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

被引：49

作者：

Atanassov, Ilian ^{[1
]}

Urlaub, Henning ^{[1
,2
]}

机构：

[1] Max Planck Inst Biophys Chem, Bioanalyt Mass Spectrometry Grp, D-37077 Gottingen, Germany

[2] Univ Med Ctr, Dept Clin Chem, Gottingen, Germany

来源：

PROTEOMICS | 2013年 / 13卷 / 20期

关键词：

IPGs; PAGE; Peptide IEF; Shotgun proteomics; Technology; IDENTIFICATION; QUANTIFICATION; FRACTIONATION; OFFGEL;

D O I：

10.1002/pmic.201300035

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

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.

引用

页码：2947 / 2955

页数：9

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