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A Reference Library of Peptide Ion Fragmentation SpectraA Reference Library of Peptide Ion Fragmentation Spectra
Stephen SteinStephen Stein11; Lisa Kilpatrick; Lisa Kilpatrick22; Pedatsur Neta; Pedatsur Neta11; Jeri Roth; Jeri Roth11; Xiaoyu Yang; Xiaoyu Yang11
National Institute of Standards and Technology, National Institute of Standards and Technology, 11Gaithersburg, MD/Gaithersburg, MD/22Charleston, SCCharleston, SCOverviewOverview
• PurposePurposeCreate comprehensive, annotated mass spectral libraries from
various organisms and selected proteins to identify peptides by matching their MS/MS spectra to reference spectra.
• MethodsMethods- Acquire ‘Shotgun’ proteomics data files from diverse sources.- Identify peptides with available sequence search engines.- For each peptide ion, create a ‘consensus spectrum’ from
replicate spectra; also find best single spectrum.- Derive reliability measures and remove ambiguities.
• ResultsResults- Spectrum libraries were built by matching both m/z’s and
intensities of MS/MS peaks.- Libraries derived from widely studied organisms such as human,
yeast, M. Smegamtis, D. Radiodurans, and standard proteins. - Consensus spectra derived from reliable peptide identifications.- Library indexing leads to very fast identification (<< 1 sec) even for
very large libraries.- Sequence identification by spectrum library searching identifies far
more spectra of known peptides than sequence library searching, can be 100 times faster and yields more robust and understandable results.
IntroductionIntroduction
High throughput proteomics requires automated, fast and accurate
library search engines to identify peptide sequences from acquired
MS/MS spectra. Current peptide identification methods match each
measured MS/MS spectrum against a coarse ‘theoretical’ spectrum of
each possible peptide sequence. Since relative abundances, neutral
losses from parent and product ions, and ratios of products having
different charge states are not predictable, this rich, peptide-specific
information is not effectively used for establishing identity. Also, prior
occurrence information is ignored – each search identifies the peptide
as if for the first time. A spectrum library search matches not only the
m/z, but also the relative intensities of the MS/MS peaks and can make
use of other prior information. However, spectrum libraries can
propagate errors, so reliable searching requires high quality reference
libraries, the development of which is described here. We find that
identifying peptides by matching their MS/MS spectra to reference
spectra can be faster, more reliable and more informative than current
sequence-based methods.
MethodsMethods1. Acquire and organize ‘Shotgun’ proteomics data files from
diverse sources.
Human5347 LC-MS/MS data files from 11 labs and repositories
Boston U. (Steffen/Ahmad) GPM (Beavis)HUPO/Plasma Proteome Project/OmennHUPO/Brain Proteome Project/Meyer (not yet published)ISB/PeptideAtlas (Deutsch/King/Aebersold/…)NCI-SAIC (Veenstra) PNNL/NCRR (Smith), UC Davis (Rice/Lee)Q-tof data from USB (Pannell) and Mayo Clinic (Muddiman)
Yeast2503 LC-MS/MS data files from 12 laboratories
Online repositoriesPeptideAtlasOpen Proteomics Database
Collaborators/ContributorsBlueprint Initiative (Hogue)Harvard University (Gygi)ISB –PeptideAtlas (Deutsch/King/Aebersold/…)NIH/LNT (Markey/Maynard/Geer/Kowalak/…)University of Arizona (Haynes)University of San Francisco (Burlingame/Baker)
NIST Test Measurements
Mycobacteria Smegmatis
253 LC-MS/MS data files from the Open Proteomics Database online repository
Deinococcus Radiodurans
495 LC-MS/MS data files from the PNNL/NCRR Repository.
Standard Proteins
19 Proteins were analyzed in NIST laboratories by LC-MS/MS.
2. Identify peptides with available sequence search engines
Different search engines often give very different scores for matching a given peptide ion with a single spectrum (figure bottom left panel). To capture the largest number of identifications, the highest score of up to four different search engines was used. This increased the number of reliable identifications by over 25% compared to any single method.
thresholds
Probably wrong
Probablyright
Probably right
Confirmed
3. Create consensus spectrum and find best replicate spectrum
For all spectra matching a given peptide ion, a multi-step process aligns m/z peaks, rejects outliers and creates a consensus spectrum. It also finds the best replicate spectrum based on search engine scores and spectrum quality. A peak in a consensus spectrum must be present in a majority of the spectra that might have generated the peak.
5. Remove ambiguities and build libraryCreate annotated spectra for consensus and best matching single spectra. Resolve problems of similar spectra that appear to generate different peptide ions.
ResultsResults
• Libraries were built from different organisms.
Peptide Class # Peptides
Consensus 35,807
Singular (one ID) 2,458
Simple Tryptic 24,205
Tryptic Missed Cleavage 5,620
Semi Tryptic 5,982
1+ 3,658
2+ 22,327
3+ 9,822
ICAT 15,061
Yeast
Peptide Class # Peptides
Consensus 43,601
Singular (one ID) 1,864
Simple Tryptic 36,447
Tryptic Missed Cleavage 7,127
1+ 3,677
2+ 30,194
3+ 9,730
ICAT 6,640
Human
Peptide Class # Peptides
Consensus 3,562
Singular (one ID) 126
Simple Tryptic 3,252
Tryptic Missed Cleavage 254
Semi Tryptic 56
1+ 111
2+ 2,130
3+ 1,287
M. Smegmatis
Peptide Class # Peptides
Consensus 8,809
Singular (one ID) 284
Simple Tryptic 6,050
Tryptic Missed Cleavage 2,486
Semi Tryptic 273
1+ 1,816
2+ 5,168
3+ 1,799
D. Radiodurans
Peptide Class # Peptides
Consensus 4,095
Singular (one ID) 15
Simple Tryptic 2,097
Tryptic Missed Cleavage 1,555
Semi Tryptic 443
1+ 663
2+ 1832
3+ 1320
4+ 245
5+ 35
Standard Proteins
4. Derive reliability measures for each spectrum
A) Spectrum/Sequence Consistency
• Match theoretical spectrum, based on relative dissociation rates of adjacent amino acids (from statistical analysis of reliable spectra). Discrimination shown at right
• Fraction of unassigned abundance (peaks not originating from a known fragmentation path)
• Y/B ion continuity and Y/B correlation
B) Peptide Sequence Confirmation
• Other peptide ions with same sequence (different charge state or modification)
• Sequence contained in (or contains) another peptide
• Number of peptides per protein / protein length
C) Peptide Class (for setting acceptance threshold)
• Tryptic or semiTryptic
• SemiTryptic – In source or unexpected
• SemiTryptic – Confirmed or unconfirmed
• Missed Cleavages: None or explained, or unexplained
• Missed Cleavages: Confirmed (found contained peptide) or unconfirmed
These libraries depend oncontributors for their success.
Please contribute.All spectra cite contributors.
• Spectrum searching identifies peptides fast and reliably.Algorithms: Spectrum similarity scores have been adapted from algorithms used for electron ionization spectra. Peaks are weighted by their significance:
- Reduce significance of common impurity ions (e.g., neutral loss from parent ion)
- Reduce weight for uncertain and isotopic peaks
- Use library spectrum reliability
- Fold in sequence score for instrument dependence – uses OMSSA scoring
Speed: Straightforward indexing leads to very fast identification (<< 1 sec) even for very large libraries.
Robustness: Spectrum match scores are less sensitive to spectral details than sequence scores (see figure below, left).
Contact: Steve SteinDirector, Mass Spectrometry Data CenterNational Institute of Standards and [email protected] 301-975-2505
Input list
Matching peptide and probability scores
Reference spectrum and annotation
Query MS/MSHead to tail sample and reference spectra comparison
• Three library formats: - Simple ASCII ‘msp’ format (derived from EI MS Library) - NIST Search Software (Windows, see figure below) - Dynamic Link Library (Source & Binary)
Small Missing Peaks Can Have A Big Effect on Sequence Scores
Sequence Search Score
• Spectrum library performance:Several times as many spectra identified by searching against spectra than against sequence (left panel, bottom right)Test Set: Yeast analysis files from the Open Proteomics Database (OPD40, 12 LC-MS/MS runs).Spectrum Library: Consensus spectra in current yeast library. Radiodurans library for false ids.Sequence Library: Search forward and reverse yeast library using relative homology scores or expectation values. Search Speed: Spectrum searching was about 100 times faster than sequence searching. May be accelerated by more peak indexing.
• A reference spectrum library provides a sensitive, reliable, fast, and comprehensive resource for peptide identification.
• A peptide mass spectrum library can be used for:• Direct peptide identification• Validating peptides identified by sequence search programs • Organizing and identifying recurring, unidentified spectra.• Sensitive, high reliability detection of internal standards, biomarkers, and target proteins• Subtracting a component from a mixture spectrum
ConclusionConclusion
Current sequence search methods yield divergent scores for the same spectrum due to use of incomplete spectrum information.
CollaboratorsCollaboratorsN. King et al, ISB - “Annotation of the Yeast Proteome with PeptideAtlas” (Poster WP 27/522)
H. Lam et al., ISB – “SpectraST: An Open-Source MS/MS Spectra-Matching Library Search Tool for Targeted Proteomics” (poster WP27/530)
L. Geer et al, NIH “Reducing false positive rates in MS/MS sequence searching and incorporating intensity into match based statistics” (Poster TP34/638)
HUPO PPP and BPP Projects
Repositories and dozens of labs who directly and indirectly provided MS/MS data for public use
Similarity of Measured vs. Theoretical Spectra (Dot Product x 100)