LC-MALDI Analysis of a ICPL-labeled Protein Digest Mixture
Contents
1. Figure 2 2 Selection of a MALDI sample plate Most used and recommended sample plate type for LC MALDI experiments is the MTP AnchorChip 800 384 The next step of the wizard is the selection of the sample spots covered by the LC fractions by drag and drop WARP LC Application Tutorial Revision 2 Page 14 of 34 2 LC MALDI SILE Workflow Bruker Daltonik GmbH z Jew WARP LC R E Calibration and sample spot selection page Select positions by using a rectangle clicking on a row column name or on the appropriate spot using the CTRL Key adds new positions to eA I te selected positions are considered as prepared and are directly added to the list of positions to measure Measuring 122 A ATR ECT 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 no calibration spots NA TITTIITTLIIITITITITTITITE Cera al SERRE Ree eee pees CHESS SSSR RS own template ERE EERE EERE RRR EEE Order ER REE ERE RRR eee eee eee Vertical ZigZag SRR RBBB EBB ER Bees a ew eee eee a H BBR RRR ERR ERE EERE eee 1 ee0e00e0000000000000000600080 etereres 1 ee0e000000000000000000080 K e e 0000000000000000000080 CHOTTON OT L e eeOSOOOSOSSSSSSeSeeeeee Start Position Ao M O OOOCOCCC0C000000000000080 N ee oe ee 020000000000000000080 Number of Postions 132 H 0 OOO 0000000OOCCCCOSEES S P e0e000200000000000000080 Figure 2 3 Definition of LC MALDI fractions and preparation measuring order Preparation order has to2. k mtm S Be E Quantitation E Protein Infor E Glycc Project Navigator 22 2 B 7 O G Main View 2 a500 D Tutorial Data 15 Info Proteins amp Peptides Glycan ESI Row OK Accessi Protein MW kDa pl Al Scores Peptides SC RMS90 ppm Rank Median L H L H CV L t cea 1 l ALBU_ Serum albumin 69 2 1 967 0 19 23 334 1 1 05 7 325 F 2 F CAH2_ Carbonic anhydr 291 1 382 3 319 373 2 046 2 11 Glycoprotein MALDI 1 4 2 Ge 1 3 T _UBIQ _ Ubiquitin Bost 86 17 2119 4395 691 3 0 50 2 56v FTMS 1 2 m J gt lon trap 3 Row OK Cmpd Mrcalc Am z ppm Rt min Sequence M Protein OK L H WH lon trap 2D LC 1 14 416 1282 7034 830 28 75 R HPEYAVSVLLR L Serum albumin precursor Bos ta Ion trap ETD 3 2 M 390 14388045 364 2750 R RHPEVAVSVLLR L Serum albumin precursor Bos ta LC MALDI 3 3 M528 1783823 841 36 75 R MPCTEDYLSLIL C Serum albumin precursor Bos ta A maXis 1 a 206 1299 6030 329 2200 R CASIQKFGER A L Serum albumin precursor Bos ta Z 0 99 maXis ICPL 1 5 H 241 13056231 0 07 22 00 R CASIQKFGER A C u Serum albumin precursor Bos ta WJ 0 99 maXis iTRAQ 1 6 W 88 921 5032 444 1800 R SLGKVGTR C L Serum albumin precursor Bos ta 101 Seton ap Tetes p 7 W 89 927523 228 1775 R SLGKVGTR C L Serum albumin precursor Bos ta 19 g h abbas SILE 5 s M 352 1743 9519 213 25 50 R KVPQVSTPTLVE L Serum albumin precursor Bos ta 1
3. 8 Tutorial hbeswprotein2 Figure 2 1 Definition of a new AutoXecute Run in WARP LC Start the wizard by selecting File gt New AutoXecute Run The next step is the selection of a MALDI sample plate type For short or medium long LC gradients an MTP AnchorChip 800 384 is recommended This sample plates provides 384 sample spots and 96 calibration spots on different chips For longer LC gradients MTP AnchorChip 1536 TF is recommended Page 13 of 34 WARP LC Application Tutorial Revision 2 Bruker Daltonik GmbH 2 LC MALDI SILE Workflow New WARP LC Run B Gole es Target geometry page Select target geometry for new AutoXecute Run MSP 48 polished steel a MSP 96 T MSP AnchorChip 600 96 MSP AnchorChip 96 MSP BigAnchor 24 MSP BigAnchor 96 MSP MALDI Biotarget 48 MTP 384 glass MTP 384 ground steel MTP 384 massive Au coated MTP 384 massive MTP 384 polished steel MTP 384 Slide Adapter MTP 384 target plate matt steel MTP AB Adapter MTP AnchorChip 1536 TF MTP AnchorChip 200 1536 MTP AnchorChip 200 384 MTP AnchorChip 400 1536 MTP AnchorChip 400 384 MTP AnchorChip 600 1536 S MTP AnchorChip 600 384 MTP AnchorChip var 384 MTP BigAnchor 384 MTP Bio Rad 3 Chip Adapter MTP Bio Rad 6 Chip Adapter MTP MSP 48 polished steel Adapter MTP MSP 96 Adapter MTP MSP AnchorChip 600 96 Adapter MTP MSP AnchorChip 96 Adapter MTP MSP BigAnchor 24 Adapter z m V Start with wizard
4. LC MALDI analysis is available as tutorial data set LC MALDI SILE on the WARP LC installation DVD WARP LC Application Tutorial Revision 2 Page 5 of 34 Bruker Daltonik GmbH 1 Introduction 1 Introduction 1 1 General In Stable Isotope Labeling Experiments SILE quantitative information about proteins in complex mixtures is obtained All current protein and peptide label chemistries such as ICPL iTRAQ SILAC ICAT and 180 C terminal labeling can be used in this workflow using WARP LC 1 3 Typically 2 states of a proteome or a protein peptide mixture are compared in SILE If more than two mixtures are compared in a single LC MS MS experiment it is called multiplex SILE Each mixture is modified with a different isotopomer of the labeling reagent Identical proteins or peptides that are present in each mixture can now be distinguished in an MS or MS MS measurement and subjected to relative quantification The mixtures are combined and typically analyzed by LC MS MS As one of the isotopomers acts as an internal standard relative quantification can be achieved with high accuracy using WARP LC 1 3 Current label chemistries provide technical quantification errors of less than 10 20 which is a lot better than the typical biological variation in these experiments Using a non isobaric label like ICPL or SILAC the quantitative information is read out from the MS data and MS MS is used to identify those peptides that are regulated
5. 0 MS MStol 08 Day E Second Round Peptide charge 1 X Monoisotopic Average Configure Rights Instrument type CID MALDI TOF TOF Z Instrument type ETD l ETD TRAP z E Peptide Decoy Mascot Use Percolator Figure 2 17 Database search parameter for SILE samples labeled with ICPL2plex Serva The method is part of the ProteinScape default setup Note The ICPL modifications must be set to variable Although digestion was performed with trypsin cleavage only occured at Arginine residues because all Lysines were labeled Therefore the best suitable enzyme is ArgC For SILE quantitation the biological states have to be addressed and the regulation ratio has to be defined Page 29 of 34 WARP LC Application Tutorial Revision 2 Bruker Daltonik GmbH 2 LC MALDI SILE Workflow Se a MS MS database search method ProteinScape LC MALDI ICPL2plex ArgC x Isotope Purity Correction Biological Experiment Biological State ICPL Labels assigned to Biological States Elemental Formula Mono lsotopic Mass Biological State HG C 6 N 1 OQ 105 021464 HG 13C 6 N OQ 111 041593 Reported Ratios ii Ratio Name _ Numerator ow _Denominator y Figure 2 18 SILE chemistry is selected according to modifications defined in the ProteinScape search method LC MALDI_ICPL_2plex_ArgC Biological states can be defined according to individual projects SILE quantitation can be performed on
6. 3 proteins have been identified and ratios of ICPL light ICPL heavy L H labeled samples have been calculated WARP LC Application Tutorial Revision 2 Page 32 of 34 3 ProteinScape Bruker Daltonik GmbH For bovine serum albumin the theoretical value of L H is 1 0 the calculated is 1 05 CV of 3 2 The theoretical L H values of carbonic anhydrase and ubiquitin were 0 5 the experimental value were 0 46 and 0 5 respectivley Page 33 of 34 WARP LC Application Tutorial Revision 2 Bruker Daltonik GmbH 3 ProteinScape Protein Regulation L H experimental Regulation L H theoretical Serum albumin 1 05 median Carbonic anhydrase 0 46 median Ubiquitin 0 50 median When the database search is started by the user from ProteinScape and not from WARP LC the SILE quantitation is not triggered automatically After the search is finished go to page Info and start the SILE quantitation DS Main view 53 For Info Proteins amp Peptides Errored v Search Result Info Owner Tutorial Tutorial Date Friday November 11 2011 12 53 47 PM Duration 165 Identified compounds 27 594 Accepted proteins 3 20 Note zi g Show Parameters 9 Repeat Search Show Protein Report Show Spectrum Report Show Search Parameter Report E SILE Quantitation Normalization ma 8 Manual validation Figure 3 2 Press SILE Quantitation on the Info page to start quantitation in WARP LC Important a WARP LC me
7. WARP LC Application Tutorial Revision 2 Bruker Daltonik GmbH Contact Limitations on Use For Research Use Only RUO Not for use in diagnostic procedures Hyperlink Disclaimer Bruker Daltonik GmbH makes no express warranty neither written nor oral and is neither responsible nor liable for data or content from the linked Internet resources presented in this document Contact Contact your local Bruker Daltonics representative for service and further information USA Germany Bruker Daltonics Inc Bruker Daltonik GmbH 40 Manning Road FahrenheitstraRe 4 Billerica MA 01821 28359 Bremen USA Germany Phone 1 978 6633660 Phone 49 421 2205 450 Fax 1 978 6675993 Fax 49 421 2205 370 Internet www bdal com Internet www bdal de Service Support Service Support Phone 1 978 6633660 1445 Phone 49 421 2205 350 E mail service support bdal com E mail service bdal de WARP LC Application Tutorial Revision 2 Page 3 of 34 Table of Contents Bruker Daltonik GmbH Table of Contents Legal and Regulatory Notices 2 00 0 0 eee c cece cece cece ccc cceccececceceeceeeeeeeeeeeees 2 Contact orcad ctor nessa tae te ade Sele pees eed aaa ues eae aero Table of Contents ck a ote as cod cded ae ulgyseunacneas ob eee weeded esha eerecess 4 Introductory Remarks 2 0 00 002 c0ccccecceeeeceecceecceeeccecececceeececeeeeteceeeeeeeeeees 4 1 Introduction oaaao ceccecccecceccecececccecceceeecceccceceececcseceecetec
8. be defined according to LC fractionation order measuring order can be defined independently of preparation order It is recommended to use Peptide calibration standard II Part No 222570 Bruker Daltonics for external near neighbor calibration Page 15 of 34 WARP LC Application Tutorial Revision 2 Bruker Daltonik GmbH 2 LC MALDI SILE Workflow New WARP LC Run B belea Run parameter page Select methods for the measuring positions If the Calibration mode is activated the respective methods are automatically assigned to calibration spots MS and MS MS methods are assigned to the regular measuring spots The retention time is calculated based on the previously selected preparation order MS Data Destination Calibration Data Directory D Data Tutorial Data WARP LC Calibration Sample Name LC MALDI SILE AutoXecute Method LC MALDI MScalibrate v Edit Comments flexAnalysis Method none Comment 1 Comment 2 AutoXecute Method 1C MALDIICPL2plex MSmeasur v Edit ProteinScape flexAnalysis Method none Experiment ID 281474976716977 m WARP LC Method LC MALDI SILE Tutorial e ESI Analysis Retention time Delay 00 25 00 a hmins Time Slice 15 00 5 s E Stop after MS measurement E Clean source after measurement tee C nees ta Figure 2 4 Selection of AutoXecute Methods for the LC MALDI analysis In the Run parameter page Figure 2 4 of the A
9. on the MALDI target represents a chromatographic fraction The MS and MS MS data acquisition of such a prepared LC run requires the definition of an AutoXecute Run Thus the AutoXecute Run is the central data source for the measurement of the LC MALDI based workflow 2 1 WARP LC New AutoXecute Run Start WARP LC 1 3 and log in ProteinScape 3 0 using your personal account and password see also ProteinScape 3 0 user s manual Under File you can start a wizard under New AutoXecute Run leading you through the different steps of the AutoXecute run definition WARP LC Application Tutorial Revision 2 Page 12 of 34 2 LC MALDI SILE Workflow Bruker Daltonik GmbH Tutorial_LC MALDI SILE ICPL2plex Bruker Daltonics WARP LC E EA File Edit Acquisition Compounds ProteinScape Help b New AutoXecute Run Ctrl N i ARP LC LC MALDI SILE Tutorial_LC MALDI SILE ICPL2plexxml cs f Eait ERTE thods LC MALDI ICPL2plex Default WarpLCMethod Recent WARP LC Method poate aes cea hod LC MALDI MSMSmeasure axe saad method ProteinScape LC MALDLICPL2plex_ArgC Interactive MALDI Workflow Control gt Start MS or MS MS m E Calculate Compound List C Display Compound List 43 Add MS MS jobs to run 9 Re B Export to ProteinScape VJ Start search k Current Automatic WARP LC Experiments No WARP LC experiments running Status Workflow MALDI SILE
10. peak area values or on peak intensities Additionally the mass tolerance of label recognition can be set here Depending on the label chemistry peptides of same sequence but with different isotopic labels show slightly different retention time This is e g the case for ICPL 4plex labeled samples To address this phenomenon Coeluting Isoforms should be unchecked and the retention time tolerance of 2 peptides should be defined WARP LC Application Tutorial Revision 2 Page 30 of 34 2 LC MALDI SILE Workflow Bruker Daltonik GmbH i LC MALDI SILE Tutorial WarpLCMethod WARP LC Method Editor E xa Ratio calculation based on Peak area Peak intensity Label Recognition Mass Tolerance 0 1000 Da Coeluting Isoforms Retention Time Tolerance 15 00 a 5 Extracted lon Chromatogram Settings Charge States from 1 to 4 x V Smooth _ Subtract Spectral Background savers seve 0K cancel Figure 2 19 Parameter settings for SILE quantitation Page 31 of 34 WARP LC Application Tutorial Revision 2 Bruker Daltonik GmbH 3 ProteinScape 3 ProteinScape After MS and MSMS acquisition has finished the data are automatically sent to ProteinScape 3 0 In the case no ProteinScape experiment number was selected in the AutoXecute run the data export can be triggered manually from WARP LC Pe ProteinScape Tutorial hbeswprotein2 File Edit Window Help
11. 03 an ee 9 353 1749 9721 111 25 50 R KVPQVSTPTLVE I Serum albumin precursor Bos ta 1 03 A a v 180 2221 8351 114 20 00 R ETYGDMADCCE 1 Serum albumin precursor Bos ta g 1 06 ight uM 162 22278552 030 20 00 R ETYGDMADCCE C Serum albumin precursor Bosta Z 106 Lp Tutorial LC MALDI SILE ICPL2plex 2 12 W 334 11056033 989 25 00 RALKAWSVAR L L Serum albumin precursor Bosta Z 1 06 L LC MALDI ICPL2plex ArgC Mascot 2011 11 13 335 11116234 752 2500 RALKAWSVAR L L Serum albumin precursor Bosta Z 106 Li Compounds 594 14 W 386 3301 5682 318 2235 R LCVLHEKTPVSE L Serum albumin precursor Bosta Z 107 15 W 387 33196286 165 2700 R LCVLHEKTPVSE C Serum albumin precursor Bosta Z 107 1s U 2 1270507 254 14 50 R CCTKPESERM L Serum albumin precursor Bosta Z 110 c 21 12765272 154 14 50 R CCTKPESERM C Serum albumin precursor Bosta Z 110 Sequence Ll Spectrum 3 gt 08 Processing View simi 54 c0 D ILs Ii 3 m Ey 0 N L 1 L L y D E T ce LC MS Survey X N E Gel ca 4 y min 1000 J 194 gt J z J J y y yaa y 12 yao yan wap T d T T T 200 400 600 800 1000 1200 1400 1600 1800 m z Cmpd_528 MS MS_ 1724 82 r Figure 3 1 Database search and quantitation results in ProteinScape 3 0 The first protein in the list serum albumin was selected The peptide list of serum albumin was sorted on L H quantitation results Results
12. 1 maxis ICPL 1 maxis iTRAG 1 xxx lon trap Test E coli 5 LC MALDI SILE 8 2 2 2 2 2 2 2 D Tutorial Data S LC MALDI SILE Figure 2 5 Selection of ProteinScape Experiment ID You have to choose your ProteinScape project and sample Note The settings for Calibration AutoXecute Calibration Method AutoXecute MS Method Data Directory Sample Name Comments WARP LC Method and Retention Time Offset and Interval can be changed before it started Page 17 of 34 WARP LC Application Tutorial Revision 2 Bruker Daltonik GmbH Figure 2 6 i New WARP LC Run Ba Loe a Finish page The following infomation has been specified for creating the new AutoXecute Run Target Geometry MTP AnchorChip 800 384 Number of positions selected 240 Preparation order HORIZONTAL_ZIGZAG AutoX Calibration method LC MALD MScalibrate AutoX MS method LC MALDI ICPL2plex MSmeasure Data Directory D Data Tutorial Data WARP LC SampleName LC MALDI SILE Comment1 Comment2 ProteinScape Experiment ID 281474976716977 WARP LC Method LC MALDI SILE Tutorial HCT Analysis Retention time offset 00 25 00 Retention time interval 00 00 15 2 LC MALDI SILE Workflow Overview of the selected methods and other settings specified for the new AutoXecute Run WARP LC Application Tutorial Revision 2 Page 18 of 34 2 LC MALDI SILE Workflow Bruker Daltonik GmbH AutoXecute
13. 977 a 0 LC MALDIICPL2plex MSmeasure none D Data Tutorial DatalWARP LC LC MALDI SILE 400_P 48 00 37 00 281474976716977 D1 0 LC MALDIICPL2plex MSmeasure none D Data Tutorial DatalWARP LC LC MALDI SILE 400_P 35 00 48 45 281474976716977 D2 0 LC MALDI ICPL2plex MSmeasure none D Data Tutorial Data WARP LC LC MALDI SILE 400_P cS 00 48 30 281474976716977 c2 0 LC MALDIICPL2plex MSmeasure none D Data Tutorial DatalWARP LC LC MALDFSILE 400_P 49 00 37 15 281474976716977 E1 1 LC MALDI MScalibrate none D Data Tutorial DataiWARP LC LC MALDI SILE 500_C 281474976716977 4 4 ii 01 b2 _ G2 _ E1 HE or is x 0 g HAHEN HAEHAE as f ky THEHEHEHE Target Geometry MTP AnchorChip 800 384 Measuring Order Calibration Sample Spots Count 240 WarpLC Method LC MALDI SILE Tutorial Prepared Mi For Calibration MEMS Measured MSMS Measured I Flatline Spect Save Save As 0k Cancel Figure 2 7 Complete overview of the new AutoXecute Run The change of settings in the AutoXecute Run such as Methods Data directory Sample Name or Retention Time is possible before the acquisition was started 1 A different Method is selected or the DataDirectory Sample Name or Comment is changed 2 Thesample spots for which settings should be changed have to be selected either in the target view or in the spreadsheet 3 The new settings are assigned to the selected sample spots in the Spreadsheet by pressing the button Assign Met
14. Application Tutorial BRUKER LC MALDI Analysis of a ICPL labeled Protein Digest Mixture ESI MS MS PROJECT B KNOWLEDGE MALDI Target TOF TOF TOF Revision 2 November 2011 Legal and Regulatory Notices Bruker Daltonik GmbH Legal and Regulatory Notices Copyright 2011 Bruker Daltonik GmbH All other trademarks are the sole property of their respective owners All Rights Reserved Reproduction adaptation or translation without prior written permission is prohibited except as allowed under the copyright laws Document History LC MALDI Analysis of a CPL labeled Protein Digest Mixture Revision 2 November 2011 Part number 283918 First edition November 2011 Warranty The information contained in this document is subject to change without notice Bruker Daltonik GmbH makes no warranty of any kind with regard to this material including but not limited to the implied warranties of merchantability and fitness for a particular purpose Bruker Daltonik GmbH is not liable for errors contained herein or for incidental or consequential damages in connection with the furnishing performance or use of this material Bruker Daltonik GmbH assumes no responsibility for the use or reliability of its software on equipment that is not furnished by Bruker Daltonik GmbH Use of Trademarks The names of actual companies and products mentioned herein may be the trademarks of their respective owners Page 2 of 34
15. Current Automatic WARP LC Experiments No WARP LC experiments running Status Workflow MALDI SILE 88 Tutorial hbeswprotein2 Figure 2 9 AutoXecute run with WARP LC method in WARP LC 1 3 before start of the run Start the AutoXecute run from the main page of WARP LC within Interactive MALDI Workflow Control When all the methods are selected properly as described in this tutorial MS and MS MS acquisition in flexControl spectra processing in flexAnalysis and database search and quantitation in ProteinScape will be performed automatically Note Before starting the AutoXecute run FlexControl methods for MS and MSMS acquisition have to be tested Typically the methods RP_700 3200 Da par and Lift 1ft are optimized for LC MALDI applications Please check settings like laser power mass range detection gain and calibration before starting the analysis Page 21 of 34 WARP LC Application Tutorial Revision 2 Bruker Daltonik GmbH 2 LC MALDI SILE Workflow 2 2 AutoXecute Methods For a complete LC MALDI analysis 3 different AutoXecute Methods are required to control the automatic MS and MS MS data acquisition and to perform a proper calibration of the MS Before you can use the default AutoXecute methods you should open them and select the flexControl methods valid at your instrument For calibration and MS measurement select a flexControl method for MS acquisition in reflectron mode for MSMS acquisition select a
16. ILE Compound Definition MALDI Mass tolerance 50 00 Merge compounds separated by less than 6 4 fractions F Identify a compound as background if 70 of all fractions empty fractions disregarded compound mass occurs in more than Figure 2 15 Compound definition the mass tolerance for compound calculation is set to 50 ppm Background peaks are not considered for MSMS acquisition For successful MSMS acquisition a valid AutoXecute method has to be selected The default method for LC MALDI experiments is LC MALDI MSMSmeasure axe S N threshold of 10 limits the selection of precursors to those with a minimum S N value of 10 Page 27 of 34 WARP LC Application Tutorial Revision 2 Bruker Daltonik GmbH 2 LC MALDI SILE Workflow Additionally co eluting compounds and maximum number of MS MS spectra acquired per fraction spot are taken into account when scheduling MS MS applications fa LC MALDI SILE Tutorial WarpLCMethod WARP LC Method Editor g Ea Workflow Compound Definition MALDI MS MS SILE Chemistry SILE Data acquisition AutoXecute Method LC MALDI MSMSmeasure axe x Precursor selection S N threshold 10 00 Min mass distance to co eluting 20 Da compounds of higher intensity Max no of MS MS per fraction 106 Allow more MS MS if no alternative fractions are available Acquisition order within fraction Acquisition Score descending ascending
17. Precursor m z descending ascending Scheduled Precursor List mam Remove Edit included compounds Data processing MS MS database search method ProteinScape LC MALDI ICPL2plex ArgC X Figure 2 16 The parameter settings for MS MS acquisition are pooled on the MALDI MS MS page Database searches are performed using ProteinScape 3 0 A proper search method has to be defined there WARP LC Application Tutorial Revision 2 Page 28 of 34 2 LC MALDI SILE Workflow Bruker Daltonik GmbH r be Protein Searches E n Search Parameters Search Parameter Configuration MS MS PFF G General Settings Mascot Select search method _ LC MALDLICPL2plex ArgC m 96 Database SwissProt z Method name LC MALDLICPL2plex_ ArgC Taxonomy a Other mammalia gt Version 10 2 gt x Enzyme Allow up to E missed cleavages Submit All spectra x Modifications Acetyl K Carbamidomethyl C Fixed Variable Acetyl N term s A Search engines V Mascot pirr aiaia APE ICPL K Fixed Variable Precursor E Use peptide Mr from glycopeptide classification Amidated C term ICPL lt 13C 6 K Fixed Variable Amidated Protein C term i d i Max no of listed proteins is d m Oxidation M D Fixed Variable Protein list compilation By search engine By ProteinExtractor z uga J paces Peptide tol 30 0 c
18. Run Editor Version 3 493 0 a sees File Edit View Help OSE oo MMZIZ x es Ms Data Destination Calibration Data Directoy DAData Tutorial Data WARP LC aa 7 Calibrati E sol Sample Name LC MALDI SILE AutoXecute Method LC MALDI MScalibrate m Ea Comments ee Gna Comment 2 AutoXecute Method LC MALDI ICPL2plex MSmeasut v Edt ProteinScape flexAnalysis Method Experiment ID 281474976716977 Cams WARP LC Method LC MALDI SILE Tutorial ESI Analysis Retention time Delay 002500 2 hmins Time Slice 1500 2 s O Assign Methods to Run Clear MS Status Clear MS MS Stat Stop after MS measurement ie o E Cean source after measurement Pos on Scout Chip o AutoX MS FAMS AutoX MS Data Directory Sample Name Status Comment Chr Index Retention time PSExperimentID Parentmass gt At 1 UC MALDI MScalibrate none D Data Tutorial DatalWARP LC LC MALDI SILE 500_C 2814749767 16977 m Al 0 LC MALDI ICPL2plex MSmeasure none D Data Tutorial Data WARP LC LC MALDI SILE 400_P 0 00 25 00 281474976716977 B1 0 LC MALDIICPL2plex MSmeasure none D Data Tutorial DatalWARP LC LC MALDISILE 400_P 47 00 36 45 281474976716977 B2 0 LC MALDIICPL2plex MSmeasure none D Data Tutorial DatalWARP LC LC MALDI SILE 400_P 46 00 36 30 2814749767 16977 a2 0 LC MALDIICPL2plex MSmeasure none D Data Tutorial DatalWARP LC LC MALDI SILE 400_P 1 00 25 15 281474976716977 a 1 LC MALDI MScalibrate none D Data Tutorial DatalWARP LC LC MALDI SILE 500_C 2814749767 16
19. Using an isobaric label like iTRAQ all MS peaks need to be MS MS analyzed as the quantitative information is unraveled only in the MS MS spectrum by the reporter ions of the different forms of the iTRAQ label see Figure Peptides occurring in all labeled mixtures are identified as pairs SILE pairs The area or intensity ratio of the two peaks e g L H ofa SILE pair is a measure of the change in the peptide abundance in the mixtures The ratio of the peptide abundances is called regulation WARP LC Application Tutorial Revision 2 Page 6 of 34 1 Introduction Bruker Daltonik GmbH iITRAQ isobaric label ICPL non isobaric label Protein samples E r O Hae wat aa Tryptically digested proteins OD D age La N Peptides labeled with iTRAQ p PPA SH oom Oa Mixed sample Mixed sample 4 2D LC FFE 1D Tryptically digested proteins mz esd MSIMS MS mz mz light label heavy label Figure 1 1 Typical workflows for common label chemistries iTRAQ peptides are labeled post digest and quantification is performed based on reporter ions in MS MS spectra All peptides need to be MS MS analyzed ICPL permits protein pre fractionation because labeling is done prior to protein digests Page 7 of 34 WARP LC Application Tutorial Revision 2 Bruker Daltonik GmbH 1 Introduction Peptide separation WARP LC flexControl flexAnalysis HyStar Compound determination WARP LC MS MS data
20. arpLC Method Ic maldi sile tutorial Prepared Ii For Calibration MEMS Measured MS MS Measured ll Flatline Spectrum Save Save As ok Cancel Figure 2 8 Change of settings in the AutoXecute Run before starting the data acquisition This example shows how to change or add ProteinScape Experiment ID to the run Select the sample spots choose new experiment ID and assign the new parameter to the run Save the AutoXecute Run either to the default directory d Methods AutoXSequences or directly to the data directory of the analysis After saving the AutoXecute Run the Editor can be closed and the initial screen of the WARP LC software appears that displays the new AutoXecute Run together with the selected WARP LC Method WARP LC Application Tutorial Revision 2 Page 20 of 34 2 LC MALDI SILE Workflow Bruker Daltonik GmbH Tutorial_LC MALDI SILE ICPL2plex tutorial Bruker Daltonics WARP LC amp EA File Edit Acquisition Compounds ProteinScape Help AutoXecute Run DAData Tutorial Data WARP LC LC MALDI SILE Tutorial_LC MALDI SILE ICPL2plex_tutorial xml a WARP LC method DAMethods WarpLCMethods LC MALDI SILE Tutorial WarpLCMethod Scheduled Precursor List AutoXecute MS MS method LC MALDI MSMSmeasure axe MS MS database search method ProteinScape LC MALDI ICPL2plex ArgC Interactive MALDI Workflow Control gt Start MS or MS MS a E Calculate Compound List E a5 5 w Start search
21. d is mandatory The path of the tutorial data on your computer should be D data Tutorial Data WARP LC LC MALDI SILE ICPL2plex_ standard protein mix Page 9 of 34 WARP LC Application Tutorial Revision 2 Bruker Daltonik GmbH 1 Introduction 1 3 Sample Preparation A mixture of 3 proteins bovine serum albumin carbonic anhydrase and ubiquitin was labeled using ICPL kit Serva Germany as described in the manual An aliquot of the tryptic digest was diluted by a factor of 20 and 5 uL of this dilution was used for LC separation For the LC MALDI analysis of IC PL labeled protein digest mixtures the following LC conditions are recommended used for the data presented in this tutorial Easy nLC Bruker with a flow rate of 0 3 uL min Column 75 um id x15cm PepMap LC Packings Solvent A water 0 05 trifluoroacetic acid Solvent B acetonitrile 0 05 trifluoroacetic acid Gradient 0 48 min 45 B 48 60 min 95 B 60 70 min 5 B Fraction collection every 15 sec MALDI target MTP AnchorChip 800 384 A continuous flow of 100 uL h matrix solution was spotted during fractionation via a syringe pump connected to the capillary coming from the LC resulting in 0 4 uL matrix solution per spot Matrix solution 748 uL 90 Acetonitrile water 36 uL saturated solution of HCCA in 95 Acetonitrile water 8 uL 10 TFA in water 8 uL 100 mM NH H PO in water Calibration spots Peptide calibration
22. d LC MALD CPL4plex MSprocessing FAMethod contain specific parameter enabling the peak picking algorithm to properly annotate the SILE pairs If you want to check or change parameters of the processing methods open the method in flexAnalysis and press Edit Parameters For more details please read the flexAnalysis User Manual 7 44 Edit Processing Method Active Settings E E E Mass List g Lind Mass List Find Edit Processing Peak Detection Algorithm Snap v Signal to Noise Threshold 10 Relative Intensity Threshold 0 Minimum Intensity Threshold 0 Maximal Number of Peaks 200 Quality Factor Threshold 100 SNAP average composition l Averagine A Edt V Fragment Peak Width 0 75 mz V SILE Mass Difference s H 1 6 021 SILE Partner SN Threshold 3 Baseline Subtraction TopHat F Page 25 of 34 WARP LC Application Tutorial Revision 2 Bruker Daltonik GmbH 2 LC MALDI SILE Workflow Figure 2 13 FlexAnalysis method for ICPL2plex processing Expected mass difference of the SILE partners is defined 2 4 WARP LC Method The WARP LC Method defines all required parameters for the MS and MS MS data acquisition of a chromatographic separated sample After the MS data acquisition of all fractions the WARP LC Method controls the generation of a non redundant list of chromatographically separated precursors compounds according to the sett
23. eeereeaeecerenees 6 sc t SR Oe ee ne OTe RR eee Iva te See a eee a Eii 6 12 TWO a atest coi tote eens eed a a tee 9 13 Sample Preparation 255g acess hice ee LLALL ect todos 10 2 LC MALDI SILE Workflow 0 00 22 c cece cece cece ccc ccc cececeeeeeeeeeececeeeeeteeeeneneees 12 2 1 WARP LC New AutoXecute RUN 00 00 cece ceecceeceeececeeeeeeceeeceeeeeeeeeeee 12 2 2 AutoXecute Methods 22 0 coco eee cee cence cece eee edoa oaao ar raoin rrean 22 23 FlexAnalysis Method oc ucecos cece c sraueen cca 0000000000 a0 aao aaa aoa aoaaa aoa ainaani 25 2 4 WARP LC Method coos oc ona lcs uc necbsid sce nananana n ALADA AL L E EDADA a aranana nana 26 3 ProteinScape 0 a aaao aa aaao aaan aoaaa A LLALLA LLALLA LLALLA DLADLA LLAD LLALLA LLa aoaaa 32 Introductory Remarks Related software or higher Compass 1 3 Patch 5 for flexSeries flexControl flexAnalysis Mascot 2 3 ProteinScape 3 0 Note In contrast to Warp LC 1 2 this new Warp LC version does not support BioTools for database searches All searches have to performed with ProteinScape 3 0 This tutorial describes the setup of aLC MALDI SILE analysis for peptide quantification of a low complex protein sample Exemplary shown in this tutorial is the LC MALDI SILE analysis of a 3 protein digest mixture labeled with the ICPL duplex kit The data set of this Page 4 of 34 WARP LC Application Tutorial Revision 2 Bruker Daltonik GmbH Introductory Remarks
24. flexControl flexAnalysis Database search quantitation ProteinScape Figure 1 2 The LC MALDI SILE workflow using WARP LC WARP LC Application Tutorial Revision 2 Page 8 of 34 1 Introduction Bruker Daltonik GmbH SILE labeled protein digest samples are separated via LC and eluting peptides are directly deposited onto a MALDI target The MS and MS MS data acquisition is controlled by flexControl triggered by WARP LC As soon as all MS data are available WARP LC determines compounds from all peak lists MS MS data acquisition is triggered by WARP LC according to MALDI MS MS parameters defined in the selected WARP LC method After the MS MS measurement is finished and all peak lists from flexAnalysis are available WARP LC triggers a Mascot search via ProteinScape of the complete data set Finally quantitation of the peptides of each identified protein is performed and results are displayed on the Proteins amp Peptides page of ProteinScape 3 0 1 2 Tutorial Data Tutorial Data for this tutorial are provided on the WARP LC installation DVD The tutorial data need to be copied to your computer before you can use them The data are collected in the directory LC MALDI SILE Note To use the tutorial data you need ProteinScape 3 0 and compass 1 3 Patch 5 or higher flexControl 3 3 108 and flexAnalysis 3 3 80 or compass 1 4 Please refer to manuals and release notes in order to install the programs maldi sw support bdal de Drive
25. hods to Run Page 19 of 34 WARP LC Application Tutorial Revision 2 Bruker Daltonik GmbH 2 LC MALDI SILE Workflow File Edit View Help De o Se 212 x es MS Data Destination a He ra ra ral ra a a a ra Calibration Data Directoy D Data Tutorial Data WARP LC ama BORRERERARAAD Atattraton Sample Name TCMALDISILE 10220012A A A E As ase mares ea oo0o0o00 200001000000 mn f ma G man G oma G oaa G a n g m G oma G o G ma G m G a f m mafa a ay MS Data Destination WARP LC Comment 2 F AtoXecute E Data Directory E Method k E a m ProteinScape exAnalysis Sample Name JESI Analysis 3 Experiment ID 281474976716977 E Retention time WARP LC ProteinScape E Comment 1 7 Experiment ID Method LC MALDI SILE Tutorial E Comment 2 ESI Analysis m Retention time Select All Select None Assign Delay 00 25 00 HH hmins Time Slice 1500 2 s Clear MS MS Status E Stop after MS measurement El Cean source after measurement FAMS AutoX MS Data Directory Sample Name Status Comment Chr Index Retention time PS ExperimentID Parentmass D Data Tutorial DatalWARP LC LC MALDI SILE 500_C 281474976716977 DOO O O O OOO OOOO OOO OO OOOO OO O88 OOOO OOOO 8 O88 OO a O O O OOOO O O O OOOO OOO OOOO OOOOF Target Geometry MTP AnchorChip 800 384 Measuring Order Calibration Sample Spots Count 240 W
26. ings in the WARP LC Method This list is called compound list In addition parameters for a Mascot database search are referenced in the WARP LC method which automatically triggers the database search of all acquired MS MS data la LC MALDI SILE Tutorial WarpLCMethod WARP LC Method Editor 5 sl Workflow Compound Definition MALDI MS MS SILE Chemistry SILE Type LC MALDI D LC ESI LC MALDI SILE Stable Isotopic Labeling Experiment LC ESI SILE Description Comment WARP LC method for the LC MALDI analysis of ICPL2plex labeled samples on the Bruker flex instruments ICPL modifies the Lys side chain epsilone amines and the alpha amino group of the PROTEIN s N terminus The ICPL label SERVA Heidelberg is used for quantitation on the proteome level An adequate ProteinScape Method considering the ICPL labels related to the local Mascot Server settings has to be selected WARP LC Application Tutorial Revision 2 Page 26 of 34 2 LC MALDI SILE Workflow Bruker Daltonik GmbH Figure 2 14 Workflow description of LC MALDI SILE Tutorial WarpLC Method The compound definition is mainly depending on the mass accuracy of your instrument Background peaks can be detected and excluded from the list of scheduled MS MS precursors r LC MALDI SILE Tutorial WarpLCMethod WARP LC Method Editor 5 E3 a Workflow Compound Definition MALDI MS MS SILE Chemistry S
27. mix Bruker was dissolved in 125 uL 30 Acetonitrile water 0 1 TFA 10 uL of this solution was mixed with 90 uL of the matrix solution and 0 5 uL were manually deposited to each calibration anchor on chip 1 of the target WARP LC Application Tutorial Revision 2 Page 10 of 34 1 Introduction Bruker Daltonik GmbH AutoXecute Method for i LC MALDI MScalibrate axe calibration Autoxecute MENON 1a LC MALD CPL2plex MSmeasure axe MS acquisition AutoXecute Method lor LC MALDI MSMSmeasure axe MS MS acquisition MERANA MENOR Op LC MALDI MScalibrate FAMSMethod calibration nexAnalysis Memamo LC MALD CPL2plex MSprocessing FAMSMethod MS acquisition flexAnalysis Method for 7 ae LC MALDI MSMSprocessing FALIFTMethod MS MS acquisition 2 WARP LC Method LC MALDI SILE Tutorial WarpLCMethod ProteinScape Method LC MALDI ICPL2plex ArgC database search The methods are all installed during WARP LC 1 3 and ProteinScape 3 0 installation You will also find them on the CD despite the ProteinScape method Page 11 of 34 WARP LC Application Tutorial Revision 2 Bruker Daltonik GmbH 2 LC MALDI SILE Workflow 2 LC MALDI SILE Workflow The LC MALDI SILE workflow in WARP LC supports the MS and MS MS data acquisition of isotopic labeled protein peptide samples A SILE labeled sample is LC separated and fractions of the eluting peptides are deposited on a MALDI target Each sample spot
28. ower The correct laser power value can be achieved by acquiring MS and MSMS spectra of a typical sample spot Also find the best laser power value for the calibration spots Page 23 of 34 WARP LC Application Tutorial Revision 2 Bruker Daltonik GmbH 2 LC MALDI SILE Workflow Note The laser power for MSMS acquisition should be optimized directly in the flexControl method Open your valid name ft method optimize laser power for Parent and Fragments using your sample and save the method Every AutoXecute method refers to a specific default processing method in flexAnalysis AutoXecute Method Editor Version 3 4 92 0 Method LC MALDIICPL2plex MSmeasure E Ex AutoXecute Method LC MALDI ICPL2plex MSmeasure General Laser Evaluation Accumulation Movement Processing MS MS flexAnalysis Method LC MALDI ICPL2plex MSprocessing FAMSMethod BioTools MS Method none none Figure 2 12 Reference to processing method in flexAnalysis WARP LC Application Tutorial Revision 2 Page 24 of 34 2 LC MALDI SILE Workflow Bruker Daltonik GmbH 2 3 FlexAnalysis Method The flexAnalyis methods define all required parameter for the MS and MS MS data processing including peak picking of MS and MS MS data and calibration of MS data In the case of ICPL labeled samples peak picking of the SILE pairs is critical The specific flexAnalysis methods LC MALD CPL2plex MSprocessing FAMethod an
29. thod for ICLP SILE has to be assigned to the run in WARP LC WARP LC Application Tutorial Revision 2 Page 34 of 34
30. utoXecute Run Wizard the AutoXecute Methods for data acquisition of the calibrants and sample spots are defined as well as the data directory and the sample name relevant for storage of the spectra The recommended data directory is d data folder name Additonally the ProteinScape Experiment ID has to be selected in order to properly load the data into ProteinScape The WARP LC method contains important settings for MS MS acquisition database search and quantitation Note The FlexAnalysis Method is defined directly in flexControl AutoXecute the ProteinScape Method and MS MS AutoXecute Method are defined in the WARP LC Method The calculation of the Retention time for each fraction is based on WARP LC Application Tutorial Revision 2 Page 16 of 34 2 LC MALDI SILE Workflow Bruker Daltonik GmbH a Delay between injection of the sample onto the LC column and the start of fraction collection b the Time Slice used collection time per fraction These values have to be defined by the user either here or before in HyStar LC and ProteineerFC fraction collector control software To enable data export to ProteinScape a ProteinScape Experiment ID has to be selected a Ps Export to Proteir spe on hbeswprotein2 mi3 Tutorial Data 15 Z _Glycan ESI _Gilycan MALDI Z Glycoprotein ESI A Glycoprotein MALDI 1 C 2D Gel 1 FTMS 1 lon trap 3 lon trap 2D LC 1 lon trap ETD 3 LC MALDI 3 maxis
31. validated Lift method Save the methods with this selection AutoXecute Method Editor Version 3492 0 Method LC MALDI MSMSmeasure Ss AutoXecute Method LC MALDI MSMSmeasure General Laser Evaluation Accumulation Movement Processing MS MS flexControl Method D Methods flexControl Methods Lift ft m Description Save Saves ok Cancel Help Figure 2 10 Selection of a FlexControl Method As the laser power differs from instrument to instrument you have to select the proper laser power for each AutoXecute method Save the methods with this new value WARP LC Application Tutorial Revision 2 Page 22 of 34 2 LC MALDI SILE Workflow Bruker Daltonik GmbH AutoXecute Method Editor Version 3 4 92 0 Method LC MALDI MScalibrate x AutoXecute Method LC MALDI MScalibrete x General Laser Evaluation Accumulation Movement Processing MS MS Laser Power Fuzzy Control MS Parent Mode On Off 1 00 On Off H Initial Initial Laser Power 15 or from Laser Attenuator 40 x Matrix Blaster 0 25 Figure 2 11 Selection of a proper laser power in the AutoXecute methods for MS measurements This should be done for LC MALDI MScalibrate axe and for LC MALDI MSmeasure axe Note Before you start an LC MALDI run open the AutoXecute methods and edit the laser p
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