Cleavable Biotin Reagent for Cys-Peptide Enrichment research articles Figure 2. Relationship between IBB enrichment effect and Cys-peptide abundance for detected proteins. Proteins are ordered on the x-axis (“protein index”) in order of increasing fractional Cys-peptide content (Cys-peptide fraction), which is shown on the right y-axis of each plot. The red curves in (A) and (C) represent the theoretical Cys-peptide fraction (predicted Cys-peptides/predicted tryptic peptides) for each protein, whereas those in (B) and (D) represent the detected Cys-peptide fraction (detected Cys-peptides/detected tryptic peptides) for each protein. The black data points represent log2-transformed ratios of spectral counts for detected proteins in the eluted (E) fraction to the global (G) fraction and are plotted as log2 (E/G) (left y-axis). The listed R values are Spearman correlation coefﬁcients for proteins with at least one predicted or detected Cys-peptide. Table 1. Summary of Identiﬁed Yeast and RKO Cys-Peptides, Total Peptides and Proteins LC-MS/MS IEF-LC-MS/MS total Cys-peptides total peptides Cys-peptide % total proteins Cys-peptides total peptides Cys-peptides % total proteins RKO yeast G E FT G E FT 233 1757 5 181 1669 9 3470 1932 3664 4122 2014 4086 6.7 91 1.4 4.4 83 0.22 932 915 988 685 810 683 2786 7482 569 1165 4565 191 18121 9081 18986 12939 6052 15554 15.4 82 3.0 8.3 75 1.2 4390 3571 4455 2705 2057 2791 chemoselective Cys-peptide capture and fractionation effects, we did two additional analyses. First, we asked whether enrichment tracked with detection of methionine-containing peptides (Met-peptides). In silico tryptic digestion of the SGD and IPI databases using the rules outlined above yielded theoretical percentages of 23.3 and 23.9% Met-peptides for yeast and RKO, respectively, which were similar to the percentages for Cys-peptides (15.5% in yeast and 23.9% in RKO cells) (Figure S4, Supporting Information). Nevertheless, Figure S5A and B (Supporting Information) showed that there was no signiﬁcant correlation between the detected Met-peptide fraction and log2 (E/G) in either yeast or Figure 3. Venn diagram representation of proteins uniquely identiﬁed in yeast and RKO cells by Cys-peptide capture. Proteins identiﬁed in global (G) and eluted (E) fractions are shown. Note that 287 and 569 proteins were identiﬁed only in the E fractions and thus indicate the beneﬁt of Cys-peptide capture. Journal of Proteome Research Vol. 9, No. 10, 2010 5467