPYING EXPORTING AND PRINTING selection dialog box from which you may choose a basic color or define a custom color Color Jei x Basic colors r Em Em Eo E EE ZS 6 Custom colors Ens E g ERER Hue f150 Red f0 m Sat 240 Green E 64 Jenne Caston Colors ColorlSolid turm fi20 Blue 255 Cancel Add to Custom Colors 4 Click OK to close the Polymerix Options dialog box The user interface will be updated immediately to reflect any changes ll IE88 7 Copying to the clipboard The graphics in any of the spectrum panes may be copied to the Windows clipboard as a bitmap picture or the spectrum details shown in that plot can be copied as a spectrum peak list The currently selected spectrum
109. x to display the Select End Group dialog box As with the repeat formulas there is a predefined list You may choose one of these if appropriate or define a new one using the same process as for repeat formulas Click the OK button to close the dialog and add the formula to the end group parameters Select End Group 3 x Choices OK Butane Cancel Ethane Ethanol Methane Methanol Propane Water x New Edit Delete i 9 Finally if there is an adduct with an unusual isotopic pattern such as lithium or silver you may wish to specify that also Click the button to the right of the Adduct edit box to display the Select Adduct dialog box As with the previous two there is a predefined list or you may define a new one After making your choice click the OK button to close the dialog box and put the adduct in the parameter set 44 DATA IMPORT AND PROCESSING Select adduct ee Crocs Ag Cancel H K Li Na New Edit Delete 10 Finally to perform the deisotoping click the Apply button The effect of deisotoping is shown in the example below You can see in the zoomed view that the higher isotopes have been completely removed and merged into the monoisotopic peak 3220 13180 13200 1322 0 OU to TEN i t317 761 37 759 1025 tigi VATS SE 1318 254 nays t320 66t 378 256 A ae 19 TE 700 tD 19220 sreo 1320 0 t3220 11 It is important to note that the deisotoping algorithm n
110. xipeg1500_cal raw Monoaisotopic mass mode Match tolerance 0 100 Da Assignments for 1 Page 6 of 8 Maximum SIR Gay 131564 R32 17369 1850 4 100 0 31 RET 1 Ratana 12742 629 1601 51 RA 1230 0 1339R gi R33 494 4 2487 51 RAE 1185 0 hrii SH R34 1533 7 6as5 1682 5 9595 51 Ra 1142 798 50 0 oH R36 1626 5 51 Rad pow 1098 g1 R37 572 1670 8 5432 the H R38 1053 6 Iray 4077 keie 51 R39 1010 piz 2519 51 RJ FEAH 966 51 A262 0 0 oa BLS spar b sate 42189 1000 0 2000 0 116 RELEASE NOTES Polymenx Processing Report Page fof 8 Homopolymer Analysis Results Printed Wed Mar 06 2013 15 42 07 Data file C Polymerixipeg1500_cal raw Monoisotopic mass mode Match tolerance 0 100 Da Homopolymer Results Summary Series Percent Percent Label Mn bir he Fo DOP DP yw DOPs Series Spectrum Total Average 1362 742 1350 581 1415 450 1 021 30 544 31 185 31 833 100 00 34 83 51 1362 742 1350 581 1415 450 1 021 30 544 31 185 31 833 100 00 34 83 Label End Group Repeat End Group Total Average Hag C2540 51 Hag C2540 Label State Adduct Charge Loss Fomala Total Are rage 1 Ha H20 C2H40 n Na 51 1 Wa H20 C2H40 n Na Homopolymer Analysis Details Series Label 51 Repeat Percent Series Percent Commuted Found Error Found Count Series Weight Spectrum Mono m z Mono m z na Intensity 18 0 07 0 04 0 02 33 472 0 000 0 000 124 29 19 0 11 0 07 0 04 877 498 577 486 11 547 1539 43 20 0 34 0 22 0 12 321 52
111. ystem v2 45 1 The first page of the setup wizard is shown above The item labeled Application is already selected In order to be able to read raw data directly from mass spectrometry vendor format files you must also select one or more of the formats listed under the Data access section In this example the full set of supported formats is available and has been selected Depending on the details of your purchase you may have a different set of data access selections If your copy of Polymerix was purchased through an MS instrument vendor only that vendor s formats will be available INSTALLATION LICENSING AND REGISTRATION a Polymerix Setup Choose Components Choose which features of Polymerix you want to install hasian YOUT mose aver a companent bo See Ibs description WOnNSOnE Install System v2 45 2 Select the desired data access option s then click the Next gt button At any time you may click the Cancel button to stop the installation and remove any installed files from your computer You Must select at least one of the data access items if you wish to read raw data from vendor format files INSTALLATION LICENSING AND REGISTRATION a Polymerix Setup Choose Install Location Choose the folder in which to install Polymerix WUNSORE Install System v2 45 3 Next choose the installation location By default Polymertx will be installed in the folder C Polymerix If this folder does not exist
112. yzed to different series based upon mass ranges theoretical isotope clusters and the match tolerance When a data point in the spectrum being analyzed matches multiple series its intensity values are equally distributed to each of the matching series Centroid cluster matching also does special accounting for theoretical isotope cluster monoisotopic peaks The m z value for the most abundant peak which matches each monoisotopic peak is determined and used for calculating error values When series assignments are shown in data plots the series annotations are positioned at m z values corresponding to computed monoisotopic masses What was covered in this chapter In this chapter we 74 HOMOPOLYMER ANALYSIS Described the statistics calculated by Polymerix to describe homopolymer series Showed how to define a homopolymer series and set matching parameters Explained the result tables and plots produced by Polymertx in the series assignment process Explained the algorithms used for quantitative analysis In the next chapter In the next chapter we will present the procedures for defining copolymer series and visualizing copolymer assignment results 75 COPOLYMER ANALYSIS Copolymer Analysis Defining and reviewing copolymer seres assignments homopolymer analysis with one crucial difference Instead of one repeating unit there are two and as a consequence the assignments for a series form a two dimensio

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