Homogenous Phase Enrichment of Cysteine-Containing Peptides for Improved Proteome Coverage

Anal Chem. 2015 Jul 7;87(13):6861-7. doi: 10.1021/acs.analchem.5b01215. Epub 2015 Jun 10.


We describe a proteomic reactor-based homogeneous phase enrichment of cysteine-containing peptides in a filter aided sample preparation (FASP) format. In this approach thiol-reduced proteins are derivatized with thiol-activated polyethylene glycol (TAPEG) before protein cleavage. Consecutive digestion with endoproteinase LysC and trypsin allows isolation of two fractions of nonderivatized peptides. After reduction of disulfide bonds between cysteine-containing peptides and the polyethylene glycol moieties, a third fraction of peptides is collected. LC-MS/MS analyses revealed that on average this fraction consists of 95% cysteine-containing peptides. Since 85-93% of all peptides are unique to a single subfraction, the combination of TAPEG and FASP offers an efficient peptide separation strategy. Analysis of whole cell lysates of mouse brain, liver, red muscle fibers, and CaCo-2 cells using the TAPEG FASP approach allowed identification of 6,900, 5,800, 4,200 and 7,900 proteins, 10-30% more than were identified using two-step digestion without isolation of Cys-containing peptides. The fractionation also increased the protein sequence coverage by 10-30%.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Brain / metabolism
  • Caco-2 Cells
  • Chromatography, Liquid
  • Cysteine / chemistry*
  • Humans
  • Liver / metabolism
  • Muscles / metabolism
  • Peptides / chemistry*
  • Peptides / metabolism
  • Proteome*
  • Tandem Mass Spectrometry


  • Peptides
  • Proteome
  • Cysteine