Cleavable Biotin Reagent for Cys-Peptide Enrichment research articles Figure 5. Impact of chemoselective fractionation with IBB on detection of yeast proteins annotated with TAP tag copy numbers. Proteins are grouped by copy number into four bins on the x-axis. One-way ANOVA analyses indicate signiﬁcant differences in IBB enrichment effect based on protein spectral counts in global (G) fraction. Data points represent log2 (E/G) for detected protein spectral counts in the eluted (E) fraction to the global (G) fraction. Means for each group are indicated by a long black line, and standard error of the mean (SEM) are indicated by short lines. *** Indicates p < 0.001. Figure 6. Venn diagrammatic representation of enhanced iden- tiﬁcation of low abundance, Cys-containing proteins in yeast. The number of identiﬁcations from the Cys-peptide-containing eluted (E) fraction is inversely proportional to protein copy number. whereas 470/711 (66%) of the proteins in the lowest spectral count bin were enriched. One way ANOVA of log2 (E/G) values indicated signiﬁcant differences in enrichment between each group (p < 0.001). Application of the same analysis to the RKO data set yielded similar results (Figure 8). The degree of enrichment by IBB capture was inversely proportional to protein abundance level. All groups were signiﬁcantly different (p < 0.001) except the highest and second highest abundance levels (Figure 8). We found that only 25/923 (2.7%) of the proteins in the highest spectral count bin were enriched, whereas 666/1047 (64%) of the proteins in the lowest spectral count bin were enriched. These data demonstrate that chemoselective Cys-peptide fractionation with IBB provides a selective enrichment of low abundance proteins in both yeast and in RKO cells. We consistently observed that Cys-peptide enrichment was most effective for low abundance proteins. This raised the possibility that cysteine content was proportionately higher in low abundance proteins. We addressed this possibility by calculating the theoretical Cys-peptide fraction (Cys-peptides/ tryptic peptides) for the TAP tag copy number annotated proteins reported by Ghememaghami et al.43 (Figure S7, Supporting Information). We found no relationship between protein copy number and theoretical Cys-peptide fraction. Classiﬁcation Analysis of RKO Cell Proteins Identiﬁed by Cys-Peptide Enrichment with IBB. We asked whether chemoselective fractionation with IBB exhibits selectivity for proteins based on subcellular distribution or functional clas- siﬁcation. We compared the distributions based on cellular component (Figure S8, Supporting Information), biological process (Figure S9, Supporting Information) and molecular function (Figure S10, Supporting Information) for identiﬁed proteins in the G and E fractions in RKO cells using the WebGestalt gene annotation tool, which allows facile compari- son for large protein data sets.46 The IPI protein accession numbers generated by our analyses were converted to Entrez gene accession numbers, resulting in 4709 (96.3%) proteins represented in this analysis out of 4887 proteins in the primary data set. We note that a protein could be categorized in different locations, biological process or molecular function. For example, 41 proteins were found in both the nucleus (1482 proteins) and mitochondrion (465 proteins). No signiﬁcant differences were noted in the distributions of proteins identiﬁed from the E fraction and the G fraction based on cellular location, biological process or molecular function. This sug- gested that chemoselective fractionation with IBB exhibits no bias for proteome components. Discussion Our objective in these studies was to assess the impact of Cys-peptide capture for global proteome analyses. We em- ployed a novel biotinylating reagent for Cys-peptides that is cleavable under mildly basic conditions and is easily integrated Journal of Proteome Research Vol. 9, No. 10, 2010 5469