Peptide separation with immobilized pI strips is an attractive alternative to in-gel protein digestion for proteome analysis

Proteomics. 2008 Dec;8(23-24):4862-72. doi: 10.1002/pmic.200800351.


Complex protein mixtures have traditionally been separated by 2-DE. Görg introduced IPGs as the first dimension of protein separation. In recent years, MS-based proteomics has increasingly become the method of choice for identifying and quantifying large number of proteins. In that technology, to decrease analyte complexity, proteins are often separated by 1-D SDS-gel electrophoresis before online MS analysis. Here, we investigate a recently introduced device for peptide separation with IPGs (Agilent OFFGEL). Loading capacity for optimal peptide focusing is below 100 microg and--similar to 2-D gels--IEF is more efficient in the acidic than the basic pH region. The 24-well fractionation format resulted in about 40% additional peptide identifications but less than 20% additional protein identifications than the 12-well format. Compared to in-gel digestion, peptide IEF consistently identified a third more proteins with equal number of fractions. Low protein starting amounts (10 microg) still resulted in deep proteome coverage. Advantages of the in-gel format include better reliability and robustness. Considering its superior performance, diminished sample and work-up requirements, peptide IEF will become a method of choice for sample preparation in proteomics.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Buffers
  • Chemical Fractionation
  • Chromatography, Liquid
  • Electrophoresis, Polyacrylamide Gel
  • Gels
  • HeLa Cells
  • Humans
  • Isoelectric Focusing
  • Isoelectric Point
  • Isotope Labeling
  • Mass Spectrometry
  • Peptides / isolation & purification*
  • Proteins / metabolism*
  • Proteome / analysis*
  • Proteomics / methods*
  • Reagent Strips*
  • Saccharomyces cerevisiae / metabolism


  • Buffers
  • Gels
  • Peptides
  • Proteins
  • Proteome
  • Reagent Strips