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Increased proteome coverage by combining PAGE and peptide isoelectric focusing: Comparative study of gel‐based separation approaches
Author(s) -
Atanassov Ilian,
Urlaub Henning
Publication year - 2013
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201300035
Subject(s) - proteome , shotgun proteomics , isoelectric focusing , proteomics , chromatography , fractionation , bottom up proteomics , peptide , shotgun , chemistry , two dimensional gel electrophoresis , mass spectrometry , biochemistry , tandem mass spectrometry , protein mass spectrometry , enzyme , gene
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 3 D workflow for shotgun proteomics that relies on protein separation by 1 D 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.