User Manual - biosciences-labs.bham.ac.uk
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1. cccccccccseccsecceeeeeeeceneceuecseeceueceueceeecsuessuesseesseessueseuesseeseeeseeesaees 43 3 4 1 In silico Library amp Input Real Spectral cccceccsccceeeseeceeeeeeeeeeeeseeeeeeeeeesseneeeseeteeeaeees 43 342 CONSO MOda E eames 43 343 Interactive MOa G rnani E E ieee R RR 44 A ARESU aoe E A stn 44 Chapter 4 Update History and Troubleshooting cccceecceeceseeeeeteeeeeeeeeeeeeeeeenseeaeeeeeees 46 ANI OG Ale IMNSIONY eaesiestst casera dic n A Ta a 46 AZ 1 TOUDISSIOOUIN O sotisa a E Uamaleutie haus ietudte a a 46 HAMMER Automated Operation of Mass Frontier to Construct In silico Mass Spectral Fragmentation Libraries Chapter 1 Overview Experimental MS mass spectral libraries currently do not adequately cover chemical space This limits the robust annotation of metabolites in metabolomics studies of complex biological samples n silico fragmentation libraries would improve the identification of compounds from experimental multi stage fragmentation data when experimental reference data is unavailable Here we present HAMMER a freely available software package to automatically control Mass Frontier software to construct in silico fragmentation libraries Chapter 2 Installation 2 1 Dependent Software 1 System requirements HAMMER supports Windows 7 Windows Vista and Windows XP both 32 and 64 bit versions Windows 8 is not fully supported The software is developed and tested on 32 bit Windows 72. 21 HAMMER Automated Operation of Mass Frontier to Construct In silico Mass Spectral Fragmentation Libraries This message appears when no fragment can be generated from the original structure This pattern is searched within region mf_operate_reg File no frag mol is provided to produce this situation Hh Structure Editor 1 aK Generation of Fragments amp Mechanisms Status No Fragments ess Already Generated m m z 58 028740 Info Mass Frontier 7 0 is continue using othe generated Pause Finish Cancel 31 mf_fragres_nofrag_ok Click to close the No Fragments message box This pattern is searched within region mf_operate_reg E Structure Editor 1 Status No Fragments Progress o Mass Frontier 7 0 is 4 continue using othe generated Pause Finish Cancel 32 mf_close_cross Click to close all sub windows in the Mass Frontier This pattern is searched within region mf_operate_reg repeatedly until no such image pattern can be found File Edit View Tools Search Library Options Help X Microsoft Office e Ha o X BOE R TOSHPOQRD kell P oo i cae oh A Q Precursors List X Structure Editor 1 R H l oor s Bex e HCAl Sv AK Fragments amp Mechanisms 1 alam xv d Fragments Mechanisms Q gt 7 a A a a oO Qa 22 2 5 4 Regions mf_toolbar_reg 1 115 Regio
3. JE ae Se PicPick Capture Region A Microsoft Office Mm N A WW W Structure Editor 1 Click on the left mouse button to select area Fe or rape BO E rie ae View C o qx S ka p tom lay 7 gt Eee RERET THERE 4 gt Ba com H reame AX BA 2 TNN a m Soe monn Paste Koan Select neues Effects A 4 KA Z oO ae 100 N More a ard Image T Image 4 ot 100 el a 200 1400 PEPEE ir 4o0 parr a aoo 200 i Gi o a NH at 4 P J o OH pa oO 300 woo 606 218 1O 242 x 226 100 Q 4 Move the image file into the Resources folder and then change the content in res index to use it in the InSilicoFragmentation 21 HAMMER Automated Operation of Mass Frontier to Construct In silico Mass Spectral Fragmentation Libraries Chapter 3 Using HAMMER 3 1 Using the RetrieveStructures 3 1 1 Target File The RetrieveStructures utilizes a plain text file namely the target file to define compounds that need to be downloaded The first line of the target file is the header which will be ignored during the download TypeSearch Database SearchTerm Other lines are used to define the target compounds Database s Comments CHEMSPIDER PUBCHEM Tri Sprintec not found in ChemSpider but found in PubChem Search Type Search Term e Search Type the type of search term RetrieveStructures can search a compound according to the databa
4. Microsoft Office Structure E v Multiple Structures Mass Spectrum GC LC MS Fragments and Mechanisms PCA Projections Neural Networks 3 mf_openfile_struct Structure aes Click to open a structure file mol This pattern is searched within region mf_openfile_reg HighChem Mass Frontier 7 0 Remaining Time 44 Days File Edit View Tools Search Library Options Help Microsoft Office Xi m O B TOLHDLAD Ste amp 4 mf_openfile_dialog Check whether the open file dialog is launched This pattern is searched within region mf_operate_reg The target offset marked with should be placed inside the File name input box Organize v New folder x Searchers Date modified W Desktop perform 2 26 2013 10 45PM File folde Downloads 1 2 didocosahexaenoyl glycerol 3 phosp 1 31 20138 05PM MOL File Dropbox 2 1 2 dioleoylphosphocholine_PCID_10350 1 31 20138 14PM_ MOL File S Recent Places 2 1 2 dioleoylphosphoethanolamine_CSID_ 1 30 20139 26AM MOL File 2 3 4 dihydroxyphenylalanine_CSID_813 mol 1 30 20139 26AM MOL File Libraries 2 3 indolepropionic acid_CSID_3613 mol 1 30 2013 9 26AM MOLFile Documents 4 hydroxyphenylpyruvic acid CSID_954 1 30 2013 9 26 AM a Music ia 5 aminovaleric acid_CSID_135 mol 1 30 2013 9 26AM MOLFile E Pictures 2 alpha hydroxyisovaleric acid_CSID_90190 1 30 20139 26 AM MOL Fil
5. ia retention ms precursor z 1 1 210 585 2 discrete 2 585 2099 58 583 4 discrete 210 1111 58 210 11 discrete 60 30 0 50 100 150 200 2 sada e dh a T a t EE E UT FTF i i T5022 81 TZE 3 HAMMER Automated Operation of Mass Frontier to Construct In silico Mass Spectral Fragmentation Libraries The CML file can be exported for each target compound containing the spectral fragment formulas and fragmentation mechanisms These files are compatible with common chemical software e g MetiTree This XML file does not appear to have any style information associated with it The document tree is shown below lt cml xmlns xsi http www w3 org 2001 XMLSchema instance gt V lt spectrumList xmlns http www xml cml org schema gt v lt metadataList gt lt metadata dictRef nmc countScans content 11 gt lt metadata dictRef nmc parentFile content example out_org CSID_2424 struct0 CSID 2424 struct0 mzxml convention mzXMLData gt lt metadata dictRef nmc elements content C8 8 H10 10 N4 4 02 2 H1 1 gt lt metadata dictRef nmc parentCompounds content InChI 1S C8H10N402 c1 10 4 9 6 5 10 7 13 12 3 8 14 11 6 2 h4H 1 383 gt lt metadataList gt lt spectrum type MS id 1 gt lt conditionList gt lt scalar dictRef nmc window dataType xsd string gt 1 lt scalar gt lt scalar dictRef nmc polarity dataType xsd string gt positive lt scalar gt lt conditionList gt v lt meta
6. 2424 struct0_layerO_fraglS mol C6H8N30 138 066188 on CSID_2424_struct0_layer0_frag10 mol 4 ya CSH7N20 111 055289 CSID_2424 struct0_layerD_frag6 mo l C4H5N2 81 044725 gt Ba CSID_ 2424 struct0_layerD_frag2 mol C9H14N402 210 11112 precmol C2H4NO 58 028740 CSID_2424_struct0_layer0_fragl mol HO 42 3 4 Using the SpectralMatching 3 4 1 In silico Library amp Input Real Spectral The SpectralMatching utilizes MSP files to constitute in silico libraries MSP files contain spectral fingerprints and formulas for both precursor and in silico fragments They can also be utilized in other spectral matching software e g LipidBlast MSSearch and NIST MSPepSearch In SpectralMatching each MSP file is exported from a unique target compound and the in silico library is a folder containing all the MSP files Users can add or remove compounds easily by moving the corresponding MSP file inside or outside the library folder The SpectralMatching accepts real spectra in MSP or plain text peak list using extension peaks format The target real spectra files should be put into the same folder in order to conduct batch matching If a real spectra does not contain m z values for the precursor first peak in MSP files and last peak in plain text files as default SpectralMatching will ask the users to assign one Please provide precursor exact mass for Caffeine HCD 80_pos peaks gt gt MatchSp
7. 4 mf_copymenu_reg HighChem Mass Frontier 7 0 Remaining Time 44 Days File Edit View Tools Search Library Options Help HA Mechanism Fp Fragment This is the region to search patterns mf_copymenu_ edit mf_copymenu_copy and mf_copymenu_listoffrags 5 mf_tablemenu_reg HighChem Mass Frontier 7 0 Remaining Time 44 Days File Edit View Tools Search Library Options Help 36 Microsc eh Structure Editor Database Manager E Chromatogram Processor Qm Spectra Classifier Fragments Comparator E Isotope Pattern I Periodic Table Formula Generator K Fragments and Mechanisms S8 Batch Fragment Generation Report Creator Structure Table fete 2 Select possible fragments with m z 52 0182 This is the region to search patterns mf_tablemenu_tools and mf_tablemenu_ structs 2 5 5 Determine Patterns and Regions Using Sikuli IDE Sikuli IDE Sikuli IDE exe provides built in methods to capture pattern images and determine search regions To capture new image patterns users should 1 Launch the Sikuli IDE and open a new script file 2 Click the Take screenshot icon and the software will enter the screen capture mode 24 exists s find findAll 8 wait s waitVanish s ae 3 Drag a rectangle to include the target image The center of the red cross indicates the focus offset of the pattern Dah
8. each fragment 2 mzxml generate mzXML file for each target compound 3 cml generate chemical markup language CML file for each target compound 4 msp generate NIST MSP file for each target compound 5 mzlist generate plain text peak list for each target compound 6 plist generate property list structure for each target compound 7 tree generate tree visualization for each target compound 8 help display the software help Here is an example to use OrganiseFragments in console mode gt OrganiseFragments exe image mzxml msp results results_org It functions to search all the mol fragments in results folder organize them according to the precursor generate 2D images generate mzXML and MSP files and store the results in results_org folder 3 3 3 Interactive Mode Besides the console mode OrganiseFragments can also be run in the interactive mode Double click OrganiseFragments exe in windows explorer to enter this mode where the parameters are set in an interactive manner gt OrganiseFragments exe Enter the path of the target folder Target path gt gt Enter the path of the export folder If users want to store the results in the original target folder leave it empty and press enter Export path gt gt 35 HAMMER Automated Operation of Mass Frontier to Construct In silico Mass Spectral Fragmentation Libraries Decide whether the 2D images should be generated Generate 2D images y n
9. enter n gt gt Decide whether the mzXML files should be generated Generate mzXML files y n enter n gt gt Decide whether the chemical markup language CML files should be generated Generate CML files y n enter n gt gt Decide whether the NIST MSP files should be generated Generate MSP files y n enter n gt gt Decide whether the property list structures should be generated Generate plist files y n enter n gt gt Decide whether the tree views should be generated Generate tree views y n enter n gt gt Decide whether the plain text peak list mzList should be generated Generate mzList files y n enter n gt gt Press enter and the OrganiseFragments will start processing 3 3 4 Results The 2D image in Portable Network Graphics format can be exported for each in silico fragment under the same filename as the original mol file and with the extension png _ 0 ro A z OH n OH C6H1005 H_struct0 mol C6H1005 H_struct0 png 36 The mzXML file can be exported for each target compound containing the encoded spectral information These files are compatible with common mass spectrometry software e g mMass Mass and MetiTree This XML file does not appear to have any style information associated with it The document tree is shown below v lt mzXML xmlns http sashimi sourceforge net schema_revision mzXML_3 1 xmlins xsi http www w3 org 2001 XMLSchema instance xs
10. folder from one computer to another For general users the Sikuli runtime is already included as part of the HAMMER package and therefore does not need to be installed separately Advanced users may want to install the full Sikuli package in order to conduct further developments Written in the Python language a Sikuli script usually consists of four major components 1 Patterns GUI images that should be searched and recognized called matched on the screen The patterns are normally status identifiers e g status bars or GUI controllers e g command buttons 2 Regions rectangular areas defined on the screen to search the patterns in order to reduce search time and improve recognition accuracy 3 Logic Codes source codes to detect the pattern status process accordingly and decide the proper actions in response 4 Actions simulated keyboard events e g press Enter and mouse events e g double click a pattern that should be sent to the automated software The Sikuli IDE is a very useful tool to develop Sikuli scripts It displays the patterns and regions intuitively along with the logic source codes The IDE also provides built in tools to capture GUI patterns and select search regions see section 2 5 5 for more information A demo script opened in the Sikuli IDE is illustrated as follows Patterns 2 FightClick ft Actions For more information and examples visit the Sikuli documentation website h
11. id KEGG C07481 id PUBCHEM 2519 id CHEMSPIDER 2424 name KEGG CHEMSPIDER caffeine formula KEGG PUBCHEM C8H10N402 pathway KEGG Phenylalanine metabolism obtain pathway compounds in KEGG 3 1 2 Console Mode In console mode the RetrieveStructures can be used as a single line command gt RetrieveStructures exe options targets export 28 where targets is the path to the target file described in the above section and export is the path to the folder wherein the results are going to be stored Options of the RetrieveStructures are 1 no split do not split multi molecule entries into separate files 2 xls generate an excel report to summarize the search results Otherwise a plain text report will be generated instead 3 outfmt format export data format feasible formats include mol default inchi smiles and formula 4 max number maximum candidates number of a target compound It should be an integer value between 0 and 100 0 means to download all the candidate compounds Default value is 10 5 help display the software help Here is an example to use the RetrieveStructures in console mode gt RetrieveStructures exe xls outfmt inchi targets txt results It functions to search the compounds listed in target file target txt convert them into inchi format export to the results folder and generate an excel report 3 1 3 Interactive Mode Besides the console
12. mf_stable_save_dialog File name Save as type SDF Format sdf a Hide Folders Check whether the Save Structures dialog is opened This pattern is searched within region mf_operate_reg The target offset should be placed inside the File name input box Wl Save Structures Organize v New folder A Favorites Fz Name MD Desktop caffeine_CSID_2424 layer0 sdf ri Downloads caffeine_CSID_2424 layer0_frag2_layerl sdf F Dropbox caffeine_CSID_2424 layer0_frag2_layerl_frag0_layer2 sdf E Recent Places caffeine_CSID_2424 layer0_frag5_layerl sdf caffeine_CSID_2424 layer0_frag5_layerl_fragl_layer2 sdf a Libraries caffeine_CSID_2424 layer0_frag9_layerl sdf C Documents caffeine_CSID_2424 layer0_frag9_layerl_frag2_layer2 sdf ad Music caffeine_CSID_2424_layer0_frag10_layer1 sdf E Pictures caffeine_CSID_2424 layer0_fragl0_layerl_fragl_layer2 sdf z Videos caffeine_CSID_2424_layer0_frag11_layer1 sdf caffeine_CSID_2424 layer0_fragl1_layerl_fragl_layer2 sdf j Computer p D _eaffeine CSM 244 lave front lavori franS lawer edf BP nnnrerace File name Save as type SDF Format sdf a Hide Folders 29 mf_stable_save_procbar Check whether the fragments saving process is finished This pattern is searched within region mf_operate_reg Q Precursors List coh trex DAST A B 230 194 079827 194 079827 30 mf_fragres_nofrag
13. total non isobaric fragments 1654 unique m z values accuracy 10 0 ppm 21 mf_copymenu_edit Click to open the copy menu This pattern is searched within region mf_copymenu_reg 18 HighChem Mass Frontier 7 0 Remaining Time 44 Days ie iew Tools Search Library Options Help a 22 mf_copymenu_copy Click to copy fragments This pattern is searched within region mf_copymenu_reg HighChem Mass Frontier 7 0 Remaining Time 44 Days File Edit View Tools Search Library Options Help X Microsoft Office 23 mf_copymenu_listoffrags Click to copy list of fragments This pattern is searched within region mf_copymenu_reg HighChem Mass Frontier 7 0 Remaining Time 44 Days File Edit View Tools Search Library Options Help E Microsoft Office xs n OL 99 PAR imi ah E IG Fragments amp Mechanisms 1 B3 Mechanism Bp Fragment ce l HCaAlwv RB 24 mf_tablemenu_tools Click to open structure table menu This pattern is searched within region mf_tablemenu_reg 19 HAMMER Automated Operation of Mass Frontier to Construct In silico Mass Spectral Fragmentation Libraries HA HighChem Mass Frontier 7 0 R De File Edit View Tools Search Library Options Help Microsoft Office Farerai O HP PLAR o i w c p p Chromatogram Processor 4 28 Spectra Classifier ce Structure E Fragments Comparator Fragments amp Mechanisms 1 lal i periodic Ta
14. 08765 C8H10N402 H 4 552e 00 4 463 109 03980 109 03964 CSH4N20 H 1 477e 00 2 180 111 05544 111 05529 CSH6N20 H 1 360e 00 0 824 81 04452 81 04472 C4H4N2 H 2 529e 00 0 042 122 07164 122 07127 C6H7N3 H 2 998e 00 report saved to file real Caffeine HCD 80 pos _ match_results txt The number of in silico compounds displayed in the Overall Results section is determined by the max parameter The explained precursor formula explained peaks number and percentage and the total m z distance are also provided In the Best Match section the explained peaks are listed in the order of relative intensity as well as the explained fragment formulas and match distances A report file containing all the matching results will be exported for each target real spectra under the filename SPEATRAL_NAME _match_results txt wherein SPEATRAL_NAME is the filename of the original spectra Contents in the report file are plain text separated with tabs t The report can be opened in Excel or any text editors 45 HAMMER Automated Operation of Mass Frontier to Construct In silico Mass Spectral Fragmentation Libraries Chapter 4 Update History and Troubleshooting 4 1 Update History 4 2 Troubleshooting 46
15. 4 00 GB 2 23 GB usable System type 32 bit Operating System Pen and Touch No Pen or Touch Input is available for this Display Computer name domain and workgroup settings Computer name Albert MacMini Full computer name Albert MacMini Computer description Workgroup WORKGROUP Windows activation 3 Click the Advanced tab and then click Environment variables HAMMER Automated Operation of Mass Frontier to Construct In silico Mass Spectral Fragmentation Libraries g System Properties scum Computer Name Hardware Advanced System Protection Remote You must be logged on as an Administrator to make most of these changes Performance Visual effects processor scheduling memory usage and virtual memory User Profiles Desktop settings related to your logon Startup and Recovery System startup system failure and debugging information Value C Users Albert AppData Roaming Ope C Program Files OpenBabel 2 3 2 C P USERPROFILE AppData Local Temp USERPROFILE AppData Local Temp ne C Program Files Thermo Foundation C COM EXE BAT CMD VBS VBE JS vr Lox cancel 5 Append the paths to the Variable value Note that the different paths must be separated using a semicolon For instance assume that the original value of the variable PATH is C Windows system32 C Windows and the JAVA_PATH is C Program Fi
16. 402 m z 194 080376 3 2 2 Parameter Settings for Mass Frontier The fragmentation results of Mass Frontier highly depends on the reaction restrictions settings Crucial settings include 1 2 Knowledge Base decide whether the fragmentation rules and or fragmentation libraries should be used The first option predicts reaction pathways based on general fragmentation and rearrangement rules The second option accesses an intelligent fragmentation mechanism knowledge base collected from scientific publications and real experiments to predict the molecule decomposition reactions Mass Frontier produces much more fragments when using the fragmentation libraries Meanwhile the computational time is significantly increased Base jonization amp Cleavage HRearangement Resonance Additional Sizes Knowledge Base Generel Fragmentstion Rules Fragmentation Litreny Bot Selected Fragmentation Library Fragmentation Library Options Actve Library Name Active records only H HighChem ESI Nex J H HighCnem ESI Pos Library tonizetion onty J B HighChem Fragme Ignore General Frag Rules in library reactons Charge Locakestion Concept only J Display this window every time Generation of Fragments amp Mechanisms is stared Restore Detcultt gt B OK Cancel lonization Method decide whether the target molecule should be ionized in electron impact El positive or negative Electrospray lonization ESI Atmospher
17. E E E 11 P P E E S E E 11 ESAR O e E E A tence sa ceeseeneaeearseenseeceaes 23 2 5 5 Determine Patterns and R GIONSGS cccccceccseeceeceeeceecceeceeeceesueeaueceeseeesaeseeeseesaeesensaeeses 24 Chapter 3 Using HAMMER cccccccceecccececeeeceeeeseeceueecaeeceuecseseeeseesacesuesseeeseeeseeesanes 28 3 1 Using the FREI SVS UIC CS iro ctec cn sercanceceateaermdnatnanecenseansuneasameameanioisswancaeemnccatactiauaseracee sd 28 SM EN ee NG E E E E E I EE E A E E O A E S E 28 a eg C ONE MOO ee E E E 28 Gy Pero ME CUVE 1 618 e E E ee ee S 29 RARO SU a eee ee ee eee eee ees ee tee ae ee ee 29 3 2 Using the A MICON PaCS MIVA OMe cceies ce sesencacenasetsucmane yocnecenate cst unasencatmanveonscnascadossuneuate ycueaceneeaaat 31 Ie TOA an tanceetene ate oe aan E cies du dananeani vein aawedemacite ss E S 31 3 2 2 Parameter Settings for Mass Frontier cccccccceecceseceeeceesceeeceeeceeeceeeseeesseesaeeseeeseeeeaeegs 31 eZee Ol ir eal Ne NAO I ate cteseisererscetae care ESE aces E EE 32 EROSO ag acces csc rec seep gn see eae sce eee gnaw E E E S 33 3 3 Using the ONG NI Sele PAGING ioe centeesce see caeecaecleancedantoctiscianceansuee EE EEEE EEE 35 a eC al a 2 2 eee ee eee eee E ener eo een nner nee eee en eee reenter ey rere 35 6G oy COE MOOO eee nee eee ee ee eee E ee 35 3 9 9 HALCL ACTIVE Mod Granec a oued eens saute daandaniwee a Gisele nates 35 IOA RESUS eT R ee 36 3 4 Using the SpectralMatcning
18. Fragmentation Libraries 2 2 Development Environment HAMMER is developed and tested under the following environment e Intel Core i5 2 5GHz CPU 4GB memory screen resolution 1680x1050 e Windows 7 32 bit professional edition e JDK 6 update 43 32 bit x86 e Mass Frontier 7 e Open Babel 2 3 1 e Python 2 7 3 32 bit x86 e Sikuli X 1 0rc3 with r930 patch 2 3 Components HAMMER includes four major components 1 2 RetrieveStructures is a batch processing based automation tool to search and download compound structures InSilicoFragmentation is a visual scripting tool to manipulate Mass Frontier and perform high throughput multi stage in Silico fragmentation on multiple compounds OrganiseFragments is a tool to organize the Mass Frontier results and export them as fragmentation data files SpectralMatching is a tool to perform batch matching of real spectral against in silico fragmentation libraries HAMMER Automated Operation of Mass Frontier to Construct In silico Mass Spectral Fragmentation Libraries 2 4 Using Sikuli for GUI Automation Sikuli is a visual scripting software package to automate GUI operations using image recognition and keyboard mouse events simulation It contains a script runtime and an integrated development environment IDE The latest version Sikuli X is available for Windows Mac OS X and Linux Sikuli can be installed easily by using the official installer or simply by copying the program
19. Java official 32 bit version Java development kit JDK Java runtime environment JRE can be downloaded from http www oracle com technetwork java javase downloads index html HAMMER works on Java 6 and or Java 7 The 64 bit version is not supported Mass Frontier 7 Mass Frontier is a scientific software package for the management evaluation and interpretation of mass spectra HAMMER has been developed for Mass Frontier 7 Previous versions will be supported in the future Open Babel Open Babel is an open source toolbox for chemical data formats conversion The latest version of Open Babel can be downloaded from http openbabel org wiki Get_Open_ Babel Python 2 7 5 Optional the official Python package can be downloaded from hitp www python org download The latest version of 32 bit Python 2 7 5 is recommended The binary version of HAMMER already contains a python runtime This step is optional Sikuli X Optional Sikuli is a popular visual scripting software package The latest version of Sikuli can be downloaded from hitp www sikuli org download html Before installation make sure that all previous versions are uninstalled The latest HAMMER already contains a standalone version of Sikuli X This step is optional If Sikuli does not work properly online support can be found here https answers launchpad net sikuli faq 2005 HAMMER Automated Operation of Mass Frontier to Construct In silico Mass Spectral
20. User Manual HAMMER Automated operation of Mass Frontier to construct in silico mass spectral fragmentation libraries Jiarui Zhou t Ralf J M Weber J William Allwood Robert Mistrik Zexuan Zhu Zhen Ji Siping Chen Warwick B Dunn Shan He and Mark R Viant College of Biomedical Engineering and Instrument Science Zhejiang University Hangzhou 310027 China School of Biosciences and School of Computer Science University of Birmingham Birmingham B15 2TT United Kingdom HighChem Ltd Leskova 11 81104 Bratislava Slovakia Shenzhen City Key Laboratory of Embedded System Design College of Computer Science and Software Engineering School of Medicine Shenzhen University Shenzhen 518060 China Oct 2013 Version 1 5 HAMMER Automated Operation of Mass Frontier to Construct In silico Mass Spectral Fragmentation Libraries Contents erante TANEN ON ane eee enn een eee eee ee eee eee ere te 4 ehaner WSC AON ean EEE EE E A 5 2 A SI ON a a S E EEE usensaieeapeeos 5 2 2 Development Environment a scsseoan cece sicnssceceaes si ceesee swede sclanndqeoueeeetanenceacensoceeeteupstesanetesdeegeeooaacian 6 PCOM ONE oe ro ee E ee ee eee A eae T 2 4 Using Sikuli for nll FAL OI AO Mics ciccseecacoreccseceasttaaecqeeenszteseseasnconueusassetmenndancmasecedsanceanseeycunmessseascees 8 Zo MVS Veale CONNU O er E E EAE A E E 9 29 1 Instal Dependent SS OTW ANS sassosonan a E N ouseeoceniearine 9 ZZ ARA MME R arer E E E E
21. ass Frontier 7 0 Remaining Time 44 Days File Edit View Tools Search Library Options Help 38 Microsoft Office Xx eB H OOHSSHAD a p ce Es i ca 4 6 amp amp Structure Editor 1 9 mf_frag_setting Base Ionization amp Cleavage Additional Sizes Check whether the structure can be fragmented This pattern is searched within region mf_operate_reg HighChem Mass Frontier 7 0 Remaining Time 44 Days File Edit View Tools Search Library Options Help 2 Microsoft Office e AGlo gt xrieaelH DO OS BLAND KX A W Structure Editor 1 v Fragmentation Library 10 mf_frag_setting_ok Click to start fragmentation This pattern is searched within region mf_operate_reg 14 i Reaction Restricts lonization amp Cleavage H Rearrangement Additional Sizes Knowledge Base General Fragmentation Rules Fragmentation Library Selected Fragmentation Library Fragmentation Library Options Active Library Name Active records only 1 E HB HighChem ESI Neg 200 2 H HighChem ESI Pos 200 E Library lonization only 3 HighChem Fragmentatic 4 MSLib ed Ignore General Frag Rules in library reactions a Charge Localization Concept only Display this window every time Generation of Fragments amp Mechanisms is started 11 mf_frag_warning There are warning messages concerning the structure Do you want to abort the ge
22. ble l xca v AS amp re Formula Generator A Fragments and Mechanisms Mechanisms QGP 82 Batch Fragment Generation Report Creator Structure Table 55 029075 56 013090 56 036900 57 020915 57 04 2 q Select possible fragments with m z 52 0182 25 mf_tablemenu_structs Structure Table Click to open precursors list dialog This pattern is searched within region mf_tablemenu_reg fa HighChem Mass Frontier 7 0 Remaining Time 44 Days File Edit View Tools Search Library Options Help X Microsoft Office e Hg G Structure Editor MDP PB OHAV ees o i Fragments amp Mechanisms 1 amp cj p Chromatogram Processor PU Spectra Classifier i w Structure E Fragments Comparator tt Formula Generator XK Fragments and Mechanisms fagments Mechanisms PD 2 Batch Fragment Generation 56029075 56 013090 56 036900 57 020915 57 04 2 2 Select possible fragments with m z 52 0182 26 mf_stable_precursor ighenMassFower70 RemsinngTne 09 File Edit View Tools Search Library Options Help 2 Microsoft Office e Hael o X ee e _O OHSS HAD 27 mf_stable_notexist No structure defined 20 Check whether the list of fragments have been pasted to the Precursors List dialog This pattern is searched within region mf_operate_reg and the default matching similarity is set to 0 9 SmaexH 0O S T B No structure defined 28
23. ched within region mf_toolbar_reg 19 mf_save_mech_dialog 17 HAMMER Automated Operation of Mass Frontier to Construct In silico Mass Spectral Fragmentation Libraries Double click to enter the export filename This pattern is searched within region mf_operate_reg The target offset should be placed inside the File name input box Search out p Organize v New folder azz v sr Favorites Date modified Type E Desktop _ KGID_C00156_struct0_layer0 mcr 5 24 2013 12 23 PM MCR File m Downloads _ KGID_C00406_struct0_layer0 mcr 5 24 2013 12 40 PM MCR File 7 Dropbox E _ KGID_C00805_struct0_layer0 mcr 5 24 2013 12 42 PM MCR File Recent Places f d SkyDrive aw Libraries E3 Documents a Music Pictures F Videos File name v Save as type Compressed Reactions mcr z Hide Folders 20 mf_save_mech_wait Check whether the saving process is finished This pattern is searched within region mf_operate_reg m i x Z Fragments Mechanisms QD age m z 50 015101 1 022927 53 002191 53 0857 54 010016 54 033826 55 005265 55 017841 55 029075 55 041651 55 054227 55 989281 56 013090 56 025666 56 049476 56 062052 56 997106 57 020 E 50 015101 51 022927 E E S E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E ggg N a a A A A A A N Q pas oo 2 i 9146
24. dataList gt lt metadata dictRef nmc scanNum content 1 gt lt metadata dictRef nmc msLevel content 1 gt lt metadataList gt v lt peakList gt lt peak id 1 xValue 195 087652 yValue 100 0 gt lt peakList gt lt spectrum gt gt lt spectrum type MS id 2 gt lt spectrum gt gt lt spectrum type MS id 3 gt lt spectrum gt gt lt spectrum type MS id 4 gt lt spectrum gt gt lt spectrum type MS id 5 gt lt spectrum gt gt lt spectrum type MS id 6 gt lt spectrum gt gt lt spectrum type MS id 7 gt lt spectrum gt lt spectrum type MS id 8 gt lt spectrum gt gt lt spectrum type MS id 9 gt lt spectrum gt gt lt spectrum type MS id 10 gt lt spectrum gt lt spectrum type MS id 11 gt lt spectrum gt lt spectrumList gt v lt moleculeList xmlns http www xml cml org schema convention cdk moleculeSet gt v lt molecule id 1 gt v lt formula concise C 8 H 10 N 4 O 2 H 1 gt lt atomArray elementType C H N O E count 8 10 4 2 1 gt lt formula gt 38 The MSP file can be exported for each target compound containing the spectra and formulas of the precursor and its in Silico fragments These files are compatible with common MS software and web services e g LipidBlast MSSearch and MassBank Name Precursor Comment MS1 Num Peaks 1 195 087652 100 0 C8H10N402 H Name CSID_2424_struct _fragmen
25. e E Videos a ATP_CSID_5742 mol 1 30 2013 9 26AM MOLFile m caffeine_CSID_2424 mol 1 30 2013 9 26 AM MOL File jE Computer m cholesterol palmitate_CSID_4937824 mol 1 30 2013 9 26 AM MOL File amp BOOTCAMP C Eil cortisone_CSID_5140966 mol 1 30 2013 9 26 AM MOL File amp Macintosh HD E tidinn OEM ENAN mnl IONIN 1 96 ARA MANI Cila Sd m r Filename 4 hydroxyphenylpyruvic acid_CSID_9 Known Structure Formats me v 5 mf_openfile_dialog open Open _ Click to open the selected mol file This pattern is searched within the region of pattern mf_openfile_dialog 6 mf_openfile_dialog error_format This error message appears when the mol file cannot be opened The pattern is searched within region mf_operate_reg File err fmt mol is provided to produce this situation the file ERR FMT MOL does not belong to a predefined set of structure formats or you are accessing the file from another program 7 mf_openfile_dialog error_format_ok 13 HAMMER Automated Operation of Mass Frontier to Construct In silico Mass Spectral Fragmentation Libraries Click to close the error message box This pattern is searched within region mf_operate_reg 8 mf_frag_icon s CaA v AA Check whether the mol file is opened in structure editor The target offset should be placed on top of the Start fragmentation button on the right hand side of the image HighChem M
26. e compound in the corresponding database 3 Structure ID ID of the substructure For multi molecule entries StructN indicates the Nth substructure molecule separated from the original compound If the candidate compound contains only one structure its value is Struct0O If the RetrieveStructures is set to no split this part is absent 4 Data Format export data format set to the RetrieveStructures A report file is generated in the name of compounds txt or compounds xls to summarize the results The search type search term candidates number candidates IDs website links and remarks of each target compound are included in the report An example of the excel report is shown as follows E 2424 2519 C07481 caffeine Phenylalanine_map00360 Candidates KGID_CO4087 KGID_C13403 PCID_2519 PCID_67294 PCID_31212 PCID_72700 KGID_CO7481 PCID_15061 PCID_76682 PCID_1326 PCID_1676 PCID_64136 PCID_79438 CSID_2424 PCID_2519 KGID_CO7481 KGID_CO7481 KGID_C13403 CSID_2424 KGID_CO7086 KGID_C00156 KGID_C02137 KGID_C19875 KGID_C00596 KGID_Co0079 KGID_C15524 KGID_Co0084 KGID_C04479 KGID_C11457 Link httpv www genome jo ddget bin www_bget cpd C04087 httov www genome_jo dbget bin www_bget cpd C 13403 htto pubchem_ncbi nim nih gov summary summary cgi cid 2519 http pubchem_ncbi nim nih govw summary summary cgi 7cid 67294 http pubchem_ncbi nim nih gov summary summary cgi cid 31212
27. ectal utilize a modified version of the pMatch algorithm for spectral matching and scoring Precursor m z values are crucial for proper matching in pMatch For more details please read the supplementary information and visit the pMatch algorithm website http pfind ict ac cn pmatch 3 4 2 Console Mode In console mode the SpectralMatching is used as a single line command gt SpectralMatching exe options database targets where database is the path to the in silico library and targets is the path to the target folder that contains all of the real spectra Options of the SpectralMatching are 1 abs measure distance in absolute value 2 tp peaks matching range default value 100 ppm 3 ift intensity filter threshold default none no limit 4 ifn intensity filter maximum peaks number default all no limit 5 pth preprocessing peaks per 100 m z default all no limit 6 cpt capital peaks intensity threshold default 0 05 7 Cpn maximum capital peaks number default all no limit 8 max maximum match candidates number default all no limit 9 help display the software help Here is an example to use SpectralMatching in console mode gt SpectralMatching exe tp 10 ifn 100 max 5 mzdb spectral It functions to perform batch matching on real spectral files in spectral folder against the in silico library mzdb using maximum m z matching distance 10 ppm u
28. er py 4 If users want to change the focus offset or similarity click the pattern to open the Pattern Settings window 5 Click the Matching Preview tab to change the similarity value was aes Tami purt a hoan ee oh ee a et iaie rn de hoh ee ail wn 2 e EIL a Se Similarity A i 7 Number of matches Max 1 8 OK Cancel 6 Click the Target Offset tab to change the offset value File Matching Preview PIE 5 Se N sloader Target offset x bO T a 8 25 HAMMER Automated Operation of Mass Frontier to Construct In silico Mass Spectral Fragmentation Libraries 7 Save the script file and then open it in Windows explorer The captured image is in the script folder te Region Bun Runin slow motion oN TO rmos HTML PYTHON x DataPOrtpy png sikuli html sikuli py 8 Copy the image file into the Resources folder and then change the content in res index to use it in the InSilicoFragmentation If the focus offset or the similarity is changed add this information into the pattern value as well see the Patterns section To determine a new search region users should 1 Launch the Sikuli IDE and open a new script file 2 Click the Create region icon and the software will enter the screen capture mode ake screenshot Insert image A exists s find s findAll s wait s 3 Drag a rectangle t
29. er than the center of the pattern Negative offset values indicate that the click point is on the top left side of the center and positive values indicate it is on the bottom right side More details of Target Offset can be found here http doc sikuli org pattern html Pattern targetOffset 4 Similarity minimum similarity to match the pattern image Its value should be between O and 1 The smaller the similarity value the easier an image can be matched to the pattern More details of Similarity can be found here http doc sikuli org pattern html Pattern similar The Image File Name Target Offset and Similarity are separated using semicolons If users want to specify the Similarity but not the Target Offset the second part of the pattern value should be set to None For instance mf_openfile_dialog OpenFilename png None 0 7 The patterns used in HAMMER are 1 mf_check_opened File Edit View Tools Search Library Options Help eg a W np m a S a Check whether Mass Frontier has launched and is sitting on top of all other software The automation tool will search the whole screen for this pattern 2 mf_openfile_icon a 7J Click to open a file This pattern is searched within region mf _openfile_reg see the regions section ee pa HighChem Mass Frontier 7 0 Remaining Time 44 Days File Edit View Tools Search Library Options Help 34
30. file log txt that contains the fragmentation time of each compound and the total process time is also provided Following is an example of the log file log txt Notepad File Edit Format View Help Layer 0 Target alpha hydroxyisovaleric acid_CSID_90190 258 407 sec Target caffeine_CSID_2424 160 371 sec 418 803 sec Total time 418 813 sec 34 3 3 Using the OrganiseFragments 3 3 1 Input Data The OrganiseFragments utilizes the InSilicoFragmentation results as its input data and processes the mol files according to the COMPOUND NAME LAYER_ID and FRAGMENT_ID in their filenames To perform OrganiseFragments the Split SDF option step 6 in section 3 2 2 must be set to Yes All the fragments must be stored in the same folder The folder can contain fragments exported from more than one compound For the results generated by the InSilicoFragmentation the export folder should be provided 3 3 2 Console Mode In console mode the OrganiseFragments is used as a single line command gt OrganiseFragments exe options targets export where targets is the path to the target folder that contains all the mol fragments and export is the path to the folder wherein the results are going to be stored If an export folder is not provided the results will be saved in the same folder as targets Options of the OrganiseFragments are 1 image generate 2D image for
31. he generation again 16 mf_fragres_fm eag Fragments amp Mechanisms Fam xv Check whether the fragmentation is finished This pattern is searched within region mf _operate_reg 16 HighChem Mass Frontier 70 Remaining Time 44 Days File Edit View Tools Search Library Options Help E Microsoft Office oo xX m B e OSPSPAD o i Ep 2a E ing hh S 3 Fragments amp Mechanisms 1 N N N Ib N H Kor ib Z Lb Z Lo LT OY m z 81 044725 m z 81 044725 m z 54 033826 m z 52 0181 i t DEANNA NN ANNAN EEE n n E E E E fv 63 60 60 D iy ae es e eSpoes S Results for m z 52 018175 Fragment No 1 17 mf_fragres_fmcheck a Check whether all the fragments have been listed in the Fragments amp Mechanisms dialog This process may take a few seconds or even minutes to finish for large compounds The InSilicoFragmentation needs to confirm it before moving to the next step This pattern is searched within region mf_operate_reg HighChem Mass Frontier 7 0 Remaining Time 44 Days File Edit View Tools Search Library Options Help E Microsoft Office N N N lb m N Lb lb lt 4 A gG te ye Yi m z 81 044725 m z 81 044725 mz 54 033826 mz 52 0181 s7652 GEER NAA NEN E N E E Ree a MEJ 18 mf_save_mech Click to save the fragmentation mechanisms This pattern is sear
32. https pubchem_nebi nim nih gov summary summary cgi cid 72700 http Awww genome_jp ddget biniwww_bget cpd C07481 http pubchem_ ncbi nim nih gov summary summary cgi 7cid 15061 http pubchem_ncbi nim nih gov summary summary cgi 7cid 76682 http pubchem_ncbi nim nih gov summary summary cgi cid 1326 http pubchem_ncbi nim nih gov summary summary cgi 7cid 1676 http pubchem_ncbi nim nih gov summary summary cgi cid 64 136 https pubchem_nebi nim nih gow summary summary cgi cid 79438 http www chemspider com Chemical Structure 2424 Atm http pubchem_ ncbi nim nih gov summary summary cgi cid 2519 http www genome jo dbget bin www_bget cpd C07481 https Wwww genome jo dbdget bin www_bget cpd C07481 httpv Wwww genome jpo ddget biniwww_bget cpd C 13403 https www chemspider com Chemical Structure 2424 htm htto www genome jo dbget binwww_bget cpd C07086 htto www genome jo dbget binwww_bget cpd C00156 httpv www genome jo ddget binwww_bget cpd C02137 htto www genome _jo dbget binwww_bget cpd C 19975 httpv Wwww genome jp ddget bin www_bget cpd CO6866 httpv Wwww genome jp ddget biniwww_bget cpd C00596 https www genome jo ddget binwww_bget cpd C00079 httov www genome jo dbget bin www_bget cpd C 15524 htto www genome jo dbget binwww_bget cpd C00084 httov www genome jo dbget binwww_bget cpd C04479 https www genome jo dbdget bin www_bget cpd C 11457 Remark 3 structures splited from the original compound remove Na 3 structures splited from the origi
33. i schemaLocation http sashimi sourceforge net schema_revision mzXML_3 1 http sashimi sourceforge net schema_revision mzXML_3 1 mzXML_idx_3 1 xsd gt lt msRun gt lt parentFile fileName example out_org CSID_ 2424 struct0 CSID 2424 struct0 mol fileType processedData fileShal 8c666bc9fa4f95966027ee4d946424199b8eb042 gt v lt dataProcessing centroided 1 gt lt software type processing name HAMMER version v1 0 0 gt lt dataProcessing gt lt scan num 1 msLevel 1 peaksCount 1 polarity lowMz 195 087652 highMz 195 087652 gt lt peaks precision 32 byteOrder network contentType m z int compressionType none compressedLen 0 gt QO0MWcELIAAA lt peaks gt v lt scan num 2 msLevel 2 peaksCount 11 polarity lowMz 58 028740 highMz 195 087652 gt lt precursorMz precursorScanNum 1 gt 195 087652 lt precursorMz gt v lt peaks precision 32 byteOrder network contentType m z int compressionType none compressedLen 0 gt QmgdbkLIAABCohbmQsgAAELaFExCyAAAQtwkf kLIAABC3hxPQsgAAELOJHSCyAAAQwoQ8kLIAABDJxe QsgAAEMoE6ZCyAAAQ2M lt peaks gt lt scan num 3 msLevel 3 peaksCount 4 polarity lowMz 83 060375 highMz 138 066188 gt lt precursorMz precursorScanNum 2 gt 138 066188 lt precursorMz gt lt peaks precision 32 byteOrder network contentType m z int compressionType none compressedLen 0 gt QqYe6ULIAABC3CR QsgAAELeHESCyAAAQwoO8kLIAAA lt peaks gt lt scan gt lt sca
34. ic Pressure Chemical lonization APCI mode Fragments produced in El mode are not yet supported in HAMMER may cause unexpected error Although the Mass Frontier supports both ESI APCI positive and negative ionization much fewer fragments will be generated in the negative mode i e based largely upon fragmentation rules since there is a lack of information in the literature on negative mode fragmentation 31 HAMMER Automated Operation of Mass Frontier to Construct In silico Mass Spectral Fragmentation Libraries Base lonizetion amp Cleavage H Rearrangement Resonance Additional Sizes lonization Method lonization on M Electron Impact El 7 Non bond el H M H Protonation ESL APCI Pibond 7 M H Deprotonetion ESI APCI Cluster lon Formation NH4 Cleavage Alkali Metal Adducts Ne mr a Vi inductive 1 Chemical lonization Display this window every time Generation of Fragments amp Mechanisms is started Restore Defautts fe a OK Cancel 3 Reaction Steps amp Reactions Limit the reaction steps number gives the maximum size of cascaded fragment reactions i e the maximum reaction generations in each fragmentation rules libraries prediction The reactions limit defines the maximum total reaction steps in the entire fragmentation process The number of fragments produced in a given reaction path increases exponentially as the number of reaction steps increases To avoid e
35. les Java jdk1 6 0 43 The modified PATH variable value should be C Windows system32 C Windows C Program Files Java jdk1 6 0_43 bin Edit User Variable Path Sikuli X Vibs C Program Files Java jre6 bin Loc cancel 6 Click OK to save the changes In Windows 7 and Vista Mass Frontier needs to be run as administrator Therefore the HAMMER should also be run as administrator to function correctly 10 1 Find the Sikuli IDE exe in folder InSilicoFragmentation Sikuli or in the path where Sikuli X is installed 2 Right click Sikuli IDE exe and then click Properties Create shortcut Delete Rename Sikuli IDE exe Sikuli IDE ini sikuli ide jar 3 Check the Run this program as an administrator option amp Sikuli IDE exe Properties F you have problems with this program and it worked correctly on an earlier version of Windows select the compatibility mode that matches that earlier version Help me choose the settings Compatibility mode E Run this program in compatibility mode for Windows XP Service Pack 3 Settings E Run in 256 colors Run in 640 x 480 screen resolution Disable visual themes E Disable desktop composition Disable display scaling on high DP settings 9 Change settings for all users 4 Click OK to save the changes 2 5 2 Install HAMMER The RetrieveStructures OrganiseFrag
36. list file mzList can be exported for each target compound containing the m z intensity fingerprints These files are compatible with common MS software and web services e g mMass Mass MassBank and MetFrag Neutral 54 033826 100 0 58 028740 100 0 81 044725 100 0 83 060375 100 0 109 039639 100 0 110 071274 100 0 111 055289 100 0 122 071274 100 0 138 066188 100 0 167 092738 100 0 168 076753 100 0 m z 179 092738 100 0 195 087652 100 0 54 033826 58 02874 81 044725 83 060375 109 039639 110 071274 111 055289 122 071274 138 066188 40 The property list structure can be exported for each target compound containing the spectral mechanisms and metadata of the fragments including the exact mass formula InChi code SMILES code and corresponding mol file name Plist is a special xml data type that can be accessed directly in Python Users can also open it in any xml editor This XML file does not appear to have any style information associated with it The document tree is shown below v lt plist version 1 0 gt lt dict gt lt key gt CSID_2424 struct0 mol lt key gt Wv lt dict gt lt key gt fragments lt key gt v lt dict gt lt key gt CSID_2424 struct0_layer0_frag0 mol lt key gt W lt dict gt lt key gt fragments lt key gt lt dict gt lt dict gt lt key gt metadata lt key gt W lt dict gt lt key gt exact_mass lt key gt lt real gt 58 02874021809075 lt real gt lt key gt filename l
37. me This is the time interval between two operations in Mass Frontier More cool down time is needed for large compounds to ensure stability of the software A positive float value is expected here Please enter cool down time sec 1 ox 6 Select whether the exported sdf file should be split into separate mol files Split SDF into separate MOLS 1 CONFIG Max Stages 1 Target Path I AutoFrag 1 0 5 In SilicoFragmentation_Win7 example 7 mols found Export Path l AutoFrag 1 0 5 In SilicoFragmentation_Win7 example Cool Down Time 1 000000 sec Split SDF True 2 PREPARE a Make sure Mass Frontier is opened and not minimized to taskbar b Close all sub windows in Mass Frontier c Set Fragmentation settings Menu gt Options gt Settings d Minimize any other running software e Try to avoid any possible popup windows including system applications notifications 3 PERFORM Click OK 8 The InSilicoFragmentation will wait 5 seconds to allow users to bring the Mass Frontier window on top of all other open software windows The in silico fragmentation will start soon after that Do not operate the computer when the InSilicoFragmentation is still running otherwise the process will be interrupted 9 A message box popups when the fragmentation process is finished Click OK to quit InSilicoFragmentation Finished with success 3 2 4 Results Result files a
38. ments and SpectralMatching work out of the box as long as the dependent software is correctly installed However the InSilicoFragmentation requires configuration before it can be used The InSilicoFragmentation is developed based on Sikuli which uses image recognition to search and operate GUI elements HAMMER provides two versions of InSilicoFragmentation for Windows 7 and XP users separately They should be able to work directly without any setup However the automation tool may sometimes need to be configured for different screen sizes resolutions or GUI styles themes In that case users should modify the contents in res index and Resources before using the tool Plain text file res index includes the index of pattern images and search regions and the folder Resources contains the corresponding pattern image files The index file res index consists of two sections patterns and regions see section 2 4 2 5 3 Patterns Image File Name Similarity mf_openfile_dialog OpenFilename png 90 20 0 7 Pattern ID Target Offset 1 Pattern ID identity of the GUI image pattern Should not be changed by users HAMMER Automated Operation of Mass Frontier to Construct In silico Mass Spectral Fragmentation Libraries 2 Image File Name name of the pattern image file stored in the Resources folder 3 Target Offset focus offset of the target GUI element Users can specify a different click point oth
39. mode RetrieveStructures can also be run in the interactive mode Double click RetrieveStructures exe in windows explorer to enter this mode wherein the parameters are set in an interactive manner gt RetrieveStructures exe Enter the path of the target file Targets file gt gt Enter the path of the export folder Export path gt gt Decide whether the multi molecule entries should be split into separate files y for yes n for no Split multi molecule entries into separate files y n enter y gt gt Decide whether the excel report should be generated y for yes n for no Generate excel report y n enter n gt gt Enter the export data format mol inchi smiles or formula Export data format enter mol gt gt Enter the maximum candidates number between 0 to 100 Maximum candidates number enter 10 gt gt Press enter and the RetrieveStructures will start the search and download process 3 1 4 Results Downloaded compounds are stored under the following format 29 HAMMER Automated Operation of Mass Frontier to Construct In silico Mass Spectral Fragmentation Libraries Candidate ID Data Format KGID_C07481_structO mol Database ID Structure ID 1 Database ID ID of the database CSID indicates the compound is downloaded from ChemSpider PCID indicates PubChem and KGID indicates KEGG 2 Candidate ID entry ID of the candidat
40. n ID Region Offset 1 Region ID identity of the search region Should not be changed by users 2 Region Offset x position y position width and height of the region rectangle Negative value usually 1 indicates the screen s width height isx used as that of the region rectangle The regions used in HAMMER are 1 mf_toolbar_reg ooa PSeOexelecalwvli ask Fragments amp Mechanisms 1 Ham xv d Fragments Mechanisms amp gt wa G A m i O J Q ates Qao This region contains the menu and toolbar of the Mass Frontier 2 mf_operate_reg File Edit View Tools Search Library Options Help E Microsoft Office ermal o X sBB B TOHHOAD A amp a Precursors List vaji pe xE z Calvas Fragments amp Mechanisms 1 Hiem xv e Fragments Mechanisms ie ft 2l ebpoe Ssss One This is the region wherein all the sub windows of the Mass Frontier are shown Most of the patterns listed above are searched within this region Therefore its values must be defined carefully 23 HAMMER Automated Operation of Mass Frontier to Construct In silico Mass Spectral Fragmentation Libraries 3 mf_openfile_reg File Edit View Tools Search Library Options Help 34 Microsoft Office ae E I Eai C E BMH LD PB jeoere tak lt a This is the region to search pattern mf_openfile_icon It must contain this pattern image
41. n num 4 msLevel 3 peaksCount 1 polarity lowMz 83 060375 highMz 83 060375 gt lt precursorMz precursorScanNum 2 gt 111 055289 lt precursorMz gt lt peaks precision 32 byteOrder network contentType m z int compressionType none compressedLen 0 gt QqYe6ULIAAA lt peaks gt lt scan gt lt scan num 5 msLevel 3 peaksCount 2 polarity lowMz 122 071274 highMz 179 092738 gt lt precursorMz precursorScanNum 2 gt 179 092738 lt precursorMz gt lt peaks precision 32 byteOrder network contentType m z int compressionType none compressedLen 0 gt QvQkfkLIAABDMxe QsgAAA lt peaks gt lt scan gt lt scan num 6 msLevel 3 peaksCount 1 polarity lowMz 81 044725 highMz 81 044725 gt lt precursorMz precursorScanNum 2 gt 81 044725 lt precursorMz gt eorasbo nrantiotane 29 hurk aNvrdare natwnrl anntantTmase m aiink caamnracct anTmas nana File View Processing Sequence Tools Libraries Links Window Help QeS 2 POR SF BOD iA BX 2 Bee B E Documents ooo eu in Ey CSID_2424_struct0 2 58 0287 100 CSID_2424 struct0 3 CSID_2424 struct0 P 81 0447 100 CSID_2424 struct0 3 109 0396 100 110 0713 100 j CSID_2424_struct0 2 111 0553 100 CSID_2424_struct0 3 J 1220713 100 138 0662 100 120 a i On N 8 167 0927 100 fF 56 8 D D 168 0768 100 Ae g 2a 179 0927 100 D ow Se e N LO O 210 1111 100 90
42. nal compound remove Na 30 3 2 Using the InSilicoFragmentation 3 2 1 Before Starting Make sure no popup dialogs or notifications show up e g Dropbox notification messages It will interfere with the process and block the screen capturing It is recommended to quit all other software besides Sikuli and Mass Frontier to avoid interference Place all the target mol files MDL MOL format in the same folder Note that the Unicode paths are not supported Open Mass Frontier Change parameter settings if needed e g change the in silico fragmentation parameters The InSilicoFragmentation will not modify the parameters for users ma Hight r File Edit View Tools Search Library O ptions Help Emi icrosot ft Office S W g EA KES ce p e co 6 iB w Structure Editor 1 k H amp key Structure Editor 1 X Reaction Restrictions Base Ionization amp Cleavage H Rearrangement Resonance Additional Sizes Knowledge Base General Fragmentation Rules Fragmentation Library Selected Fragmentation Library Fragmentation Library Options ive Library Name V Active records only H HighChem ESI Neg 200 H HighChem ESI Pos 200 Library lonization only aA HighChem Fragmentatic N 5 sty ar Charge Localization Concept only v Display this window every time Generation of Fragments amp Mechanisms is started Unspecified Charge Site none bd Restore Defauts B B als CeH10N
43. neration and review the messages or do you wish to ignore the warnings and continue the generation This warning message appears when the structure cannot be fragmented The pattern is searched within region mf_operate_reg File err frag mol is provided to produce this situation There are warning messages concerning the structure Do you want to abort the generation and review the messages or do you wish to ignore the warnings and continue the generation 12 mf_frag_warning_abort Click to abort the fragmentation process This pattern is searched within the region of pattern mf_frag_warning 13 mf_frag_warning_ok Click to confirm the process abortion This pattern is searched within region mf_operate_reg 15 HAMMER Automated Operation of Mass Frontier to Construct In silico Mass Spectral Fragmentation Libraries Structure Check Results Errors Warnings 1 Carbens are energetically unfavorable 14 mf_frag_error Generation Stopped The structure contains error s This message appears when errors occurred during the fragmentation The pattern is searched within region mf_operate_reg File gen fail mol is provided to produce this situation 15 mf_frag_error_ok Click to close the error message box This pattern is searched within region mf_operate_reg x The structure contains error s Please correct the structure and then start t
44. o include the target region J ChemSpiderPort py chemspipy py CompDownloader py 4 Move mouse arrow on top of the captured region The x position y position width and height of the region rectangle will be displayed in a tooltip had Create Region Region 188 222 624 110 5 Change the region values in res index to use them in the InSilicoFragmentation see the Regions section 26 Using Third Party Software users can utilize other software to capture the pattern images One of the most popular free screen capture software is the PicPick website http www picpick org en To obtain new image patterns using PicPick users should 1 Launch the PicPick Select one of the methods in the Screen Capture pane the Region capture is recommended ri ae T a Home View 92 Pick a task New task Graphic Accessories Z Color Picker Pick a color code on the screen Color Palette nss Find and tune the color that you want Q Magnifier p geen Capture Give a closer look at pixels on screen OR rm Pixel Ruler Full screen z i Measure the size of desktop objects and images CA Window Control 2 CrossHair El Scrolling Window S ae Figure out the relative coordinate i Pa Protractor Measure any angle on screen L WhiteBoard O Repent laiche For presentation or drawing something m Do not show this window when program starts
45. re placed in the previously defined export folder These files are exported for each compound 33 HAMMER Automated Operation of Mass Frontier to Construct In silico Mass Spectral Fragmentation Libraries e COMPOUND _NAME _ layer LAYER_ID txt unique m z values exported from the list of fragments The COMPOUND_NAME is the filename of the target compound LAYER_ID is the number of fragmentation stages For multi stage fragmentation the filename contains more than one LAYER_ID parts separated by FRAGMENT_ ID see the 3rd point For instance file Caffeine_layer0 txt is the m z values exported form Caffeine mol on the first stage of fragmentation and file Caffeine _layerO_fragO layer1 txt is the m z values from the MS MS fragmentation layer1 whereas its precursor is the first fragment fragO generated by the MS fragmentation layer0 of Caffeine mol e COMPOUND NAME layer LAYER_ID sdf in silico fragments results generated by Mass Frontier The fragment structures are stored in sdf structure data file format e COMPOUND _NAME _ layer LAYER_ID mer in silico fragmentation mechanisms and results generated by Mass Frontier e COMPOUND _NAME layer LAYER_ID _frag FRAGMENT_ID mol Optional structure of each fragment split from the sdf file These files are generated only when the lIs split option step 7 in the above section is set to True An overall log
46. se ID search type id the compound name search type name or the empirical formula search type formula It can also obtain all of the compounds within specific pathways search type pathway The search type databases and search term are separated using one or more tabs t Note that the spaces should not be used for separation e Database s databases to search the compounds within RetrieveStructures supports searching in ChemSpider PubChem and KEGG Only one database should be provided to the id search type and the pathway search type supports the KEGG database only e Search Term the key words to search a compound in online databases Its value varies for different search types e id the search term is the entry ID of the target compound in the corresponding database e name the search term is the name of the target compound formula the search term is the empirical formula of the target compound pathway the search term is the name of the target pathway RetrieveStructures will search the KEGG database for candidate pathways ChemSpider and PubChem do not support pathway searching All the compounds within each candidate pathway will be downloaded separately e Comments Optional comments starts with the hash character All the contents in a line behind will be ignored An example of the target file is shown as follows TypeSearch Database SearchTerm
47. sing the top 100 real peaks with the highest intensity for matching and export the top 5 matching results with the highest scores for each real spectra 43 HAMMER Automated Operation of Mass Frontier to Construct In silico Mass Spectral Fragmentation Libraries 3 4 3 Interactive Mode Besides the console mode SpectralMatching can also be run in the interactive mode Double click SpectralMatching exe in the windows explorer to enter this mode where the parameters are set in an interactive manner Enter the path to the n silico library Enter the path to the real spectral target folder Decide the matching range Decide the intensity filter threshold Decide the intensity filter maximum peaks number Decide the maximum preprocess peaks per 100Th Decide the capital peaks intensity threshold Decide the maximum capital peaks number Decide the maximum match candidates number Decide whether the m z distance should be measured as an absolute value Press enter and the SpectralMatching will start processing 3 4 4 Results Overall scores and the best matching in silico compound will be displayed 44 Entry CSID_ 2424 struct0 C8H1O0N402 H 13 peaks Score 0 45712 Rel Intensity Experimental m z In silico m z Formula Distance in ppm 100 000 138 06664 138 06619 C6H7N30 H 3 274e 00 29 818 110 07142 110 07127 CSH7N3 H 1 326e 00 9 548 83 06017 83 06037 C4H6N2 H 2 468e 00 9 028 195 08854 195
48. t key gt v lt string gt Users 030bug Repository HAMMER example out_org CSID_2424 struct0 CSID 2424 struct0_layer0 frag0 mol lt string gt lt key gt formula lt key gt lt string gt C2H4N0 lt string gt lt key gt inchi lt key gt lt string gt InChI 1S C2H4NO c1 3 2 4 h1H3 H 3 4 q 1 lt string gt lt key gt smiles lt key gt lt string gt CN C 0 lt string gt lt dict gt lt dict gt lt key gt CSID_2424 struct0_layer0_fragl mol lt key gt gt lt dict gt lt dict gt lt key gt CSID_ 2424 struct0_layer0O fragl10 mol lt key gt e lt dict gt lt dict gt lt key gt CSID_2424 struct0_layer0_ fragll mol lt key gt gt lt dict gt lt dict gt lt key gt CSID_2424 struct0_layer0_ fragl2 mol lt key gt gt lt dict gt lt dict gt lt key gt CSID_2424 struct0_layer0_ fragl3 mol lt key gt gt lt dict gt lt dict gt lt key gt CSID_2424 struct0_layer0_ fragl4 mol lt key gt 41 HAMMER Automated Operation of Mass Frontier to Construct In silico Mass Spectral Fragmentation Libraries The tree visualization can also be exported for each compound It is stored in pdf format and provides a more intuitive way to display the fragmentation mechanisms Ti St 4 APHeeciem 2 hy eom de S E a lt CSH7N20 111 055289 CSID_2424_struct0_layer0_frag7 mol C8H11N40 179 092738 CSID_2424_structO_layer0_frag20 mol 1 C6H8N30 138 066188 CSID_
49. ts PRECURSORMZ 195 087652 Comment MS2 Num Peaks 11 58 028740 100 0 C2H4N0 81 044725 100 0 C4H5N2 109 039639 100 0 C5H5N20 110 071274 100 0 C5H8N3 111 055289 100 0 C5H7N20 122 071274 100 0 C6H8N3 138 066188 100 0 C6H8N30 167 092738 100 0 C7H11N40 168 076753 100 0 C7H10N302 179 092738 100 0 C8H11N40 195 087652 100 0 C8H11N402 Name CSID_2424 struct _layer _fragql3_fragqments PRECURSORMZ 138 066188 Comment MS3 Num Peaks 4 83 060375 100 0 C4H7N2 110 071274 100 0 CSH8N3 111 055289 100 0 CSH7N20 138 066188 100 0 C6H8N30 Spectral Browser ver 1 07 1 044 935 087 3 0603 3 0403 10 0712 8 0287 1 0447 3 i 22 0712 38 0661 GWARE 179 0927 95 0876 22 9712 79 9927 3 060 11 05 68 076 38 0661 if tt showallm z show match m z changecolor Ord Que Name Hit Match Disable Peak Precursor ID CSID_2424_structO_layerO_frag20_fragments 2 us000005 4 USO00009 4 2 G CSID_2424 structO_layerO_frag19_fragments a USO00003 us000011 6 I5 s 4 CSID_2424_struct0_layer0_fragl13_fragments 3 aa 2 US CSID_2424_structO_layerO_frag12_fragments UUU CSID_2424_structO_layerO_frag11_fragments J 1 CSID_2424_struct0_fragments Copyright C 2006 MassBank Project 39 HAMMER Automated Operation of Mass Frontier to Construct In silico Mass Spectral Fragmentation Libraries The plain text peak
50. ttp doc sikuli org 2 5 Install amp Configure 2 5 1 Install Dependent Software The dependent software can easily be installed using the official installers Users should check the software status using following commands in cmd console Note that the version numbers may be different depending on which packages are installed and Python is optional If a Command Not Found error occurs add the following path to the system environment variable PATH 1 Java JAVA_PATH bin JAVA_PATH is the path where JDK JRE is installed 2 Open Babel BABEL_PATH BABEL PATH is the path where Open Babel is installed 3 Python PYTHON PATH PYTHON _PATH is the path where Python is installed To add the paths into system environment variables 1 Right click My Computer on the desktop and then click Properties Open cls Manage Map network drive Disconnect network drive Create shortcut Delete Rename Properties 2 In Windows 7 and Vista click Advanced system settings on the left pane Ignore this step in Windows XP ex o om S Jee gt Control Panel All Control Panel Items System 7 Search Control Panel 2 View basic information about your computer Windows edition Windows 7 Professional Copyright 2009 Microsoft Corporation All rights reserved Rating 595 Windows Experience Index Processor Intel R Core TM i5 2520M CPU 2 50GHz 2 50 GHz Installed memory RAM
51. xcessive computational resources usage the most important fragmentation reactions are conducted first and are followed by lesser important fragmentation rules until the reactions limit is reached In HAMMER we suggest the users to set the reactions limit to its maximum value i e 20 000 Base lonization amp Cleavage H Rearrangement Resonance Additional Sizes Reaction Steps Mass Range Mex Number 5 Erom 30 mz Resonance reactions are not included in this number Io 3000 m z Reactions Limit Yalue 10000 Reactions limit means number of temporary generated internal reactions You can reasonably increase this number for larger input structures Display this window every time Generation of Fragments amp Mechanisms is started Restore Defaults 8 OK Cancel Other settings may cause minor changes to the fragmentation results See Mass Frontier User Guide Chapter 6 the Reaction Restrictions section for more details 3 2 3 Perform Fragmentation 1 Double click InSilicoFragmentation cmd 2 Enter fragmentation stages A positive integer value is expected here Please enter fragment stages il 3 Enter the path of the target folder 4 Enter the path of the export folder If the export folder does not exist the InSilicoFragmentation will create it 32 a j a Please enter export folder 5 Enter cool down ti
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