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

Proteomics. 2013 Oct;13(20):2947-55. doi: 10.1002/pmic.201300035.

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.

Keywords: IPGs; PAGE; Peptide IEF; Shotgun proteomics; Technology.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatography, Liquid
  • Electrophoresis, Polyacrylamide Gel / methods*
  • HeLa Cells
  • Humans
  • Isoelectric Focusing / methods*
  • Mass Spectrometry
  • Peptides / isolation & purification*
  • Proteome / metabolism*
  • Proteomics / methods*
  • Reproducibility of Results
  • Trypsin / metabolism

Substances

  • Peptides
  • Proteome
  • Trypsin