Lian Yang 2 Baozhen Shan 1 Bin Ma 2 1 Bioinformatics Solutions Inc Canada 2 University of Waterloo Canada Problem Complete protein sequence coverage antibody confirmation ID: 931436
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Slide1
In-depth Analysis of Protein Amino Acid Sequence and PTMs with High-resolution Mass Spectrometry
Lian Yang2; Baozhen Shan1; Bin Ma21Bioinformatics Solutions Inc, Canada 2University of Waterloo, Canada
Slide2Problem
Complete protein sequence coverageantibody confirmationbiomarker discovery Database search software along is insufficientProtein sequence analysis
Slide3Possible reasons for incomplete coverage
“non-database” peptidesunexpected modificationsmutated residuesnovel peptidedatabase errors Meanwhile Large amount of high-quality spectra are not matched.
Protein sequence analysis
Slide4A workflow to identify
both the database and “non-database” peptidesObjectiveMaximize protein sequence coverageExplain more high-quality MS/MS spectraProposed workflow for in-depth analysis
Slide5Workflow
Proposed workflow for in-depth analysis
Multiple enzyme
Multiple
protein digests with different
enzymes
High accuracy MS
for both
precursor
and
fragment
ions
Slide6Workflow
Proposed workflow for in-depth analysisPEAKS: powerful software for peptide de novo sequencing by tandem mass spectrometry. Rapid Commun Mass Spectrom
. 2003;17(20):2337-42.
Multiple enzyme
Identify
de novo
sequence tags
Reveal
a set of high quality spectra
Slide7Workflow
Proposed workflow for in-depth analysisPEAKS DB: De Novo sequencing assisted database search for sensitive and accurate peptide identification. Mol Cell Proteomics 2012; 11:10.1074, 1–8.
Multiple enzyme
Identify
database peptides
.
Database search result validated by
de novo
tags
Reveal
a set of confident proteins
Slide8Workflow
Proposed workflow for in-depth analysisPeaksPTM: Mass spectrometry-based identification of peptides with unspecified modifications.Journal of Proteome Research 10.7 (2011) : 2930-2936
Multiple enzyme
Identify peptides with
unexpected modifications
Peptides from the set of confident proteins are “modified” in-
silico
by trying all possible modifications in UNIMOD.
Speed up by
de novo
tags
For input spectra with
+
highly confident de novo tags
-
no significant database matches
Slide9Workflow
Proposed workflow for in-depth analysisSPIDER: software for protein identification from sequence tags with de novo sequencing error. J Bioinform
Comput Biol. 2005 Jun;3(3):697-716.
Multiple enzyme
Identify peptides with
mutation
,
such as residue
insertion
,
deletion
, and
substitution
.
Screen the protein database to find short sequences similar to
de novo
tags
Use both the
de novo
tags and database sequence to reconstruct the most probable sequences that match the spectrum
For input spectra with
+
highly confident de novo tags
-
no significant database matches
Slide10Workflow
Proposed workflow for in-depth analysis
Multiple enzyme
Unassigned
de novo
sequence tags
are reported as
possible novel peptides
Slide11Result integration
Proposed workflow for in-depth analysis
Slide12Test the workflow with the standard bovine serum albumin
SampleWorkflowIn-depth analysis of BSA
Pure ALBU_BOVIN from SIGMA3 digests with Trypsin
, LysC
, GluC.
LC-MS/MS with Thermo LTQ-Orbitrap XL.
Workflow implemented in PEAK 6
3 digests in one project
Searched database: Swiss-Prot
Trypsin
LysC
GluC
Workflow
LC-MS/MS
Slide13More PSMs are identified in each additional step:
Result5,152 MS/MS spectra1,737 PSMs906 PSMs44 PSMs
38 MS/MS spectra
Filtered
at 1% FDR
1,737
->
2,687
PSMs
PEAKS ALC score > 70%
Slide14BSA coverage
ResultThe uncovered 4% is in the protein N-terminal region, which is mostly likely cleaved-off and not in the purchased sample1.1specific binding site (Asp-Thr-His-Lys) for Cu(II) ions. T. Peters Jr., F.A. Blumenstock. J. Biol. Chem., 242 (1967), p. 1574
Slide15Contaminants
Identified with at least 3 unique peptides. Human keratin proteins (K2C1_HUMAN and K1C_HUMAN) Bacteria protein (SSPA_STAAR)Trypsin (TRY1_BOVIN)Result
Slide16PTMs
Unsuspected modifications identified by PTM searchThree PTMs specified in database searchCarbamidomethylation (C)Oxidation (M)Deamidation (NQ)Result
Slide17Mutation
214th amino acid A TBrown 1975, Fed. Proc. 34:591Result
Slide18Unexplained de novo tags
Might be…Novel peptides outside of the searched databaseResult
KK.QTALVELLK.HK |||||||
DPALVELLKK
Slide19A software workflow proposed for in-depth protein sequence analysis
Found many things in a “pure” sampleContaminantsUnsuspected PTMsMutationsImproved protein sequence coverageBSA coverage: 87% -> 96%Explained more high-quality MS/MS spectraIdentified MS/MS spectra: 1,737 -> 2,687
Summary
Slide20Q / A