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Increased proteome coverage by combining PAGE and peptide isoelectric focusing: comparative study of gel-based separation approaches.

ABSTRACT: The in-depth analysis of complex proteome samples requires fractionation of the sample into subsamples prior to LC-MS/MS in shotgun proteomics experiments. We have established a 3D workflow for shotgun proteomics that relies on protein separation by 1D PAGE, gel fractionation, trypsin digestion, and peptide separation by in-gel IEF, prior to RP-HPLC-MS/MS. Our results show that applying peptide IEF can significantly increase the number of proteins identified from PAGE subfractionation. This method delivers deeper proteome coverage and provides a large degree of flexibility in experimentally approaching highly complex mixtures by still relying on protein separation according to molecular weight in the first dimension.

SUBMITTER: Atanassov I

PROVIDER: S-EPMC4285200 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Increased proteome coverage by combining PAGE and peptide isoelectric focusing: comparative study of gel-based separation approaches.

Atanassov Ilian I Urlaub Henning H

Proteomics 20131001 20

The in-depth analysis of complex proteome samples requires fractionation of the sample into subsamples prior to LC-MS/MS in shotgun proteomics experiments. We have established a 3D workflow for shotgun proteomics that relies on protein separation by 1D PAGE, gel fractionation, trypsin digestion, and peptide separation by in-gel IEF, prior to RP-HPLC-MS/MS. Our results show that applying peptide IEF can significantly increase the number of proteins identified from PAGE subfractionation. This meth ...[more]

PMID: 23943586

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