Cysteinyl peptide capture for shotgun proteomics: global assessment of chemoselective fractionation.
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.
Lin
De
D
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-8575, USA.
Li
Jing
J
Slebos
Robbert J C
RJ
Liebler
Daniel C
DC
D000704:Analysis of Variance; D019921:Biotinylation; D045744:Cell Line, Tumor; D005591:Chemical Fractionation / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods*; D003110:Colonic Neoplasms / Q000378:metabolism / Q000473:pathology; D003545:Cysteine / Q000032:analysis / Q000737:chemistry; D006801:Humans; D007525:Isoelectric Focusing; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods*
D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; D003545:Cysteine