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ProteoRed Multicentric Experiment 6 Kerman Aloria Hospital La Paz. Madrid November 18, 2010

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Page 1: ProteoRed Multicentric Experiment 6 - CSICestrellapolar.cnb.csic.es/proteored/Biblioteca/Ficheros/502075564... · ProteoRed Multicentric Experiment 6. Kerman Aloria. ... A.I. and

ProteoRed Multicentric Experiment 6Kerman Aloria

Hospital La Paz. MadridNovember 18, 2010

Page 2: ProteoRed Multicentric Experiment 6 - CSICestrellapolar.cnb.csic.es/proteored/Biblioteca/Ficheros/502075564... · ProteoRed Multicentric Experiment 6. Kerman Aloria. ... A.I. and

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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%)

Page 3: ProteoRed Multicentric Experiment 6 - CSICestrellapolar.cnb.csic.es/proteored/Biblioteca/Ficheros/502075564... · ProteoRed Multicentric Experiment 6. Kerman Aloria. ... A.I. and

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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

Page 4: ProteoRed Multicentric Experiment 6 - CSICestrellapolar.cnb.csic.es/proteored/Biblioteca/Ficheros/502075564... · ProteoRed Multicentric Experiment 6. Kerman Aloria. ... A.I. and

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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

Page 5: ProteoRed Multicentric Experiment 6 - CSICestrellapolar.cnb.csic.es/proteored/Biblioteca/Ficheros/502075564... · ProteoRed Multicentric Experiment 6. Kerman Aloria. ... A.I. and

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DDA vs. MSE: Identified peptides

• Number of unique peptides

MSE: 2095 peptidesDDA: 582 peptides

165579%440142

24%

DDA

MSE

Page 6: ProteoRed Multicentric Experiment 6 - CSICestrellapolar.cnb.csic.es/proteored/Biblioteca/Ficheros/502075564... · ProteoRed Multicentric Experiment 6. Kerman Aloria. ... A.I. and

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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

Page 7: ProteoRed Multicentric Experiment 6 - CSICestrellapolar.cnb.csic.es/proteored/Biblioteca/Ficheros/502075564... · ProteoRed Multicentric Experiment 6. Kerman Aloria. ... A.I. and

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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

Page 8: ProteoRed Multicentric Experiment 6 - CSICestrellapolar.cnb.csic.es/proteored/Biblioteca/Ficheros/502075564... · ProteoRed Multicentric Experiment 6. Kerman Aloria. ... A.I. and

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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

Page 9: ProteoRed Multicentric Experiment 6 - CSICestrellapolar.cnb.csic.es/proteored/Biblioteca/Ficheros/502075564... · ProteoRed Multicentric Experiment 6. Kerman Aloria. ... A.I. and

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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.

Page 10: ProteoRed Multicentric Experiment 6 - CSICestrellapolar.cnb.csic.es/proteored/Biblioteca/Ficheros/502075564... · ProteoRed Multicentric Experiment 6. Kerman Aloria. ... A.I. and

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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

Page 12: ProteoRed Multicentric Experiment 6 - CSICestrellapolar.cnb.csic.es/proteored/Biblioteca/Ficheros/502075564... · ProteoRed Multicentric Experiment 6. Kerman Aloria. ... A.I. and

Proteomics Core Facility - SGIKERKerman Aloria

Biochemistry and Molecular BiologyMiren Josu OmaetxebarriaJesus Mari Arizmendi

Genetics, Physical Anthropology and Animal PhysiologyAsier Fullaondo

Electronics and TelecommunicationsGorka Prieto