1. Material Measurement Lab Material Measurement Laboratory Mass Spectrometry Data Center Biomolecular Measurement Division Q. Dong; M. Lorna A. De Leoz; Y. Liang; X. Yan; X. Yang; S.E. Stein Mass Spectral Libraries for Biomanufacturing

2. Material Measurement Lab Our Expertise Identification by MS Fragmentation – Match observed and reference spectra – Provide confidence ‘score’ – Types of IDs: Exact, Class, Recurrent – Fragmentation rules & chemical structures Raw Data → Chemical Identity → Quantity/Variability – MS Data Analysis (a.k.a. MS Bioinformatics) – Proteomics, Metabolomics, … Measurements – QTOF, Orbi Elite, Qexactive, GC/MS – Reference Spectra & Materials – Quality Metrics

3. Material Measurement Lab Ion Fragmentation is Reproducible O’Neal et al. Anal. Chem. 1951 NIST 2012 A mass spectrum is an energy-dependent property of an ion Hexadecane

4. Material Measurement Lab Sep. 4, 2012

5. Material Measurement Lab Content NIST-evaluated spectra for 212,961 compounds Accompanying chemical name(s) and identifiers Chemical structures and retention information Software for spectrum extraction & matching The NIST/EPA/NIH Mass Spectral Library (SRD1A) The NIST/EPA/NIH Mass Spectral Library (SRD1A) Reference Standard Mass Spectra for Chemical Identification Fragments of charged molecules provide reproducible, discriminating fingerprints for molecules in complex mixtures in: Usage World’s most widely used MS library >5,000 new installations / year Integrated into instrument vendor software systems Common element in GC-MS ‘gold standard’ identifications Metabolomics Environmental Analysis Homeland Security Forensics Chemical Manufacturing Food Analysis

6. Material Measurement Lab # Precursor Ions 2005 2008 2011 2012 NIST Tandem Mass Spectral Library 2012 Fragmentation Type Precursor Ions Ion Trap >10,000 Beam Collision Cell (QTOF, QQQ, HCD) >8,000 Classes: Metabolites, Drugs, Sugars, Phospholipids, Peptides, Surfactants, Pesticides, etc. Precursors: [M+H] +, [M+2H] 2+, [M­-H]- ­, [M+Na] +, [M+NH4] +, [Cat] +, [An]- ­, [p-H2O], [p-­NH3], etc. New Software Features: Exact or isotopic precursor mass & fragment ions. Formats: mzXML, mzData, mgf, msp, dta, pkl, JCAMP, …. Compatible with NIST EI & Peptide Tandem Libraries. New methods for finding targets in the presence of noise. New Scoring: Compounds with few dominant peaks. Compensates for m/z tuning errors. Compounds7,020 Precursor Ions15,517 Spectra123,781

7. Material Measurement Lab Starter Glycan MS/MS Library NGA4 2 G0 NGA2F 1 G0F NGA3B 1,2 G0B NA2G1F 1 G1F A1F 1 G2FS NA2F 1 G2F A2F 1 G2FS2 NGA2B 2 G0B NGA2 1 G0 NGA4B 2 G0B 1 Purchased; 2From the Consortium for Functional Glycomics through Dr. Jim Paulson Typical Precursor ions  [M+2H] 2+  [M+H+K] 2+  [M+H+Na] 2+  [M+2Na] 2+ Additional precursor ions for sialylated glycans  [M+2Na-4H] 2-  [M+K-3H] 2-  [M-2H] 2-  [M+Na+K-4H] 2- Fragmentation Methods  Ion Trap (Resonant)  ‘HCD’ at 12 energies (5-70eV)

8. Material Measurement Lab Content Eight species, including human 324,352 human peptide ions (24% coverage of human proteome) compiled from >30M replicates Spectrum matching methods Quality Metrics: “NISTMSQC” “Performance Metrics for Liquid Chromatography- Tandem Mass Spectrometry Systems in Proteomics Analyses”, MCP, 9, 225, 2010. NIST Library of Peptide Ion Fragmentation Spectra Usage Most widely used peptide tandem MS database Freely distributed and accessible on the web Integrated into major widely used data systems peptide.nist.gov

9. Material Measurement Lab NIST Mass Spectral Libraries NIST/EPA/NIH Mass Spectral Library EI spectra of 212K compounds Peptide Library 500,000 peptides for 8 species Small Molecule Tandem MS 7,020 compounds Ion Trap/Collision Cell IgG Library with NIST reference material glycans glycopeptides peptides intact & large fragments?

10. Material Measurement Lab Single Protein Digest Pipeline Raw Data Translate, Annotate XTandem Prospector OMSSA Comet Integrate, Class FDR Consensus Spectrum Six Filters Library Build Library InSpect TagRecon MS1, MS2, RT

11. Material Measurement Lab Start with Human Serum Albumin (HSA)

12. Material Measurement Lab HSA Library ClassPeptide DescriptionInitialFinal 1Simple Tryptic152145 2Tryptic with Expected Missed-Cleavage173154 3Common Modifications9274 4In-Source Semitryptic332266 5In-Solution Semitryptic788575 6Artifacts and PTMs1143699 7Unexpected Missed-Cleavage404295 8Under/Over Alkylation419259 9Unidentified Modifications2464456 Total59672921

13. Material Measurement Lab IgG Tryptic Peptide Libraries 6 IgG Drugs + NIST “Refimab” -1 Constant Region + Variable Region Libraries Adalimumab Example – Spectra for 1,236 Ions ClassType*Peptide DescriptionIonsIon % Abund % 1-2ESimple Tryptic1631461.9 3ECommon Modifications3831.2 4EIn-Source Semitryptic128105 5UIn-Solution Semitryptic186155.5 6UArtifacts and PTMs210174.5 7U Unexpected Missed- Cleavage 1791411.9 8UUnder/Over Alkylation3931.1 9UUnidentified Modifications293248.9 Total1236100 Oxidation Formyl Cation:Fe Cation:Ca Cation:Na Dioxidation Dehydrated Carbamyl Glu->pyro-Glu Aluminum Deamidated Methyl Gln->pyro-Glu N-terminal pyroglutamate formation C-termical Lys clipping Asn-linked glycosylation * E – Expected, U - Unexpected

14. Material Measurement Lab IgG Digest Peptides Annotation FNW(O)YVDGVEVHNAK/3+ Library Consensus Spectrum

15. Material Measurement Lab Dark Matter

16. Material Measurement Lab Identified and Unidentified Sampled Ions

17. Material Measurement Lab “Proteomics” Minor Constituents, Digest Quality Glycopeptide Peptide Sampled/No ID Unsampled

18. Material Measurement Lab Variability

19. Material Measurement Lab

21. Variability: Digestion Conditions

22. Material Measurement Lab Metabolomics

23. Material Measurement Lab Plasma Metabolites by LC-MS/MS (Anal. Chem., in press)

24. Material Measurement Lab Ions from Plasma Metabolites Identified Sampled/No ID Unsampled

25. Material Measurement Lab Metabolites by GC/MS in E-coli (GC-MS direct injection, column DB-200)

26. Material Measurement Lab NIST MS Data Center Computer