Narrow-Range Peptide Isoelectric Focusing as Peptide Prefractionation Method Prior to Tandem Mass Spectrometry Analysis

  • Maria Pernemalm
Part of the Methods in Molecular Biology book series (MIMB, volume 1023)


High sample complexity is one of the major challenges in mass spectrometry-based proteomics today. Despite massive improvement in instrumentation, sample prefractionation is still needed to reduce sample complexity and improve proteome coverage. Isoelectric focusing (IEF) has been traditionally used as a first-dimension protein separation technique in two-dimensional gel electrophoresis-based proteomics. Recently, peptide IEF has emerged as appealing alternative for anion exchange chromatography in multidimensional LC-MS/MS workflows. The rationale behind using narrow-range peptide isoelectric focusing as a prefractionation method prior to ms/ms is to reduce the complexity induced by tryptic digestion. This is done by selectively analyzing a sub-fraction of peptides with an acidic pI. The pI range is chosen as it has previously been shown that 96 % of human proteins have at least one tryptic peptide between pH 3.4 and 4.9. This ensures high proteome coverage while reducing the number of peptides with 2/3. In addition the focusing precision is optimal in this range. Therefore, by analyzing this sub-fraction of peptides the complexity of the sample can be reduced without significant loss of proteome coverage. As the theoretical pI of peptides can be calculated, the pI of the identified peptides can be used to validate the peptide sequence (identified peptides with pI outside the pH range 3.4–4.9 are more likely to be false positives). In addition, this approach is compatible with iTRAQ labelling as the different iTRAQ labels migrate similarly in IEF.

Key words

Narrow-range peptide isoelectric focusing Prefractionation Mass spectrometry Proteomics pI iTRAQ 


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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Maria Pernemalm
    • 1
  1. 1.Science for Life Laboratory, Department of Oncology–PathologyKarolinska InstitutetStockholmSweden

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