1. 1 Welcome! Mass Spectrometry meets ChemInformatics Tobias Kind and Julie Leary UC Davis Course 3: Mass spectral and molecular database search Class website: CHE 241 - Spring 2008 - CRN 16583CRN 16583 Slides: http://fiehnlab.ucdavis.edu/staff/kind/Teaching/http://fiehnlab.ucdavis.edu/staff/kind/Teaching/ PPT is hyperlinked – please change to Slide Show Mode

2. 2 Molecules and mass spectra Close relationship between molecular structure and mass spectra Molecular structure is reflected in mass spectral features (peaks, peak heights and peak combinations) Mass spectra reflect a state of gas phase ion physics and chemistry (rearrangements, fragmentations, bond cleavages) Electron impact (70 eV) mass spectra; Source: NIST05

3. 3 Molecules and mass spectra Similar structures may or may have not similar mass spectra Electron impact (70 eV) mass spectra; Source: NIST05; Created using structure similarity search in NIST MS Search program

4. 4 Molecules and mass spectra Similar mass spectra may or may have not similar structures Electron impact (70 eV) mass spectra; Source: NIST05; Created using spectral similarity search in NIST MS Search program

5. 5 Mass spectral databases I Name Spectra countType NIST05NIST05 200,000 electron impact spectra (EI 70 eV) Wiley 8Wiley 8400,000electron impact spectra (EI 70 eV) Palisade 600K Palisade 600K 600,000electron impact spectra (EI 70 eV) NIST MS/MSNIST MS/MS5,200MS/MS (ESI, +/-, 30-100V CID) MassFrontierMassFrontier7,000MS n, ESI, (Spectral Tree Library )Spectral Tree Library Important is data quality Annotation with CAS and Structure and Formula Link to literature or publication useful Currently no large ESI,APPI,APCI libraries available (free or commercial)

6. 6 Mass spectral databases II Smaller specialized libraries Pfleger Maurer Weber (Drugs) MS+RI, 70eV MassFinder (Volatiles) MS+RI, 70eV RIZA DB (Toxicants) MS+RI, 70eV Golm DB (primary Metabolites) MS+RI, 70eV Fiehnlib (primary Metabolites) MS+RI, 70eV MassBank (Metabolites) ESI, MS n, accurate masses AAFSAAFS (Drugs, Forensic,Toxicology), MS+RI, 70eV ChemicalSoft (Drugs), MS/MS, MS E _____________________________________________________________ In case of electron impact (EI) same GC-Column (DB-5, RTX-5, DB-1, OV-1) and temperature program must be used for matching retention indices In case of ESI, APPI spectra (LC-MS) same mass spectrometer design and setup should be used (triple-quad, ion-trap, TOF, Q-TOF), collision energy

7. 7 Mass spectral search algorithms PBM - Probability Based Matching (McLafferty & Stauffer) – since 1976 Dot Product (Finnigan/INCOS) – since 1978 Weighted Dot Product (Stein) – since 1993 Mass Spectral Tree Search (Mistrik) – since 21 st century Source: Stein S.E. see notes A u and A r : are the abundances of peaks in the user and reference mass spectra m: m/z values w: weighting term Weighted Dot Product:

8. 8 NISTMS mass spectral search The NIST MS Search program is the “gold standard” for EI spectral search Used for all types of unit resolution spectra MS/MS, APCI, ESI-MS spectra

9. 9 NIST MS Search program 2.0 Search everything: A) Library Search: Reverse, Normal, Similarity, Neutral Loss B) Structure Similarity Search: find molecules similar to C) Formula Search: find C 11 H 13 N 3 O 3 S D) Constrained peak search: find peaks with m/z 122 and 188 and 266 E) Name search: find Stuntman (maleic hydrazide) Search Connections: Import/Export molecular structures: (msp, hpj, sdf) Interpret Structures (MSInterpreter.exe) Find substructures (expert algorithm) Import spectra from other programs (AMDIS, Chemstation, ChromaTOF) [Download] – freely available (NIST05 MS Library is licensed ~ $1200)Download

10. 10 Mass Spectral Trees in Mass Frontier MassFrontierMassFrontier searches MSn and CID mass spectra Source: MassFrontier Helpfile

11. 11 Mass Frontier MS search MS Tree Hitlits

12. 12 Mass spectral search Library search is always the first step during the identification process. Usually library search is not enough to assign unique isomer structures. Mass spectra must be clean and background free before search. For LC-MS and GC-MS this requires peak picking and deconvolution. Additional orthogonal information has to be used: restriction of compound space to certain species or material use of isotope pattern information use of retention index if derived from GC-MS data use of retention – logp or logD correlations in case of LC-MS additional fragmentation at different voltages (MS E ) Only certain mass spectra can be in-silico predicted (calculated) (peptides, lipids, carbohydrates) – this is not the rule for other molecules

13. 13 MALDI MS based proteomics Clinical Science www.clinsci.org Clin. Sci. (2005) 108, 369-383 Clinical Science www.clinsci.org Clin. Sci. (2005) 108, 369-383

14. 14 LC-MS based proteomics approach Source: Paul Rudnick / NISTPaul Rudnick

15. 15 Picture Source: Paul Rudnick / NISTPaul Rudnick Proteomics data analysis (pipeline) General approaches A) database search (Sequest, Mascot, OMSSA) B) de-novo sequencing (Peaks, Lutefisk, Pepnovo) C) hybrid methods (GutenTag, Popitam, Inspect)

16. 16 OMSSA- Open mass spectrometry search algorithm Source: OMSSA (NCBI)OMSSA submit spectra to MS/MS search in-silico digestion of proteins matching of experimental vs. calculated MS n hit score computation inspection and review of results Download OMSSAOMSSA

17. 17 Mass spectral search of peptides (new) Source : Paul Rudnick / NISTPaul Rudnick See also ProMEX (MPIMP Golm)ProMEX

18. 18 Conversion of mass spectral libraries Usually a hassle. Keep a copy of libraries always in non-proprietary format. Request export functions or converters from your mass spec producer. XCalibur LibraryManager.exe Thermo Electron Fisher Finnigan MAT ICIS/GCQ/ITS 40 (*.lib, *.lbr) AutoMass (*.spr, *.prs, *.nam, *.hdr, *.fsf, *.cfs) MassLab (*.idb) to NIST and vice versa NIST LIB2NIST.exe [LINK]LINK Spectral files *.msd, *.hpj, *.sdf HP LIB (*.LIB), NIST LIB, JCAMP-DX, (*.jdx *.hpj)

19. 19 How to search molecules Exact searchSubstructure searchSimilarity searchLigand search R-group/Markush search

20. 20 NIST MS DB has structure similarity search Good for comparing mass spectra of similar compounds (may have similar mass spectra)

21. 21 Searching Molecules on PubChem Goto PubChem Structure SearchPubChem Structure Search 18 million compound DB (++)

22. 22 CAS SciFinder 33 million molecules and 60 million peptides/proteins largest reaction DB (14 million reactions) and literature DB substructure and similarity search of structures a must for chemists and biochemists/biologists no bulk download, no good Import/ Export, no Link outs Download Scifinder

23. 23 Structure search in SciFinder Retrieved 4000 papers (refine search only MS and MALDI)

24. 24 How scientist publish mass spectra (*) OCR – optical character recognition DB – database (*) – and structures and other spectral data PD F A B Scientist A Runs MS Publication on paper as bitmap graphic OCRDB CurationDB CreationSell DB Scientist B Needs DB A B DB Better: Central and Open Repository Electronic Publishing in XML Computerized Free or Paid Curation Today:

25. 25 DB Open data repository for mass spectra Submit spectra before publication (ticket system) No loss of information (high resolution spectra) No truncated data (report five peaks only) No hamburger to cow algorithm needed (OCR) Fast and instant use with no restrictions New synergism for data interpretation Can still cost money (curation) Works in genomic sciences (GenBank) Commercial use may be possible Central and Open Repository … checkout the BlueObeliskBlueObelisk

26. 26 The Last Page - What is important to remember There are different search types for mass spectral data  similarity search, reverse search, neutral loss search, MS/MS search There are large libraries for electron impact spectra (EI) from GC-MS  There are no large open/commercial libraries for spectra from LC-MS For creation of mass spectral libraries a holistic approach is important  Mass spectral trees can give further information (MS E or MS n ) There are different types of searching structures  Exact search, similarity search, substructure search Before you start a research project, create target lists of possible candidates  Collect mass spectra or structures in libraries with references

27. 27 Reading list (20 min) Additional reading list for very diligent and interested pupils (30 min) (*) An MS/MS Library on an Ion-Trap Instrument for Efficient Dereplication of Natural Products. Different Fragmentation Patterns for [M + H]+ and [M + Na]+ Ions The History of the NIST/EPA/NIH Mass Spectral Database (WO2006040622) DETERMINATION OF MOLECULAR STRUCTURES USING TANDEM MASS SPECTROMETRY [Link] [PDF]LinkPDF Chemical derivatization and mass spectral libraries in metabolic profiling by GC/MS and LC/MS/MS The critical evaluation of a comprehensive mass spectral library Development and validation of a spectral library searching method for peptide identification from MS/MS (*) Edison: “Two per cent is genius and 98 per cent is hard work” “Bah. Genius is not inspired. Inspiration is perspiration” [SOURCE]SOURCE

28. 28 Tasks (7 min): Should be solved and may be graded 1)Goto PubChem [LINK] or Chemspider [LINK] and perform the 3 differentLINK structure searches using benzene; report on the number of results (use the sketch function to draw benzene (6 ring with 3 aromatic bonds)) 2) Download NIST MS Search [LINK] and perform the 3 different mass spectral searches on cocaineLINK (download JAMP-DX from NIST [link])link 3) Use Instant-JChem [LINK] from last course session and create a local demoLINK database with PubChem data. Perform 3 different structure searches with benzene by double-clicking on the structure search field. Report number of results. Additional task for proteomics candidates: 4) Download the NIST peptide search [LINK] and perform a search on the given examplesLINK

29. 29 Link List http://www.google.com/search?hl=en&q=rearrangements%2C+fragmentations%2C+bond+cleavage&btnG=Search High-resolution mass spectral database http://www.massbank.jp/http://www.massbank.jp/ http://www.google.com/search?hl=en&q=mistrik+highchem&btnG=Search http://www.google.com/search?hl=en&q=stein+se+peptide+search&btnG=Search http://fields.scripps.edu/sequest/ http://books.google.com/books?lr=&as_brr=0&q=EDISON+Genius++ +inspiration+++perspiration+++date%3A1800-1898&btnG=Search+Books http://allured.stores.yahoo.net/idofesoilbyg.htmlhttp://allured.stores.yahoo.net/idofesoilbyg.html (fragrances, terpenoid mass spectra SE-52 column + RIs) http://kanaya.naist.jp/DrDMASS/DrDMASSInstruction.pdf http://www.google.com/search?q=mass+spectral+libraries+NIST05&hl=en&start=10&sa=N http://books.google.com/books?id=7IUVi06u0TQC&pg=PA114&lpg=PA114&dq=cid+mass+spectra http://www.google.com/search?hl=en&q=cid+mass+spectra+library+pbm+dot+product&btnG=Google+Search http://www.google.com/search?hl=en&q=%22similarity+search%22+Substructure+search%22+%22exact+search%22&btnG=Search http://mmass.biographics.cz/ http://pubchem.ncbi.nlm.nih.gov/omssa/browser_help.htm#RunOMSSASearchLocalDialog http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1906842 http://www.google.com/search?hl=en&q=proteomics+sequest+mascot++mudpit+OMSSA&btnG=Search http://www.google.com/search?hl=en&q=de+novo+sequencing+peaks+sequit+lutefisk&btnG=Search