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Informatics for proteomic inventories. [email protected] Biomedical Informatics Vanderbilt University. Overview. Explaining the whys and hows of proteomics Matching peptides from protein sequence databases to MS/MS spectra

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informatics for proteomic inventories

Informatics for proteomic inventories

[email protected]

Biomedical Informatics

Vanderbilt University

  • Explaining the whys and hows of proteomics
  • Matching peptides from protein sequence databases to MS/MS spectra
  • Filtering peptide-spectrum matches (PSMs) to an acceptable false discovery rate (FDR)
  • Inferring proteins parsimoniously and scalably
methods capture only part of story
Methods capture only part of story

Genomics and epigenetics describe state of “catalog.”

Transcriptomics describes current “purchase orders.”

Proteomics measures current inventory of cell capabilities.

Metabolomics examine cell state most directly.

J_Alves: glycinetRNA

J_Alves: glucose and cholesterol

ElaineMeng: H-ras, PDB 121P

what does proteomics include
What does proteomics include?





1D and 2D Gel






Gerald_G scales, Gsagri04: gel,

AB SCIEX tissue image

discovery proteomics
Discovery Proteomics












Mass Spectrometry


Ions of Peptide

Collide Ions to


Collect Fragments

in Tandem MS

Two types of measurements for each peptide: intact m/z (mass/charge) and a list of fragment m/zs.

collision induced dissociation cid
Collision-induced dissociation (CID)
  • “Tickle” energizes peptide, causing varied conformations and proton movement.
  • A mobile proton associates with a carbonyl adjoining a peptide bond, drawing electrons.
  • Electrons of the prior carbonyl attack, forming a ringed intermediate that quickly dissociates.

Wysocki et al, Anal. Chem. (2000) 35: 1399-406.

Paizs and Suhai, Rapid Comm. Mass Spectrom. (2002) 16: 1699-1702.

broken peptide bonds yield fragments
Broken peptide bonds yield fragments



b4 ion


y6 ion

hfiselek 2 charge state
HFISELEK, +2 charge state

Neutral loss of

water from peptide






same spectrum compared to fheikels instead of hfiselek
Same spectrum compared toFHEIKELS instead of HFISELEK

Neutral loss of

water from peptide

-EIKELS has same

mass as -ISELEK

FH- has same

mass as HF-

disassembly and reassembly
Disassembly and reassembly

After AI Nesvizhskii, Mol Cell Proteomics (2005) 4: 1419-40.

database search overview
Database search overview

Eng et al (1994) J. Amer. Soc. Mass Spectrom. 5: 976-989.

Yates et al (1995) Anal. Chem. 67: 1426-1436.

emulating proteases in silico
Emulating proteases in silico

N Edwards and R Lippert. Lecture Notes In Computer Science (2002) 2452: 68-81.

dynamic ptms grow search space
Dynamic PTMs grow search space

Because multiple PTMs may be in each peptide, adding PTMs to a search creates an exponential cost.

Here, three sites lead to eight PTM variants.


peptide mass filter
Peptide mass filter
  • Sequences outside mass tolerance are not compared.
  • Many sequences may share a common mass.
  • Sequences of one mass may score differently.
  • Sequences of different mass may score the same.
fragment masses and charge segregation
Fragment masses andcharge segregation



































sequest cross correlation
Sequest cross correlation
  • Normalize observed spectrum.
  • Generate model spectrum for each candidate.
  • Convert observed and model spectrum to frequency domain by FFT.
  • Cross-correlate, reporting ratio between zero-offset alignment and nearby alignments.

J Eng et al. J. Proteome Res. (2008) 7: 4598-4602.

J Eng et al. J Amer. Soc. Mass. Spectrom. (1994) 5: 976-989.

x tandem scoring
X!Tandem scoring
  • Predict more accurate fragment intensities
  • Count matched b ions and matched y ions
  • Compute dot product of intensities
  • Generate hyperscore =
  • Build histogram of scores per spectrum
  • Report expectation value

Craig and Beavis. Rapid Comm. Mass Spectrom. (2003) 17:2310-2316.

Fenyö and Beavis. Anal. Chem. (2003) 75: 768-774.

random match probabilities
Random match probabilities
  • Imagine spectrum as jar of 100 black and 900 white marbles (peaks and voids).
  • Sample 20 marbles for a predicted peaklist, drawing 15 black and 5 white.
  • Compute probability of random match by hypergeometric distribution:

T Fridman. J. Bioinfo. Computat. Bio. (2005) 3: 455-476.

disassembly and reassembly1
Disassembly and reassembly

After AI Nesvizhskii, Mol Cell Proteomics (2005) 4: 1419-40.

the longest list problem
The “longest list” problem
  • Perceived value of early proteomics experiments was linked only to sensitivity.
  • Systems to evaluate specificity lagged behind, and false positive rates were left unchecked.
  • Two developments were needed:
    • Community consensus on reporting standards
    • New tools for evaluating identification error rates

Carr et al. Mol. Cell. Proteomics (2004) 3: 531-533.

Taylor et al. Nature Biotech. (2007) 25: 887-893

strategy i target decoy estimates fdr
Strategy I: Target/decoy estimates FDR
  • Sequence database has equal numbers of target and decoy sequences.
  • False IDs distribute evenly between target and decoy sequences.
  • Apply a threshold, and:
    • False estimate = 2 x [decoy hit count].
    • False Discovery Rate (FDR) = False estimate divided by number of passing IDs.

Elias and Gygi.

Nature Methods

(2007) 4: 207-214

decoys model false distribution
Decoys model false distribution
  • A match to targets is possibly true; a match to decoys is surely false.
  • As threshold slides to lower scores, more decoys are kept, escalating FDR.
  • Alternatively, may be used if decoys are excluded from final list.

Elias Nat. Methods (2007) 4: 207-214

strategy ii peptide prophet
Strategy II: Peptide Prophet
  • Estimates correctness probability for individual identifications
  • Combines multiple subscores from each Sequest identification through DFA
  • Fits mixed model to observed matches with expectation maximization
  • A Keller. Anal. Chem. (2002) 74: 5383-5392.
mixture model analysis separates true and false distributions
Mixture Model analysisseparates true and false distributions
  • Expectation maximization adjusts two curves to fit observed data.
  • Here, negatives are fit to a gamma distribution and positives to a normal distribution.
disassembly and reassembly2
Disassembly and reassembly

After AI Nesvizhskii, Mol Cell Proteomics (2005) 4: 1419-40.

why are peptides shared among proteins
Why are peptides sharedamong proteins?

“Orthologs are direct evolutionary counterparts derived from a common ancestor through vertical descent; whenever we speak of the ‘the same gene in different species,’ we actually mean orthologs. In contrast, paralogs are genes within the same genome that have evolved by duplication.”

Koonin. Genome Biology (2001) 2: comment 1005.1-1005.2.

protein isoforms
Protein isoforms
  • A single gene may give rise to many transcripts that overlap for one or more exons.
  • When isoforms are listed as separate proteins in the FASTA, a peptide may match a shared or distinctive part of a protein sequence.
  • VEGF incorporates eight exons, where either 6 or 7, both, or neither may be incorporated.
  • noun: “economy of explanation in conformity with Occam's razor”
    • Merriam Webster OnLine
  • “Plurality ought never be posed without necessity.”
    • William of Occam
  • Assemble maximal protein list.
  • Combine proteins that point to the same peptides, and combine peptides that point to the same proteins.
  • Find “set cover” by greedy algorithm to pick minimal protein list to explain peptides.

B Zhang et al. J. Proteome Res. (2007) 6: 3549-3557.

Z Ma et al. J. Proteome Res. (2010) 8: 3872-3881.

two proteins or seven
Two proteins or seven?
  • Sample mixes mouse and human proteins.
  • Isoforms, paralogs, and orthologs complicate protein-peptide map.
  • Untangling relationships is non-trivial.

Data from Broad Institute, CPTAC

greedy algorithm
Greedy algorithm

Data from Broad Institute, CPTAC

  • Combine peptide identification probabilities into protein identification probabilities.
  • Distribute probability for shared peptides across multiple proteins.
  • Compute protein probability by subtracting probability that all observed peptides are false from 1.
    • AI Nesvizhskii. Anal. Chem. (2003) 75: 4646-4658.
number of sibling peptides and degenerate peptides
Number of Sibling Peptides and Degenerate Peptides
  • NSP places more confidence in peptides for proteins with abundant supporting evidence.
  • Degenerate peptides match multiple potential proteins, each associated with a weight.
  • Expectation maximization determines weights that minimize proteins count and maximize protein probability.
parsimony reduces protein lists
Parsimony reduces protein lists

Maximal list

Grouping indiscernibles

Grouping + parsimony

SwissProt HUMAN

International Protein Index

SwissProt Multispecies

Zhang et al. J. Proteome Res. (2007) 6: 3549-57.

protein fdr is not psm fdr
Protein FDR is not PSM FDR
  • PSM FDR fixed at 3%
  • Two distinct peptides required per protein
  • True PSMs group together on true proteins.
  • False PSMs spread across the database.

Data from Broad Institute, CPTAC

takeaway messages
Takeaway messages
  • Tandem mass spectrometry produces lists of fragment m/z values and precursor masses.
  • Database search narrows the set of all possible peptides to plausible candidates.
  • Controlling peptide and protein FDR is essential for credible, publishable inventories.
  • Parsimony and scalable filtering are necessary to field modern data sets.