Cysteinyl Peptide Capture for Shotgun Proteomics: Global Assessment of Chemoselective Fractionation De Lin,†,‡ Jing Li,§ Robbert J. C. Slebos,‡,| and Daniel C. Liebler*,†,‡ Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, Department of Biomedical Informatics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, and Jim Ayers Institute for Precancer Detection and Diagnosis, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 Received July 7, 2010 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. Keywords: AUTHOR • PLEASE • SUBMIT • KEYWORDS Introduction The tremendous range of protein concentrations in living systems presents the greatest barrier to comprehensive pro- teome analysis. Low abundance proteins are difﬁcult to detect in the presence of much more abundant proteins, particularly in shotgun proteome analyses using liquid chromatography- tandem mass spectrometry (LC-MS/MS).1 Multidimensional protein and peptide separations are widely used to improve depth of proteome analysis.1 Another useful approach is selective afﬁnity capture or chemoselective fractionation, which samples peptide or protein subsets that share some common chemical characteristic. Afﬁnity-based techniques have been described to enrich peptides containing cysteine,2-6 arginine,7 * To whom correspondence should be addressed: Jim Ayers Institute for Precancer Detection and Diagnosis, U1213C Medical Research Bldg. III, 465 21st, Ave. South, Nashville, TN 37232-8575. Ph.: 615-322-3063. Fax: 615-936- 1001. E-mail: daniel.liebler@vanderbilt.edu. † Department of Biochemistry. ‡ Jim Ayers Institute for Precancer Detection and Diagnosis. § Department of Biomedical Informatics. | Department of Cancer Biology. 10.1021/pr1007015  2010 American Chemical Society histidine,8 methionine,9 as well as peptides that are phospho- rylated10,11 and glycosylated.12,13 These studies all have dem- onstrated that chemoselective fraction simpliﬁes complex mixtures. Chemoselective fractionation also enhances detection of lower abundance proteins in shotgun proteome analyses3-6 and in targeted analyses.14,15 Cys-peptides are attractive targets for chemoselective frac- tionation. In silico analysis of the current IPI human protein database (version 3.37) indicates that 91% of the indexed proteins contain at least one cysteine residue and that 24% of predicted tryptic peptides contain a cysteine. Similarly, for Saccharomyces cerevisiae (yeast) proteins, 88% contain cysteine and would yield 16% tryptic peptides (SGD orf_trans-all, downloaded July 17, 2007). Thus, a substantial fraction of these proteomes can be represented by a subset of tryptic peptides. The unusual nucleophilicity and redox chemistries of the cysteine thiol and thiolate anions provide high selectivity for modiﬁcation by electrophilic reagents and thiol-disulﬁde reagents. One approach to Cys-peptide capture is via thiol-disulﬁde chemistry using thiopropyl sepharose.16 In this approach, Journal of Proteome Research 2010, 9, 5461–5472 5461 Published on Web 08/23/2010