Building a Comprehensive Reference Tandem MS

Library of Peptides, Glycans and Glycopeptides

for Therapeutic Antibodies
Material Measurement Laboratory

Q. Dong; M. Lorna A. De Leoz; L.E. Kilpatrick; Y. Liang; X. Yan; X. Yang; S.E. Stein

Material Measurement Lab

 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:

• Environmental Analysis • Forensics

• Medical Research • Chemical Manufacturing
• Homeland Security • Food Analysis
Content

• NIST-evaluated spectra for 212,961 compounds

• Accompanying chemical name(s) and identifiers
• Chemical structures and retention information
• Validated software for spectrum matching

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
Biomolecular Measurement Division

Material Measurement Lab

 Ion Fragmentation is Reproducible

O’Neal et al.
Anal. Chem.
1951

NIST
2012
Hexadecane

A mass spectrum is an energy-dependent property of an ion

Material Measurement Lab

Material Measurement Lab
 NIST Tandem Mass Spectral Library 2012
16,000 2012
# Precursor Ions Compounds 7,020
14,000
2011 Precursor Ions 15,517
12,000
Spectra 123,781
10,000
8,000 Fragmentation Type Precursor Ions
6,000 2008 Ion Trap >10,000
4,000 Beam Collision Cell >8,000
2,000 2005 (QTOF, QQQ, HCD)
0

Classes: New Software Features:

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.
• 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.

Material Measurement Lab

Ion Trap & Collision Cell Spectra

HCD/Orbi
45 eV
TKPREEQYNSTYR

Ion Trap

Material Measurement Lab

 Energy Dependence
Collision
Energy [M+2Na]2+

58eV

65eV

72eV

Material Measurement Lab

 NIST Library of Peptide Ion Fragmentation spectra (SRD1C)
Reference Standard tandem Mass Spectra for peptide and protein measurements

• Biomedical Research • Bio-manufacturing

• Protein Identification • Homeland Security
• Characterization of Protein
Modifications

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

Usage

• Most widely used peptide tandem MS database

• Freely distributed and accessible on the web
• Integrated into Thermo ScientificTM Proteome
Discoverer software
• Compatible with many open-source projects
Biomolecular Measurement Division

Material Measurement Lab

Peptide Library Pipeline
Raw Data

Translate, Annotate

XTandem OMSSA Prospector Comet

Integrate, Class FDR

Consensus Spectrum

Build Library

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

Small Molecule
Peptide Library Tandem MS
500,000 peptides 7,020 compounds
for 8 species Ion Trap/Collision Cell
IgG Library
with NIST
reference
material

peptides glycans
glycopeptides
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 OMSSA Prospector Comet InSpect TagRecon

Integrate, Class FDR

Consensus Spectrum

MS1, MS2, RT

Six Filters

Build Library

Library

Material Measurement Lab

 Six Filters
  Filter Data Type Description Rejection Threshold
>= two or more unusual
1 Peptide class Peptide identity Low prior probability
classes
Median relative abundance and
2 Ion Significance MS1 peptide ion identification PIF ≤ 0.01
frequency, PIF
>=0.25 m/z for LTQ
3 m/z Error MS1 Median m/z deviation
>=5 ppm for Orbitrap
Unidentified fraction of all and Overall Score >= 0.28
Unidentified
4 MS2 top 20 abundances and number Intensity Only Score
Fragment Ions
of peaks >= 0.1
Sufficient Ions Fraction of ions * fraction
Fraction of ions and abundance
5 Above the MS2 of abundance above the
above precursor m/z
Precursor m/z precursor m/z <=0.1
Unexpected Number of basic units, NBU vs
6 Ion charge |NBU-CS|>0
Charge State charge state, CS

Material Measurement Lab

Biological Modifications
• Methionine Oxidation
– ETYGEMADCCAK

• Cysteinylation
– ALVLIAFAQYLQQCPFEDHVK

• Phosphorylation
– TCVADESAENCDK

• Acetylation
– LKCASLQK

Material Measurement Lab

 Some Analytical Modifications
•Methionine Oxidation • Transpeptidation
– ETYGEMADCCAK – TPVSDRR

•Glutamine Deamination
– QTALVELVK
• Iron
– AAFTECCQAADK
•Pyro-carbamidomethyl
– CCTESLVNR • Sodiation
•Underalkylation – LVNEVTEFAK
– CCTESLVNR
• Carbamylation
•Overalkylation – SLHTLFGDK
– LVNEVTEFAK
• 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
Modification Site Different Ions
Carbamyl N-term, K 32

Oxidation M, H, W 27

Cation:Na D, E 25

Gln->pyro-Glu Q at N-term 12

Cation:Ca D, E 12

Formyl N-term, K, S, T 10

Dehydration D 8

Pyro-carbamidomethyl C at N-term 7

Transpeptidation Arg and Lys at N or C-term 6

Glu->pyro-Glu E at N-term 2

Deamidation N, Q 2

Material Measurement Lab

 IgG Digest Peptides
Library Consensus FNW(O)YVDGVEVHNAK/3+
Spectrum

Annotation

Material Measurement Lab

 Glycopeptide Features
OXONIUM IONS NEUTRAL MONOSACCHARIDE LOSSES
Theory Our addition 1+ 2+ 3+ 4+
m/z 163.0601 m/z 138.0550 -(2H2O+CH2O) 203.0794 101.5397 67.6931 50.7698
m/z 204.0867 m/z 168.0655 -2H2O
m/z 274.0921 m/z 186.0761 -H2O 162.0528 81.0264 54.0176 40.5132
m/z 292.1026
m/z 366.1387 146.0579 73.0289 48.6859 36.5144
m/z 454.1555 291.0954 145.5477 97.0318 72.7738
m/z 657.2349
Hexose N-acetylhexosamine
Deoxyhexose (e.g. fucose) Sialic Acid (e.g. NeuAc)

Fibrinogen_20x_CID_HCD_15_to_55 #18877 RT: 92.68 P: + NL: 9.36E4

T: FTMS + c NSI d Full ms2 1443.97@hcd35.00 [100.00-2000.00]
35eV HCD MS2
1691.2653
100
90 Neutral Losses
80 Threshold: ~20ppm
Relative Abundance

70 366.1377
60
Oxonium ion peaks All z=2
1610.2410

50 204.0858
Threshold: ~60ppm 81
40
101 81
81 81
30 168.0648
20 274.0909 1164.0836
81 1508.7065 101 1873.8231
657.2297
10 1346.6636 1836.8585
435.6248 611.8971 713.3240 828.4542 1010.1514 1127.8215 1912.7704
0
200 400 600 800 1000 1200 1400 1600 1800 2000
m/z

Material Measurement Lab

 18 hr - NIST Standard IgG
Guanidine/HT : HCD fragmentation

“Proteomics”1E11

1E10 Glycopeptide
Peptide
Sampled/No ID
Minor 1E9 Unsampled
Constituents,
Digest
Quality 1E8

1E7

0 10 20 30 40 50 60 70 80 90 100 110
Retention/min
Material Measurement Lab
 Guan/RT 18 hr/Trypsin

DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
20 Length=63, 6 charge states
3 MC in 19 charge states

15
Semi-Tryptic
MC mid
MC end
Tryptic
Intensity

10

SLSLSPG

0
100 200 300 400 SLSLSPGK

Material Measurement Lab

 Guanidine/RT-Trypsin
20

15
18 hr EYKCKVSNK
10
VDKRVEPK TISKSK
5

0
25 100 200 300 400

20

15
2 hr Semi-Tryptic
10 MC mid
MC end
5 Tryptic
0
25 100 200 300 400

20

15
15 min
10

0
100 200 300 400

Material Measurement Lab

Vary Digestion Conditions
35

30
25
Urea/Trypsin
18 hr
20
15

10
5
0
30 100 200 300 400

25

20 Semitryptic
2 hr MC Mid
15
MC End
10 Tryptic
5

0
30 100 200 300 400

25

20 15 min
15

10

0
100 200 300 400

Material Measurement Lab

 Other Proteases
25
20
15
10
2 hr Trypsin
5
0
5
10
15
20
25 2 hr AspN
30
35
100 200 300 400

Position
Material Measurement Lab
 NGA42 NGA2F1 Starter
G0 G0F
Glycan
NGA3B1,2 NA2G1F1
MS/MS
G0B G1F
Library
Typical Precursor ions
A1F1 NA2F1  [M+2H]2+
G2FS G2F  [M+H+K]2+
 [M+H+Na]2+
 [M+2Na]2+
Additional precursor ions
A2F 1
NGA2B 2
for sialylated glycans
G2FS2 G0B  [M+2Na-4H]2-
 [M+K-3H]2-
 [M-2H]2-
 [M+Na+K-4H]2-
NGA21 NGA4B2 Fragmentation Methods
 Ion Trap (Resonant)
G0 G0B  ‘HCD’ at 12 energies (5-70eV)

Purchased; 2From the Consortium for Functional Glycomics through Dr. Jim Paulson
1

Material Measurement Lab

GLYCAN G0F FOUND IN RITUXIMAB SAMPLE

Rituximab

Head to Tail Comparison

Glycan Library

Material Measurement Lab

 SRM/D
SRD + SRM
SRD
• Standard Reference Materials
Data + Data
– Whole material
MS Library, + Data + Libraries
Thermodynamic Data, Chemistry
Webbook, …

• Standard Reference Materials

– Target Analytes (Cholesterol, Vitamin D)
• Often in Matrix (urine, plasma, …)
– Whole Materials (Plasma Metabolites, …)
• Identify and value-assign many components

Material Measurement Lab

SRM/DFuture:
- Metabolites
SRM/D inIgG
Plasma

http://srm1950.nist.gov
http://IgG.nist.gov

Material Measurement Lab

NIST MS Data Center

Lab

Computer

Material Measurement Lab