Daniel Liebler
Faculty Member
Last active: 2/15/2016

Cysteinyl peptide capture for shotgun proteomics: global assessment of chemoselective fractionation.

Lin D, Li J, Slebos RJ, Liebler DC
J Proteome Res. 2010 9 (10): 5461-72

PMID: 20731415 · PMCID: PMC2948434 · DOI:10.1021/pr1007015

The complexity of cell and tissue proteomes presents one of the most significant technical challenges in proteomic biomarker discovery. Multidimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based shotgun proteomics can be coupled with selective enrichment of cysteinyl peptides (Cys-peptides) to reduce sample complexity and increase proteome coverage. Here we evaluated the impact of Cys-peptide enrichment on global proteomic inventories. We employed a new cleavable thiol-reactive biotinylating probe, N-(2-(2-(2-(2-(3-(1-hydroxy-2-oxo-2-phenylethyl)phenoxy)acetamido)ethoxy)-ethoxy)ethyl)-5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide (IBB), to capture Cys-peptides after digestion. Treatment of tryptic digests with the IBB reagent followed by streptavidin capture and mild alkaline hydrolysis releases a highly purified population of Cys-peptides with a residual S-carboxymethyl tag. Isoelectric focusing (IEF) followed by LC-MS/MS of Cys-peptides significantly expanded proteome coverage in Saccharomyces cerevisiae (yeast) and in human colon carcinoma RKO cells. IBB-based fractionation enhanced detection of Cys-proteins in direct proportion to their cysteine content. The degree of enrichment typically was 2-8-fold but ranged up to almost 20-fold for a few proteins. Published copy number annotation for the yeast proteome enabled benchmarking of MS/MS spectral count data to yeast protein abundance and revealed selective enrichment of cysteine-rich, lower abundance proteins. Spectral count data further established this relationship in RKO cells. Enhanced detection of low abundance proteins was due to the chemoselectivity of Cys-peptide capture, rather than simplification of the peptide mixture through fractionation.

MeSH Terms (14)

Analysis of Variance Biotinylation Cell Line, Tumor Chemical Fractionation Chromatography, Liquid Colonic Neoplasms Cysteine Humans Isoelectric Focusing Peptides Proteomics Saccharomyces cerevisiae Saccharomyces cerevisiae Proteins Tandem Mass Spectrometry

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