MS2Analyzer User Manual - Metabolomics Fiehn Lab
Contents
1. 25 ShGKMSMSp d MS MS of 389 0654247 1 at 0 137566666666667 mins 0 26 ShEKMSMSp d MS MS of 389 0654247 1 at 0 142983333333333 mins 27 5h6kMSMSp d MS MS of 237 0834021 1 at 0 154183333333333 mins 28 5h6kMSMSp d MS MS of 237 0834021 1 at 0 15635 mins 29 Sh6KMSMSp d MS MS of 391 2845829 1 at 0 159066666666667 mins 30 5hGkMSMSp d MS MS of 391 2845829 1 at 0 161783333333333 mins 31 5h6kMSMSp d MS MS of 450 3579445 1 at 0 164683333333333 mins 32 5hGKMSMSp d MS MS of 450 3579445 1 at 0 167566666666667 mins 33 5hGKMSMSp d MS MS of 430 3896905 1 at 0 170966666666667 mins 34 5HGKMSMSp d MS MS of 430 3896905 1 at 0 174366666666667 mins 35 5h6kMSMSp d MS MS of 371 3161711 1 at 0 186133333333333 mins 36 5hGKMSMSp d MS MS of 371 3161711 1 at 0 18885 mins 37 5HGKMSMSp d MS MS of 279 1604900 0 at 0 193433333333333 mins 38 5h6kMSMSp d MS MS of 279 1604900 0 at 0 198033333333333 mins 39 5h6kKMSMSp d MS MS of 436 3415480 1 at 0 2032 mins gt hl Sheet1 Ready 2 r 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ecoocosoocoooocooocoooooo 0 0 0 0 0 0 0 4 0 0 0 ooonoocoooocooocooo eans not found 1 m SSeS gt Seer iii olololololololalolololaloEBE l l lel2 l lol lol l l la islalalslalalelsialelelelaleetealsleinielelalasiela elainele coonooocooooocooo ooonooocooooooo X The titles of spectra are presented in the row he2. 9527430249094 9 05 43 2779406334487 8 160714 277 21431070736 1499 587 740 518886551 048 923 6494 Again the above format only contains the necessary part of the data and some optional parameters are allowed If your MSP file is different from the above format but is still correct please contact us with your format We will update the software to read custom files 8 Run in batch In order to run the program in batch mode you can use the command line version of the program which can only be started in command line window or batch files From command window 5 Page 1 Open the command window cmd in windows and change the current directory to the directory of MS2Analyzer_commandline jar e g cd D Projects MS2Analyzer Commandline Or go to the folder containing the jar file and open command window here shift right click on the folder name 2 Start the java program with the following arguments java jar MS2Analyzer_commandline jar argumenti argument2 argument3 argument4 argument5 argumenti the full pathway and name of MSP MGF file e g D Projects Data lipids mgf argument2 the full pathway and name of the text query file e g D Projects Data query txt argument3 the full pathway and name of the output excel 2003 file e g D Projects Data lipids xls argument4 m z window e g 0 005 argument5 intensity threshold e g 0 05 Using batch file Open a text editor such as Notepad and put in the following te
3. 16 4 DGTS 1 M H 2 M H 3 M H 4 M H 210 1983 208 1827 206 1670 204 1514 202 1357 256 2402 254 2245 252 2089 250 1932 248 1771 eg 1497 682 5621 680 5464 678 5308 Sh6kMSMSp d MS MS of 237 0834021 0 at 0 04565 mins 2 0 0 0 i 0 Ay 5h6kMSMSp d MS MS of 237 0834021 0 at 0 0477833333333333 mins 0 0 0 5h6kKMSMSp d MS MS of 391 2845829 1 at 0 0537166666666667 mins 0 5h6kMSMSp d MS MS of 450 3579445 1 at 0 0567 mins Neutra Loss 0 Precu sor lon s 5h6kMSMSp d MS MS of 450 3579445 1 at 0 0596833333333333 mins 0 0 0 rodu ct ion Sh6kMSMSp d MS MS of 430 3896905 1 at 0 0633666666666667 mins 10 Sh6kMSMSp d MS MS of 430 3896905 1 at 0 0670333333333333 mins 11 Sh6kMSMSp d MS MS of 237 0834021 1 at 0 07835 mins 12 5hGkMSMSp d MS MS of 237 0834021 1 at 0 0806166666666667 mins 13 Sh6kMSMSp d MS MS of 391 2845829 1 at 0 0833666666666667 mins 14 Sh6kMSMSp d MS MS of 391 2845829 1 at 0 0861166666666667 mins 15 SEM SM Sp d sms a 450 3579445 1 at 0 0890166666666667 mins at0 0919333333333333 mins oo Ei N T 5 oO O i DO ONSC SOOO OOO COCO daib oo 19 SHGRMSMSp d MS MS of 371 3161711 0 at 0 110283333333333 mins 20 5h6kMSMSp d MS MS of 371 3161711 0 at 0 113266666666667 mins 21 Sh6KMSMSp d MS MS of 279 1604900 1 at 0 118 mins 22 5h6kMSMSp d MS MS of 279 1604900 1 at 0 122716666666667 mins 23 5h6kMSMSp d MS MS of 436 3415480 1 at 0 127433333333333 mins 24 Sh6KMSMSp d MS MS of 436 3415480 1 at 0 13215 mins
4. 1827 206 1670 204 1514 202 1357 256 2402 254 2245 252 2089 250 1932 248 1776 236 1497 682 5621 680 5464 678 5308 y 588 096 604 112 62 y 172 i68 gt 188 1696 204 902 P 19 l5 676 514 gt 1 446 3504333 0 0 0 0 0 0 0 0 P m z J q 296 4854 10075450 81 0 0 0 0 0 x 236 150142025144 1 0 d 1 0 0 0 0 0 0 1 o o 7 d A a o 0 T 744 594559214211 0 91 0 i 0 0 0 d 1 2 0 0 0 455 3579131065 0 07 0 0 0 0 0 d 7 Error messages Error messages will pop up when something is wrong in the searching process Below are some error messages and their meanings 1 When MSP MGF file are not found The system cannot find the MGF MSP file please make sure you select the correct file 2 When there s no keyword such as NEUTRAL LOSS in your query file The tool cannot find any query keyword in your query file Please note that key words are case sensitive 3 When there are lines without any keyword or REM in your query file Warning your query file contains contents that are not query or comments MS2Analyzer will keep processing but we recommend you to check your query file format 4 When the query format is wrong e g there are spaces in names or numbers Query format is incorrect Please make sure the there s no extra space in query line and mass is numeric 5 When the input intensity threshold is less than 0 or larger than 1 Please make sure intensi
5. MS2Analyzer User Manual Version 1 0 November 22 2013 MS2Analyzer Ver2 1 Help Input MGF MSP file D input Data MSP Browse Input query file txt D inputiquery txt Browse m z window intensity threshold 0 1 0 0 001 0 02 Output file xIs D outputiResults xls co 1 Introduction MS2Analyzer is a program developed for small molecule substructure elucidation from accurate mass MS MS spectra It was developed in Java with a graphical user interface GUI using the Open Source IDE Eclipse The main function of the software is to search mass spectral features such as neutral losses product ions m z differences and precursor ions in a large number of MS MS spectra Combining the searching results and feature substructure compound class relationships knowledge substructure or compound class can be identified If possible precursor mass information is also considered even specific compound can be annotated Examples were shown in lipid identification from green algae 2 Start the program MS2Analyzer works with all operating systems with Java Runtime Environment JRE 7 After downloading the zip file extract it first and then follow the following guidelines to start the program in different operating systems Window double click on the jar file Linux 1 Open the Terminal 2 Change the current pathway to the directory of MS2Analyzer jar file for exa
6. adings while the names followed by the masses of the spectral features are presented in the column headings Searching results are reported in integer numbers starting from 0 which represent the times that they are found For neutral loss product ion and 3 Page precursor ions the number can only be 0 and 1 However for m z difference sometimes it can be higher than 1 since the same m z difference can be found multiple times in one tandem mass spectrum Different colors are used in the names to distinguish the type of spectral features Black neutral loss Green m z difference Blue product ion Red precursor ion To help users validate the searching result manually when neutral loss m z difference or product ion are found in a spectrum a comment with detailed information is usually written to the cell neutral loss precursor m z its relative intensity often 0 0 product ion m z its relative intensity m z difference larger product ion m z its relative intensity smaller product ion m z its relative intensity product ion product ion m z its relative intensity When more than 3 m z differences are found in one mass spectrum no comment is added or else the text in the comments will be too long and file size will be too large Example B c D E F G H l J K L M N o P o DGTS30 DGTS30 DGTS30 DGTS30 14 0 H20 14 1 H20 14 2 H20 14 3 H20 14 4 H20 16 0 16 1 16 2 16 3 16 4 DGTS 1 M H 2 M H 3 M H 4 M H 210 1983 208
7. important to them 4 How long does it take to use MS2Analyzer to analyze my data The time it takes to finish processing data depends on the speed of CPU the data file size the number of queries and the parameters you are using For example with a 8 core i7 CPU 3 60 GHz it took about 1 min to search 165 neutral losses in 19329 MS MS spectra in a 26 4 MB file The m z window was 0 01 and intensity threshold was 0 02 6 Page 9 Contact us To report bugs ask questions and offer suggestions please contact us by email yanma ucdavis edu Thanks for using MS2Analyzer 7 Page
8. mple type in cd home vm Downloads MS2Analyzer 3 Start the program by typing in java jar MS2Analyzer ver2 1 jar Mac OS similar to Linux 1 Page 3 Input Files Tandem mass spectra stored in NIST Mass Search format MSP or Mascot generic format MGF can be processed MGF can be exported from raw data file using software provided by instrument manufacturer such as Agilent MassHunter software or freely available software such as _ ProteoWizard http proteowizard sourceforge net Due to the limitation of the output Excel 2003 files the maximum number of input spectra is 65534 Data files with a larger number of spectra have to be split 4 Query Files Query files are text files that contain the information of mass spectra features that users want to search It can be easily written by users as long as the following format is followed Definition Name Mass Definition NEUTRAL LOSS the m z difference between precursor ion and product ions MZ DIFFERENCE the m z difference between the product ions in a spectrum PRECURSOR ION the m z of the precursor ion PRODUCT ION the m z of the product ion Name Name of the feature Mass Mass of the feature Both integer and decimal numbers are allowed but comma is not allowed in the mass number Please note 1 Each query starts with a definition keyword and takes a single line Keyword is case sensitive 2 The separator between Definition Name and Mass can be s
9. pace single or multiple tab or a combination of space and tab 3 No space or tab is allowed inside Definition Name and Mass 4 Optional separate intensity threshold of each product ion by adding a number 0 0 1 0 after mass This will overwrite the universal threshold in GUI 5 Optional Any line that starts with REM is considered as a comment 6 If any line is written in query file without or keyword MS2Analyzer will give a warning message but it can still continue working if you click OK and ignore that line Example MZ DIFFERENCE HCOOH 46 005 NEUTRAL LOSS NH3 17 027 PRODUCT ION DGTS 236 150 0 5 PRECURSOR ION PG18 1 16 1 745 502 The maximum number of queries in one query file is 255 due to the limitation of the Excel 2003 output file 5 m z window and intensity threshold 2 Page m z window This parameter is designed for mass spectra measured with different mass accuracies The mass accuracy depends on the instrument type calibration etc Once it is set by users the mass spectral features can only be reported when the differences between the experimental values and the query values are within this window For example if the m z window is 0 005 and we want to look for product ion of 236 150 only product ion m z between 236 145 and 236 155 can be found Please note that in this example 236 145 and 236 155 cannot be found Intensity threshold This parameter can be used to reduce the effect of the noisy peaks in
10. the spectra The intensity threshold is actually a relative intensity based on the base peak i e the most intense peak in each mass spectrum Therefore it can only be a value between 0 and 1 0 and 1 included Only peaks with a higher intensity than the threshold can are processed others are ignored in the first step of searching and won t be reported even if they are close enough to the query values If it is set to be O all the peaks are processed and searched if it is set to be 1 all the peaks are excluded 6 Output Files Due to the Java Excel API we were using currently the output result can only be exported as an Excel 2003 xls file Higher Excel versions can still open it in compatible mode and users can save it as other format if they would like to Here s an example of how an output file look like Insert Page layout Formulas Data Review View Add Ins JChem Community Clips amp cut Ja 7 OF oa 4 1 E Autosum Ap a osr sumo E co hee Ssh i A Paste S aw Ae ESE HE HMegea Center 8 23 Conditional Format Cell Insert Delete Sort amp Find amp Format Painter O B 2 F Bid Mero 2 SA Formatting as Tabie v Sykes 7 _ e Fiter Selec Clipboard Font a Alignment A Number A Styles Cells Editing A3 X fe 5h6kMSMSp d MS MS of 237 0834021 0 at 0 04565 mins 5 Cc D E F G H I J K E M N O DGTS30 DGTS30 DGTS30 DGTS30 14 0 H20 14 1 H20 14 2 H20 14 3 H20 14 4 H20 16 0 16 1 16 2 16 3
11. ty threshold are within 0 0 1 0 6 When the output file pathway is not valid or the file is open in Excel 4 Page Exception occurs in writing files Please make sure your output file pathway is correct and the file is not used by another process 7 When the MGF file format cannot recognized Unrecognized MGF file format Please check the file format How to solve it Make sure the file format is correct Try to open it with other software such as NIST search or open it with NotePad or TextPad MS2Analyzer can only read MGF file in the following core format BEGIN IONS PEPMASS 740 5181274 CHARGE 1 TITLE MS MS of 740 5181274 1 at 11 1204666666667 mins 28 9527430249094 9 05 43 2779406334487 8 160714 277 21431070736 1499 587 740 518886551048 923 6494 END IONS Other optional parameters are also allowed in the beginning of the data file If your MGF file is different from the above format but is still correct please contact us with your format We will update the software to read customer files 8 When the MSP file format cannot recognized Incorrect MSP file format please make sure the file format is correct and there s no extra blank line in the file How to solve it Similar to the error of MGF file format first make sure the format is correct MS2Analyzer can only read MSP file in the following format Name MS MS of 740 5181274 1 at 11 1204666666667 mins PrecursorMZ 740 51 81274 Num peaks 4 28
12. xt java jar MS2Analyzer_commandline jar argumenti argument2 argument3 argument4 argument5 Save it as bat file in the same folder of the jar file and double click to run it If the file is not saved in the same folder the full pathway of the jar file also needs to be included 9 FAQ 1 What kind of analysis can MS2Analyzer perform Any kind of small molecule substructure identification from tandem mass spectra can be performed as long as the relationships between mass spectral features and substructures compound classes are well understood Some manual work has to be done after search by MS2Analyzer The examples of lipid identification are shown in another tutorial 2 Does MS2Analyzer only work for accurate mass MS MS spectra No Although it is designed for accurate mass MS MS spectra it can also work with low mass accuracy spectra by setting a high m z window However it is noteworthy that with a higher mass accuracy the searching results are more specific and fewer candidates will be found 3 What m z window and intensity threshold should use They are experiment dependent and have to be decided by users For example mass accuracy is not only related to the type of instrument but also how well it is calibrated Mass accuracy can be determined by measuring reference ions during the run Intensity threshold on the other hand it is more of an arbitrary decision Users can look at the data and decide which level of peaks is not
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