Cleavable Biotin Reagent for Cys-Peptide Enrichment use of a yeast reference proteome that is quantitatively an- notated for cellular expression levels43 and provides benchmark reference for comparison with spectral count data from LC-MS/ MS analyses.27 This provided a means of validating the spectral counting approach to quantify global enrichment of Cys- peptides in both yeast and human RKO cell proteomes. Our results demonstrate not only that Cys-peptide capture broadly enhances detection of Cys-proteins and expands the entire proteome inventory but that the effect is most pronounced for lower abundance Cys-proteins. The rationale for Cys-peptide capture is to simplify the analysis by selecting a smaller set of peptides that represent the larger collection. IBB-based fractionation generates a nearly pure collection of Cys-peptides and this enrichment step enhances detection of Cys-proteins in direct proportion to their cysteine content. The degree of enrichment was most typically 2-8-fold but ranged up to almost 20-fold for a few proteins. Identiﬁcations made in the Cys-peptide fraction can be added to the global inventory to expand overall proteome coverage. This is essentially the approach used by Liu et al.3 and Wang et al.,5 who used thiopropyl Sepharose for Cys-peptide enrich- ment. Our work extends their ﬁndings by quantitatively docu- menting the enhanced detection of low abundance proteins in yeast and extending this quantitative evaluation to a human cell proteome. Our results raise the question of why Cys-peptide enrichment has the greatest effect on detection of low abundance proteins with higher cysteine content. We considered the possibility that lower abundance proteins may have greater fractional cysteine content, but an in silico analysis of TAP tag data from the yeast proteome indicates no such relationship (Figure S7, Supporting Information). It seems likely that the elimination of non-Cys- peptides in the E fraction generates a mixture in which Cys- peptides from lower abundance proteins compete with fewer high abundance peptides for detection in the MS instrument. Proteins with greater numbers of cysteines have a greater probability of being detected, due to their representation by more Cys-peptides. Moreover, it appears that peptides in the E fraction map to a more diverse set of proteins. Inspection of Table 1 indicates that the E fractions invariably yield fewer peptide identiﬁcations than do the G fractions, yet both fractions yield comparable numbers of protein identiﬁcations. This observation also suggested to us the alternate possibility that enhanced detection of low abundance proteins was due to a fractionation effect. In this scenario, the E fraction yields additional identiﬁcations not because it is enriched in Cys- peptides, but simply because it represents a simpler subfraction of the global mixture. To test this hypothesis, we combined IEF subfractions from the global mixture (G fraction) to approximate the numbers of conﬁdent peptide identiﬁcations in the E fraction. However, these combined IEF subfractions failed to generate the enhanced detection of their components. In contrast, analysis of the Cys-peptide-rich E fraction signiﬁ- cantly expanded the list of identiﬁcations. This experiment clearly demonstrated that the advantage of chemoselective fractionation lies in the chemoselection per se, rather than in the fractionation effect. Chemoselective fractionation thus yields a subset of peptides that is broadly representative of the entire proteome. Physical fractionation (e.g., IEF) yields subsets of peptides in any fraction that have similar properties, but that may be much less representative of the overall composition of the proteome. research articles Our results indicate that chemoselective fractionation based on Cys-peptide capture can enhance global proteome coverage, especially for low abundance proteins that contain cysteines. Incorporation of a chemoselective fractionation step into proteome analysis workﬂows can signiﬁcantly expand the coverage of proteomes. Although we have characterized Cys- peptide capture in the context of data-dependent LC-MS/MS analyses on ion trap instruments, the rapid emergence of targeted proteomics using multiple reaction monitoring MS suggests a possible role for chemoselective fractionation in the development of sensitive and speciﬁc targeted quantitative analysis strategies. IBB or similar reagents could be used in this context to enhance targeted analysis of low abundance, cysteine-rich proteins. Abbreviations: CPTAC, Clinical Proteomic Technology Asssessment for Cancer; Cys-peptides, cysteinyl-containing peptides; Cys-proteins, cysteinyl-containing proteins; IBB, N-(2- (2-(2-(2-(3-(1-hydroxy-2-oxo-2-phenylethyl)phenoxy)acetami- do)ethoxy)-ethoxy)ethyl)-5-(2-oxohexahydro-1H-thieno[3,4- d]imidazol-4-yl)pentanamide; ICAT, isotope-coded afﬁnity tag; IEF, isoelectric focusing; IPI, International Protein Index; LC-MS/MS, liquid chromatography-tandem mass spectrom- etry; Met-peptides, methionine-containing peptides; SGD, Sac- charomyces Genome Database; TAP tag, tandem afﬁnity puri- ﬁcation tag; TFE, triﬂuoroethanol. Acknowledgment. This work was supported by a cooperative agreement award 5U24CA126479 from the National Cancer Institute through the Clinical Proteomic Technology Assessment for Cancer (CPTAC) program. We thank Prof. Ned A. 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