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

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%.