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Critical Comparison of Multidimensional Separation Methods for Increasing Protein Expression Coverage

  • Linn Antberg
  • Paolo Cifani
  • Marianne Sandin
  • Fredrik Levander
  • Peter James
Publishing year: 2012
Language: English
Pages: 2644-2652
Publication/Series: Journal of Proteome Research
Volume: 11
Issue: 5
Document type: Journal article
Publisher: The American Chemical Society

Abstract english

We present a comparison of two-dimensional separation methods and how they affect the degree of coverage of protein expression in complex mixtures. We investigated the relative merits of various protein and peptide separations prior to acidic reversed-phase chromatography directly coupled to an ion trap mass spectrometer. The first dimensions investigated were density gradient organelle fractionation of cell extracts, 1D SDS-PAGE protein separation followed by digestion by trypsin or GluC proteases, strong cation exchange chromatography, and off-gel isoelectric focusing of tryptic peptides. The number of fractions from each first dimension and the total data accumulation RP-HPLC-MS/MS time was kept constant and the experiments were run in triplicate. We find that the most critical parameters are the data accumulation time, which defines the level of under-sampling and the avoidance of peptides from high expression level proteins eluting over the entire gradient.


  • Immunology in the medical area
  • protein and peptide separation
  • methods comparison
  • off-gel isoelectric
  • focusing
  • GluC
  • trypsin
  • GelLC
  • density gradient cell fractionation
  • strong cation exchange chromatography
  • reversed-phase chromatography
  • proteome coverage


  • CREATE Health
  • ISSN: 1535-3893
Peter James
E-mail: peter [dot] james [at] immun [dot] lth [dot] se


Department of Immunotechnology

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