proteored multicentric experiment 6 -...
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ProteoRed Multicentric Experiment 6Kerman Aloria
Hospital La Paz. MadridNovember 18, 2010
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DDA vs. MSE
DDA (Data Dependent Acquisition) MSE (Data Independent Acquisition)
Sample digestion
NanoAcquitySYNAPT HDMS
VEMS ProteinLynx Global ServerData processing and protein identification
All identified proteins have been considered for DDA vs. MSE comparison (protein FDR 5%)
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DDA vs. MSE: Identified proteins• Number of identified proteins:
DDA: 167 proteins MSE: 149 proteins
DDA MSE
1056237%
4430%
• Proteins identified with 1 peptide
MSEDDA
PME6_1101
107
114
5
1
7
71
79
87
66
38
50
PME6_2
PME6_3
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DDA vs. MSE: Replicates
DDA MSE
58%
10%
32%
60%
13%
26%
Identified in 1 replicate
Identified in 2 replicates
Identified in 3 replicates
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DDA vs. MSE: Identified peptides
• Number of unique peptides
MSE: 2095 peptidesDDA: 582 peptides
165579%440142
24%
DDA
MSE
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DDA vs. MSE: quantification
• CV of the intensities of the peptides identified in 3 replicates
MSE
DDA
153 peptides39%
630 peptides79%
CV
(%)
peptide number
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DDA vs. MSE
Conclusions
• There is no significant difference in the number of identified proteins between DDA and MSE.
• MSE identifies significantly higher number of peptides than DDA
• The reproducibility of peptide intensities is significantly better in MSE
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Protein inference problem
• Protein inference: the task of assembling the sequences of identified peptides to infer the protein content of the sample
Nesvizhskii, A.I. and Aebersold, R. Mol Cell Proteomics. 2005 4: 1419-1440.
Conclusive proteins: they have at least one distinct/unique peptide
Indistinguishable proteins: all the peptides are shared and at least one of them is distinct/unique
for this group
Protein group: All peptides are shared and several combinations of proteins can explain the presence
of all peptides
Nonclonclusive proteins: All peptides are shared and their presence is explained by other proteins that must
be present in the sample
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PAnalyzer
• A software that classifies peptide and protein identifications from ProteinLynx Global Server based on the proposal of Nesvizhskii and Aebersold (with minor modifications).
• Peptides:
- Distinct/unique: peptides that belong only to one protein sequence
- Shared:
- meaningless: shared peptides belonging to proteins that have to be in the sample
- meaningful: shared peptides that are not meaningless
• Proteins:
- Conclusive: they have at least one distinct/unique peptide. These proteins have to be in the sample.
- Indistinguishable: they share the same meaningful peptides. At least one of the proteins has to be in the sample.
- Group: they do not share the same meaningful peptides. Several combinations of proteins of the group can explainthe presence of all peptides in the sample
- Nonclonclusive: they only have meaningless peptides. Their presence is not necessary to explain the presence ofall peptides in the sample.
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PAnalyzer
Peptide list Protein list
• PAnalyzer reads the results .xml file from ProteinLynx Global Server and classifies the identified proteins based on the defined criteria
Upcoming improvements:
• Definition of a standard format for data uploading
• Implementation of a quantification module
Possibility to filter peptides by score thresholds
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PME6 results with PAnalyzer
PME6_1 PME6_2 PME6_3
PLGS 106 108 121
PAnalyzer
Conclusive 84 93 87
Indistinguishable 4 (2 groups) 2 (1 group) 4 (2 groups)
Group 0 0 0
Nonconclusive 18 13 30
Filtered 0 0 1
Proteomics Core Facility - SGIKERKerman Aloria
Biochemistry and Molecular BiologyMiren Josu OmaetxebarriaJesus Mari Arizmendi
Genetics, Physical Anthropology and Animal PhysiologyAsier Fullaondo
Electronics and TelecommunicationsGorka Prieto