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Agilent Adding Retention Times to the METLIN Personal Metabolite Database to Improve Compound Identification Manual

1. C6H1004 c4H605 3 hydroxy 3 methyl Glutaric acid isoquinoline N oxide 5 6 dimethyl 1H benzoimidazole Glu Val Met Valine Carbaryl Adipic acid malic acid Mass M Delta Mass RT RT Submitted ot ppm Submitted min 12 050 11 750 12380 14 980 1 440 11 750 13 920 12910 5 900 1 230 162 05282 145 05276 146 08440 377 16206 117 07898 191 05824 266 09027 162 05253 145 05253 146 08417 377 16160 117 07884 191 05802 266 09002 201 07880 201 07898 146 05778 146 05791 134 02142 134 02152 METLIN Figure 7 Review the metabolite standard information and confirm that there are no conflicting hits Conclusions Adding retention times to metabolite databases provides a second match criterion orthogonal to and independent of compound mass Searching both accurate mass and retention time will often provide a narrower more focused list of possible metabolite identities especially for targeted metabolite Adding Retention Times to the METLIN Personal Metabolite Database searches Unlike public metabolite data bases retention times can be added to the Agilent METLIN Personal metabolite database The process of adding reten tion times to the METLIN database can be significantly simplified and accelerat ed by preprocessing data from metabo lite standards with the Agilent MassHunter Mass Profiler software 42 Agilent METLIN P
2. updating locate the compounds and save the 2 Open the data file from the standards 4 Save the method so that it can be resulting data file analysis and select Method gt Edit applied to the same type of standard Processing the standards data with the Method on the menu bar mix in the future MassHunter Mass Profiler software 3 In the Pre Analysis Filters tab 5 Export the feature table as a comma The data acquired by analyzing the choose the Special Masses option separated value file csv metabolite standards are processed in Limit to These Figure 3 Enter or the Mass Profiler software to generate a paste the exact neutral masses of Feature Summary table of the standards the metabolite standards and click and retention times This table is OK Entering the exact neutral mass uploaded to the METLIN Personal es here allows the Mass Profiler soft metabolite database and is used to add ware to filter out all non standards or update retention times for the for example chemical noise or impu metabolite entries rities that may have been detected New Project Method Parameters SynUrine_RI_ Standard PreAnalysis Filters Alignment amp Normalization Result Fiters Specity Project Name Specify the number of groups in the project Urine Standard 1 O2 Feature position s 7 Use al the avaiable data Group name Experiment Min Max prde TS Limit to these Tolerance 0 0050 RT jo a0 mn 590371 740368 750320 75 0684 760160
3. 880160 Samples Ad ee ee 9 0477 920473 1030633 104 1075 105 0426 Mee IE fa Da 1120273 1120569 1150633 1160110 1180266 Scalar File Name 0 0 ae 1180266 118 0868 1190582 1190582 1230320 126 0423 130 0266 130 0630 131 0562 131 0695 Isotope pattern Fomula Custom a 000 Da 0 00000 xmass Deviation alowed 050 Da Features of unknown mass Included Excluded Limited to these Charge state Any Mult charge requred Mult charge forbidden Abundance pet file Neutral losses Min relative abundance Min absolute abundance O Top laigest 45000 0 Count Figure 2 To begin processing the standards data in the Mass Figure 3 Filter out non standards data from the analysis by specifying the exact neutral Profiler software create a new project that includes the masses of the metabolite standards standards data mhd www agilent com chem metlin 3 Technical Overview Creating an accurate mass retention 1 In the METLIN software select 3 In the Batch Search tab Figure 5 time AMRT METLIN database using File gt New Database The original choose Optional for Retention times an imported mass RT list METLIN Personal metabolite data 4 Click File and in the dialog box that After processing the metabolite stan base is write protected so a new appears select the Summary Feature dards data in the MassHunter Mass database must be created before table csv for the metabolite stan Profile
4. 2 Required C Include cations l 131 05759 1 03 l Notes Geigy vol 3 p 108 RT tolerance 394 14658 15 56 103 0635 3 87 jia a Delta RT METLIN 2 Aceto 2 hydroxybutyric acid C6H1004 146 05791 Mevaldic acid C6H1004 14605791 541 071 alpha Ketopantoic acid C6H1004 146 05791 88 470 30 4 Isosorbide C6H1004 146 05791 88 652 675 adipate C6H1004 l 14605791 88 76465 8 3 Methylglutaric acid C6H1004 14605791 626 51 7 2 Methylglutatic acid C6H1004 14605791 Adipic acid C6H1004 C 14605791 Monomethyl glutaric acid C6H1004 O 14605791 2 2 Dimethylsuccinic acid C6H1004 O 14605791 0000000000 Figure 6 Confirm that each standard mass in the batch search list is correctly identified in the batch search results If necessary select the correct identity for a standard in the batch search results www agilent com chem metlin 5 Technical Overview 8 The Apply Retention Time tab will become active once the number of conflicting hits is set to zero Once active click Apply Retention Times in the Batch Summary tab The data base Is updated to include the retention times Figure 8 and is automatically saved Searching a custom METLIN personal database using accurate masses and retention times After an AMRT METLIN database is cre ated it is easy to incorporate retention times into subsequent searches When specifying the search parameters in the Batch Searc
5. Technical Overview p m ee ee Adding Retention Times to the METLIN Pi Personal Metabolite Database to amp 3 me l Improve Compound Identification Authors eet A key step in metabolomics research is the identification of metabolites Steven Fischer and Theodore Sana Searching accurate mass LC MS data against metabolite databases is a AUER ETRE DORES powerful technique for identifying metabolites Due to the presence of isobaric masses such as compound isomers and diastereomers database searching by mass alone is generally insufficient for positive identification Public databases do not contain retention time information and thus there is no way to resolve the issue of isobaric masses However with Agilent Technologies METLIN Personal metabolite database empirically derived method specific retention times RTs can be added to compounds in the database creating an accurate mass retention time AMRT database Retention times then provide a second match criterion orthogonal to and independent of compound mass Searching an AMRT database provides a narrower more focused list of possible metabolite identities This technical note discusses the addition of retention times to the METLIN Personal metabolite database and provides a faster batch approach for creating an AMRT database Our measure Is your SUCCESS products applications software services 3 Agilent Technologies Technical Overview Add
6. chromatographic method that produces good results for both positive and negative ion polarities and for both electrospray and APCI should be used This makes the result ing AMRT database useful for a wider range of compounds Agilent recom mends a water methanol mobile phase with approximately 5 mM ammonium acetate and 0 1 acetic acid Chromatographic methods optimized for a particular ionization polarity ionization technique or analyte can be used but the resulting retention times will only be meaningful for compounds analyzed with that specialized method RT Library csv Custom METLIN Personal Database Standard n Standard 2 Standard 1 S Import the RT library that includes RTs to a custom METLIN Personal Database Figure 1 Workflow for batch addition or update of retention times in the METLIN Personal metabolite database Adding Retention Times to the METLIN Personal Metabolite Database to Improve Compound Identification Once the metabolite standard sample s 1 Create a new project in the Mass in the metabolite standards sample have been analyzed process the data Profiler software Specify one group This significantly simplifies subse using the MassHunter Qualitative in the project and add the data file s quent METLIN database searching Analysis software Use the Molecular mhd created from the metabolite and retention time assignment or Feature Extraction MFE algorithm to standards see Figure 2
7. ersonal Metabolite Database C MassHunter databases Wetlin Urine mt Single Search Batch Search Batch Summary Report comments Edit Metabolites Molecule ened Mass list search parameters Mass list file C MassHunter Metlin Mass List Files SynUrine_Pos cs Masses 10 ppm Neutral Search Neutrals Retention time parameters Best mass match results RT s 0 1 min Optional Total hits 73 213 34 3 Conflicting hits 0 73 0 inhibitor Apply Retention Times Single matches 28 213 13 1 to Improve Compound Identification Notes Analgesic Anti Inflammatory Antipyretic platelet agaregation Batch Summary Results 66 hits 73 total hits 28 single matches 213 submitted E Name Formula gt 3 hydroxy 3 methyl Glutaric acid C6H1005 Mass M Delta Mass Submitted SSS ppm 162 05253 162 05282 Isoquinoline N oxide C9H7NO 15 05253 145 05276 Active metabolite of Acetylsalicylic acid aspirin 5 6 dimethyl 1 H benzoimidazole CSH10N2 Glu Val Met C1I5H27N3065 14 46 146 08440 Valine C5H11NO2 pera 60 377 16206 117 07884 117 07898 Desmethyldeschlorobenzoyl Indomethacin 5 3 4 Dihydroxy 15 cyclohexadien 1 yl 5 ethylbarb C12H14N205 191 a 191 05824 C12H11N02 266 09002 266 09027 201 07880 201 07898 146 05778 146 05791 Figure 8 The AMRT METLIN database is updated to incl
8. h tab simply select Optional or Required under Retention times im Agilent METLIN Personal Metabolite Database C MassHunter databasesWetlin Urine mtl a ORO Single Search Batch Search Batch Summary Report comments Edit Metabolites Molecule Mass list search parameters Mass list file C MassHunter Metlin Mass List Files SynUrine_Pos csy Masses 10 ppm Neutral Search Best mass match results Total hits Retention time parameters RT s 0 1 min Optional Conflicting hits Apply Retention Times Single matches Neutrals 13 34 3 0 73 0 28 213 13 1 Choosing Optional ranks mass matches higher that also match RT Required returns only mass matches that also have RT matches As a general rule choose Optional for general non targeted searches because not all compounds in the database will have retention times For targeted searches Required is useful because it eliminates compounds that are not of interest from the results Structure MOL Text OH inhibitor Active metabolite of Acetylsalicylic acid aspirin Notes Analgesic Anti Inflammatory Antipyretic platelet aggregation a v Batch Summary Results 66 hits 73 total hits 28 single matches 213 submitted C6H1005 C9H7NO C9H10N2 C15H27N3065 C5H11N02 Desmethyldeschlorobenzoyl Indomethacin C10H9N03 5 3 4 Dihydroxy 1 5 cyclohexadien 1 yl 5 ethylbarb C12H14N205 C12H11N02
9. ing retention times to create an AMRT database Retention times can be added to the METLIN Personal metabolite database in a manual process of running metabo lite standards selecting the proper data base entry for each standard and updating the retention time in the data base This approach is effective but can be slow A faster easier approach uses the Agilent MassHunter Mass Profiler software to preprocess the data file from the analysis of metabolite standards before database searching This approach filters out non standards and simplifies the assignment of standard matches and addition of retention times Compound gt Finding MassHunter MFE Standards Data File d in the METLIN software The workflow is outlined in Figure 1 Analyzing the metabolite standards The first step in the workflow is to acquire accurate mass data from metabolite standards As many stan dards as is convenient may be analyzed in a single sample however each stan dard must have a unique mass range around it that is free of interfering masses If two or more metabolite standards have masses within 5 mDa or 50 ppm whichever is larger of each other they should be analyzed in separate samples For the retention times to be useful the metabolite standards and experiment samples must be analyzed using the MHD File gt Mass Profiler Processing exact same chromatographic method Ideally a universal
10. r data use this procedure to cre retention times can be added dards that was exported from the ate a copy of the METLIN Personal data 9 Select an existing database from Mass Profiler software pase am age Ter Manes a which to begin Figure 4 Enter a 5 Click Find Metabolites on the toolbar metabolite standards to the database new name and a description and click Create 1E Apilent METLIN Personal Metabolite Database C Program Files Agilent MassHiunter databasesWetlin mt 1 Create a New MEILIN Database Jur oS e Oo Singe Seach BalchSesch BatchSummaryy Ed Metaboltes 1 Select an existing database Metlir Metlin_Empty Masses Structure MOL Text O MH Neutra MHF Gow fare assiRT lst From a file Mass tolerance 10 pm Onda Retenbon times Radical ion search mode Ignore F Include neutrals Optional C Include anions Required C Include cations RT tolerance 2 Enter the new database name MetinUrne 3 Enter a description of this database A database including retention times for common metabolites Batch Search Results No hits for Mass RT in urine Figure 4 Create a new METLIN Personal metabolite database from an existing METLIN database Figure 5 Upload the standards Feature Summary table csv to the new METLIN database Adding Retention Times to the METLIN Personal Metabolite Database to Imp
11. rove Compound Identification 6 One standard ata time highlight the Note If a metabolite standard is not 7 Once all of the metabolite standards standard mass in the Batch Search present in the database add the stan are assigned identities from the data list and select the correct metabolite dard in the Edit Metabolites tab In the base review the results in the Batch identity in the Batch Search Results Edit Metabolites tab click the Add New Summary tab Figure 7 When all of Figure 6 In almost all cases there button enter the appropriate metabolite the assignments are correct you are will be more than one match due to information and click Save As New ready to update the database isobaric compounds in the database Return to the Batch Search tab and but since the identity of each ana select the newly added metabolite as lyzed standard is already known it the identity of the standard should be easy to select the correct metabolite from the search results 12 Agilent METLIN Personal Metabolite Database C MassHunter databasesWetlin Urine mt ene TR ToTu a _ Batch Search Batch Summary Edit Metabolites Hie Masses Molecule Structure MOL Text 377 15915 13 84 25 O M H Neutral MH 377 1616 14 98 24 Mass tolerance 10 ppm O ma 333 11717 1 18 Retention times Radical ion search made 114 0683 10 37 Ignore Include neutrals 114 06831 11 51 Optional C Include anions 164 04742 16 7
12. ude RT information from the at Feature Summary table www agilent com chem metlin Technical Overview About Agilent Technologies Agilent Technologies is a leading supplier of life science research systems that enable scientists to understand complex biological processes determine disease mech anisms and speed drug discovery Engineered for sensitivity repro ducibility and workflow productivity Agilent s life science solutions include instrumentation microflu idics software microarrays con sumables and services for genomics proteomics and metabolomics applications Buy online www agilent com chem store Find an Agilent customer center in your country www agilent com chem contactus U S and Canada 1 800 227 9770 agilent_inquiries agilent com Asia Pacific adinquiry_aplsca agilent com Europe info_agilent agilent com This item is intended for Research Use Only Not for use in diagnostic procedures Information descriptions and specifications in this publication are subject to change without notice Agilent Technologies shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing per formance or use of this material Agilent Technologies Inc 2008 Printed in the U S A May 23 2008 5989 8546EN Our measure is your success products applications software services dg Agilent Technologies

Agilent Adding Retention Times to the METLIN Personal Metabolite Database to Improve Compound Identification Manual

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