title: Homogenous Phase Enrichment of Cysteine-Containing Peptides for Improved Proteome Coverage.
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%.
authors.affiliation: Biochemical Proteomics Group, Department of Proteomics and Signal Transduction, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.
authors.forename: Jacek R
authors.initials: JR
authors.lastname: Wiśniewski
authors.affiliation: Biochemical Proteomics Group, Department of Proteomics and Signal Transduction, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.
authors.forename: Gabriela
authors.initials: G
authors.lastname: Pruś
mesh_terms: D001921:Brain / Q000378:metabolism; D018938:Caco-2 Cells; D002853:Chromatography, Liquid; D003545:Cysteine / Q000737:chemistry*; D006801:Humans; D008099:Liver / Q000378:metabolism; D009132:Muscles / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D020543:Proteome; D053719:Tandem Mass Spectrometry
chemical_list: D010455:Peptides; D020543:Proteome; D003545:Cysteine