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Novel Peptide Identification using
ESTs and Genomic Sequence
Nathan Edwards
Center for Bioinformatics and Computational Biology
University of Maryland, College Park
Novel Peptides
• Absent from traditional protein
sequence databases
• IPI, SwissProt, TrEMBL, NCBI’s nr, MSDB
• Due to
• Deliberate “redundancy” elimination
• “Dark-side” genes
• Bias towards high-quality, high-confidence
full-length protein sequence
2
What is missing?
• Known coding SNPs
• Novel coding mutations
• Alternative splicing isoforms
• Alternative translation start-sites
• Microexons
• Alternative translation frames
3
Why should we care?
• Alternative splicing is the norm!
• Only 20-25K human genes
• Each gene makes many proteins
• Proteins have clinical implications
• Biomarker discovery
• Evidence for SNPs and alternative splicing
stops with transcription
• Genomic assays, ESTs, mRNA sequence.
• No hard evidence for translation start site
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Novel Protein
HEQASNVLSDISEFR
Evidence:
• log10(E-value) = -9.6
• 100’s of ESTs
• Full length mRNA sequence
Details:
• Peptide Atlas A8_IP (Resing et al.);
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Novel Protein
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Novel Protein
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Novel Protein
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Novel Splice Isoform
LQGSATAAEAQVGHQTAR
Evidence:
• log10(E-value) = -6.8
• 10’s of ESTs
• Full length mRNA sequence
Details:
• Peptide Atlas raftflow (von Haller, et al.);
• LIME1 gene
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Novel Splice Isoform
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Novel Splice Isoform
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Novel Splice Isoform
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Novel Frame
TAGSPLCLPTPGAAPGSAGSCSHR
Evidence:
• log10(E-value) = -3.9
• 10’s of ESTs
• Full length mRNA sequence
Details:
• Peptide Atlas raftflow (von Haller, et al.);
• LIME1 gene, downstream from LQGSA...
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Novel Frame
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Novel Frame
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Novel Frame
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“Novel” Microexon
LQTASDESYKDPTNIQLSK
Evidence:
• log10(E-value) = -6.4
• 10’s of ESTs / mRNA sequences
• SwissProt variant, absent from IPI
Details:
• Peptide Atlas raftflow (von Haller, et al.);
• SPTAN1 gene
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“Novel” Microexon
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“Novel” Microexon
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“Novel” Microexon
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“Novel” Microexon
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Novel Mutation
KADDTWEPFASGK
Evidence:
• log10(E-value) = -7.6
• 2 ESTs from same clone library
• Ala2 Deletion
Details:
• HUPO PPP 29_b1-EDTA_1 (Qian/He; Omenn et al.);
• TTR gene
• Known Mutation: Ala2-to-Pro associated with
familial amyloidotic polyneuropathy.
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Novel Mutation
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Novel Mutation
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Novel Mutation
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Novel Mutation
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Known Coding SNP
DTEEEDFHVDQ[V|A]TTVK
Evidence:
• log10(E-value) = -9.5 / -9.4
• Known dbSNP (coding): Val12-to-Ala
• Wildtype also observed
Details:
• HUPO PPP 40 (Wang; Omenn et al.);
• SERPINA1 gene
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Wildtype
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Known Coding SNP
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Known Coding SNP
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Known Coding SNP
LQHL[E|V]NELTHDIITK
Evidence:
• log10(E-value) = -6.7/-10.9
• 4 ESTs, same clone library
• Known dbSNP (coding): Glu5-to-Val
• Wildtype also observed
Details:
• HUPO PPP 28_b2-CIT
(Pounds/Adkins/Rodland/Anderson; Omenn et al.);
• SERPINA1 gene
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IPI Common Variant
Elimination
YYGGGYGSTQATFMVFQALAQYQK
Evidence:
• log10(E-value) = -5.9
• 100’s ESTs, mRNA sequence
• IPI has (rare) variant (Insertion of AS@10)
• Differ in 5’ splice site.
Details:
• HUPO PPP 29 (Qian/He; Omenn et al.);
• C3 gene
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Why don’t we see more
novel peptides?
• Tandem mass spectrometry doesn’t
discriminate against novel peptides...
...but protein sequence databases do!
• Searching traditional protein sequence
databases biases the results towards
well-understood protein isoforms!
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Why don’t we see more
novel peptides?
• Traditional protein sequence
databases
• High-quality, full-length proteins only
• Many interesting peptides are omitted
• Exclusive – peptide identifications are lost.
• ESTs, genomic & mRNA sequence
• Used as evidence for full-length protein
sequences
• Inclusive – may need to filter results
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Significant False Positives
• E-values are not enough!
• Random guessers are easy to beat.
• Post-translational modifications vs. amino-acid
substitution
• methylation (on I/L, Q, R, C, H, K, S, T, N): +14
• D → E, G → A, V → I/L, N → Q, S → T: +14
• Peptide extension z=+2 → z=+3
• Nonsense AA masses sum to precursor
• Need to ensure:
• fragment ions define novel sequence
• sequence evidence is strong
• other plausible explanations can be eliminated
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Significant False Positives
• DFLAGGLAAAISK
2.2x10-8
• DFLAGGIAAAISK
2.2x10-8
• DFLAGGVAAAISK
3.7x10-8
• 2 ESTs
• IPI (2), RefSeq, mRNA, ~ 1400 ESTs
• IPI, RefSeq, mRNA, ~700 ESTs
• DFLAGGVAAAISKMAVVPI 3.5x10-5
• Genscan exon
• AISFAKDFLAGGIAAAISK
• Genscan exon
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3.3x10-4
Significant False Positives
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How do we know they are
novel?
• How do we know they are real?
•
•
•
•
Good spectra
Good E-value
Good ion ladders
Good sequence evidence
• Lack of other explanations...
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Peptide Sequence Evidence
Gb of Sequence
Self Corrected ESTs (1)
Genome Corrected ESTs (2)
Corrected ESTs (1+2)
Genscan Exons (3)
Genscan Exon Pairs (4)
Combo (1+2+3+4)
Genomic ORFs (5)
Naïve Enumeration
7.60
2.90
5.40
1.30
13.00
4.20
0.10
0.06
2.30
1.60
28.40
10.06
6.20
1.90
• C3 Compression:
• Amino-acid 30-mers
• Complete, Correct(, Compact)
• Present at least twice (ESTs only)
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C3
0.18
0.12
0.20
0.05
0.55
0.78
1.50
SBH-graph
ACDEFGI, ACDEFACG, DEFGEFGI
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Compressed-SBH-graph
1
2
2
1
2
ACDEFGI
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Peptide Sequence Databases
• MS/MS search engine input only
• Protein context is lost
• Inclusive, rather than exclusive
• Download from http://www.umiacs.umd.edu/~nedwards
• Exact string search for gene/protein context
• Recover peptide sequence evidence
• Relational database to reassemble...
...with respect to genes & genome
• Grid Computing + Web Services + Viewer
• Work in progress
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Peptide Identification Navigator
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Peptide Identification Navigator
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Conclusions
• Peptides identify more than proteins
• Search EST sequences (at least)
• Compressed peptide sequence
databases make this feasible
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