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

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Wisniewski,  Jacek R.
Mann, Matthias / Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Max Planck Society;

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Prus,  Gabriela
Mann, Matthias / Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Max Planck Society;

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Citation

Wisniewski, J. R., & Prus, G. (2015). Homogenous Phase Enrichment of Cysteine-Containing Peptides for Improved Proteome Coverage. ANALYTICAL CHEMISTRY, 87(13), 6861-6867. doi:10.1021/acs.analchem.5b01215.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0028-3362-6
Abstract
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%.