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

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

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Biochemical Proteomics Group, Department of Proteomics and Signal Transduction, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.


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%.

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