Material Measurement Lab Material Measurement Laboratory Q. Dong; M. Lorna A. De Leoz; L.E. Kilpatrick; Y. Liang; X. Yan; X. Yang; S.E. Stein Building a Comprehensive Reference Tandem MS Library of Peptides, Glycans and Glycopeptides for Therapeutic Antibodies

Material Measurement Lab Content NIST-evaluated spectra for 212,961 compounds Accompanying chemical name(s) and identifiers Chemical structures and retention information Validated software for spectrum 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 per year Integrated into instrument vendor software systems Common element in GC-MS ‘gold standard’ identifications Environmental Analysis Medical Research Homeland Security Forensics Chemical Manufacturing Food Analysis Biomolecular Measurement Division

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

Material Measurement Lab

# Precursor Ions 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

Material Measurement Lab Ion Trap & Collision Cell Spectra HCD/Orbi 45 eV Ion Trap TKPREEQY N STYR

Material Measurement Lab 58eV 65eV 72eV [M+2Na] 2+ Energy Dependence Collision Energy

Material Measurement Lab Content Spectra for 8 species, including human 324,352 human peptide ions (24% coverage of human proteome) compiled from >30M replicates Validated software for spectrum matching NIST Library of Peptide Ion Fragmentation spectra (SRD1C) NIST Library of Peptide Ion Fragmentation spectra (SRD1C) Reference Standard tandem Mass Spectra for peptide and protein measurements Usage Most widely used peptide tandem MS database Freely distributed and accessible on the web Integrated into Thermo Scientific TM Proteome Discoverer software Compatible with many open-source projects Biomedical Research Protein Identification Characterization of Protein Modifications Bio-manufacturing Homeland Security Biomolecular Measurement Division

Material Measurement Lab Peptide Library Pipeline Raw Data Translate, Annotate XTandem Prospector OMSSA Comet Integrate, Class FDR Consensus Spectrum Library Build Library

Material Measurement Lab peptide.nist.gov NISTMSQC: Full Analysis of LC-MS/MS data Library/quality metrics “Performance Metrics for Liquid Chromatography-Tandem Mass Spectrometry Systems in Proteomics Analyses”, Molecular & Cellular Proteomics, 9, 225, 2010

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?

Material Measurement Lab Why Libraries? Contain ‘fingerprints’ of previously identified components in a sample (digest) – Store, organize, annotate, extend and re-identify Identify all known components – Independent of settings Length, MCs, charge, protease, modification, … – More reliable (use peak intensities and priors) Common resource among different labs – Data standard

Material Measurement Lab David M. Hambly, Douglas D. Banks, Joanna L. Scavezze, Christine C. Siska, and Himanshu S.G Anal. Chem. 2009, 81, 7454–7459 Detection and Quantitation of IgG 1 Hinge Aspartate Isomerization: A Rapid Degradation in Stressed Stability Studies UV intensities MS masses

Material Measurement Lab Challenges with Improvements in Instrument Performance With increase in sensitivity and mass accuracy/resolution: – More components with increasing detail revealed Document in library – view and re-use All components may be ‘annotated’ – What are they? Are they significant? “Truth in Labeling” Libraries can hold this information in reusable format

Material Measurement Lab Single Protein Digest Library All identifiable, recurring products in the digest of 1 protein Peptides – Use available MS ‘sequencing’ methods for tentative IDs – Employ wide range of digestion conditions – Apply filters to reject ‘homologies’ – For each peptide store: Spectrum (ion trap and collision cell fragmentation) Occurrence frequencies, intensity, retention Glycopeptides – Identify by Oxonium and Loss-Patterns - then extract Glycans – From known samples: See Poster (Lorna De-Leoz)

Material Measurement Lab Start with Human Serum Albumin (HSA)

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

Material Measurement Lab Six Filters FilterData TypeDescriptionRejection Threshold 1 Peptide classPeptide identityLow prior probability >= two or more unusual classes 2 Ion SignificanceMS1 Median relative abundance and peptide ion identification frequency, PIF PIF ≤ m/z ErrorMS1Median m/z deviation >=0.25 m/z for LTQ >=5 ppm for Orbitrap 4 Unidentified Fragment Ions MS2 Unidentified fraction of all and top 20 abundances and number of peaks Overall Score >= 0.28 Intensity Only Score >= Sufficient Ions Above the Precursor m/z MS2 Fraction of ions and abundance above precursor m/z Fraction of ions * fraction of abundance above the precursor m/z <=0.1 6 Unexpected Charge State Ion charge Number of basic units, NBU vs charge state, CS |NBU-CS|>0

Material Measurement Lab Biological Modifications Methionine Oxidation – ETYGEMADCCAK Cysteinylation – ALVLIAFAQYLQQCPFEDHVK Phosphorylation – TCVADESAENCDK Acetylation – LKCASLQK

Material Measurement Lab Some Analytical Modifications Methionine Oxidation – ETYGE M ADCCAK Glutamine Deamination – QTALVELVK Pyro-carbamidomethyl – C CTESLVNR Underalkylation – C C TESLVNR Overalkylation – LVNEVTEFAK Transpeptidation – TPVSDR R Iron – AAF TE CCQAADK Sodiation – LVN E VTEFAK Carbamylation – S LHTLFGDK Tris-derived N-term (=70) – Method/Sample Dependent

Material Measurement Lab Indigestible Peptides Hyper-missed cleavage peptides – DVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEK/4+ – NYAEAKDVFLGMFLYEYARRHPDYSVVLLLR/4+ – QNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSR/5+ Formed quickly, not affected by digestion time

Material Measurement Lab IgG Modifications Large-Scale Identification and Quantification of Covalent Modifications in Therapeutic Proteins – “In an LC/MS/MS analysis of a tryptic digestion of an IgG2 mono-clonal antibody, 1712 peptide ions … and 227 modifications were identified …” Zhongqi Zhang, Anal. Chem. 2009, 81, 8354–8364 From protein and processing – Most from processing

Material Measurement Lab IgG Tryptic Peptide Libraries Six Commercial Drugs – 1 Constant Region, Individual Variable Region Libraries Cetuximab Example – Spectra for 975 Different Ions – 156 Simple Tryptics – 175 Modifications – 200 ‘In-sample’ Semitryptics – 169 ‘In-source’ Semitryptics – 101 Unexpected Missed Cleavage – 41 Under-/Over-alkylation – 133 Unidentified Modifications

Material Measurement Lab Cetuximab Modifications ModificationSiteDifferent Ions CarbamylN-term, K32 OxidationM, H, W27 Cation:NaD, E25 Gln->pyro-GluQ at N-term12 Cation:CaD, E12 FormylN-term, K, S, T10 DehydrationD8 Pyro-carbamidomethylC at N-term7 TranspeptidationArg and Lys at N or C-term6 Glu->pyro-GluE at N-term2 DeamidationN, Q2

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

Material Measurement Lab Glycopeptide Features NEUTRAL MONOSACCHARIDE LOSSES OXONIUM IONS TheoryOur addition m/z m/z m/z m/z m/z m/z m/z m/z (2H 2 O+CH 2 O) m/z H 2 O m/z H 2 O Oxonium ion peaks Threshold: ~60ppm Neutral Losses Threshold: ~20ppm All z=2 35eV HCD MS 2 Hexose N-acetylhexosamine Deoxyhexose (e.g. fucose)Sialic Acid (e.g. NeuAc)

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

Material Measurement Lab

Vary Digestion Conditions

Material Measurement Lab Other Proteases

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)

Material Measurement Lab Rituximab Glycan Library Head to Tail Comparison G LYCAN G0F FOUND IN R ITUXIMAB S AMPLE

Material Measurement Lab SRD Standard Reference Materials – Target Analytes (Cholesterol, Vitamin D) Often in Matrix (urine, plasma, …) – Whole Materials (Plasma Metabolites, …) Identify and value-assign many components Standard Reference Data – MS Library, Thermodynamic Data, Chemistry Webbook, … Standard Reference Materials + Data – Whole material + Data + Libraries SRD + SRM SRM/D

Material Measurement Lab SRM/D - Metabolites in PlasmaFuture: SRM/D IgG

Material Measurement Lab NIST MS Data Center Lab Computer