research articles Figure 1. Reverse phase LC-MS total ion chromatograms for reaction products of Ac-TpepC and IBB under different conditions. The labeled peaks are I, Ac-TpepC; II, Ac-TpepC-IBB conjugate; III, IBB hydrolysis product; IV, IBB; V, S-carboxymethyl-Ac-TpepC. (A) Ac-TpepC; (B) Ac-TpepC-IBB conjugate formed by reaction of Ac-TpepC with IBB in 100 mM sodium phosphate, pH 7.0, containing 33.3% TFE at room temperature in dark for 20 min; (C) Ac-TpepC-IBB conjugate in 50 mM sodium phosphate buffer containing 3.67% TFE, pH 5.5 at 37 °C for 4 h; (D) Ac-TpepC-IBB conjugate in 50 mM sodium phosphate buffer containing 3.67% TFE, pH 6.5 at 37 °C for 4 h; (E) Ac-TpepC-IBB conjugate in 50 mM sodium phosphate buffer containing 3.67% TFE, pH 7.5 at 37 °C for 4 h; (F) Ac-TpepC-IBB conjugate pH 8.0, 50 mM ammonium bicarbonate at room temperature for 5 h; (G) Ac- TpepC-IBB conjugate in pH 4.5, 50 mM acetate buffer at room temperature in the dark overnight. sites; (iii) no missed cleavages were permitted; and (iv) cleav- ages at K-P and R-P sites were permitted (to simplify the calculations). In this study, protein enrichment or depletion is represented by the log2-transformed ratio of spectral counts for proteins in the Cys-peptide fraction (E fraction) to counts for the same proteins in the global fraction (G fraction). This ratio, log2 (E/ G) is positive for proteins that are enriched in the E fraction and is negative for proteins that are depleted in the E fraction. Only proteins with at least two spectral counts combined from triplicate analyses of the FT, E and G fractions from RKO cells were considered. In yeast studies, only proteins with at least 3 spectral counts in the combined fractions were considered. This restriction resulted in a protein level FDR of 0.034 (RKO cells) and 0.045 (yeast). In cases where spectral counts in the E or G fractions were zero, the value for that E or G fraction was adjusted to “1” to ensure validity of log2(E/G). 5466 Journal of Proteome Research Vol. 9, No. 10, 2010 Lin et al. The protein identiﬁcation data for yeast and RKO cell proteins are plotted in Figure 2. 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 left y-axis of each plot. The red curves in Figure 2A and C represent the theoretical Cys-peptide fraction (predicted Cys- peptides/predicted tryptic peptides) for each protein, whereas those in Figure 2B and D represent the detected Cys-peptide fraction (detected Cys-peptides/detected tryptic peptides) for each protein. The ﬂat segment of each red curve represents proteins with no cysteine residues. The black data points represent measured log2 (E/G) ratios for detected proteins (right y-axis). A total 3025 proteins from yeast were identiﬁed by triplicate IEF-LC-MS/MS analyses using the identiﬁcation threshold criteria described above. These proteins account for ∼44% of the proteins in the SGD database. Of these, 345 proteins generate no Cys-peptides upon in silico digestion and values of log2 (E/G) for all of these were less than zero (Figure 2A). The remaining 2736 proteins contained at least one Cys- peptide. For these, a Spearman correlation (R ) 0.40) between log2 (E/G) and Cys-peptide fraction suggested a modest en- hancement of detection of proteins with higher Cys-peptide fraction through the use of IBB capture. Similar results were found in RKO cells, as shown in Figure 2B. A total 4887 proteins were identiﬁed in triplicate analyses at the indicated threshold, representing ∼7% of the proteins in the IPI database. Of the identiﬁed proteins, 237 proteins yielded no cysteinyl peptide upon in silico digestion. Almost all of these were not enriched by IBB capture, as indicated by log2 (E/G) values less than zero. The Spearman correlation (R ) 0.44) for the relationship between log2 (E/G) and Cys-peptide fraction again indicated a modest enrichment of proteins containing more Cys-peptides by IBB capture. We further analyzed the correlation between log2 (E/G) and the ratio of detected Cys-peptides to detected total peptides (detected Cys-peptide fraction) for each protein. Only seven of 1131 proteins without Cys-peptides were found to be enriched (Figure 2C), which provides an estimate of detectable nonspeciﬁc binding to steptavidin. The correlation of log2(E/ G) with the detected Cys-peptide fraction (Spearman R ) 0.85) was much stronger than for the relationship based on theoreti- cal Cys-peptides. Figure 2D showed the similar results for RKO cells, with a Spearman r ) 0.83 for the correlation of log2(E/G) with the detected Cys-peptide fraction. Only 14 of 1569 RKO proteins without Cys-peptides were enriched. An important question is the degree to which Cys-peptide enrichment expands the number of protein identiﬁcations. Figure 3 provides a Venn diagrammatic representation of the identiﬁcation overlaps between the E and G fractions for both yeast and RKO cell analyses by IEF-LC-MS/MS. Although there is considerable overlap in identiﬁcations between the G and E fractions, the E fraction contained 287 unique identiﬁcations for yeast and 569 unique identiﬁcations for RKO cells. Thus, the content of the E fraction expanded identiﬁcations by 10.6% for yeast and 13.0% in RKO cells. Despite the chemical speciﬁcity of the IBB reagent for thiols, enrichment of Cys-proteins in the above analyses could also be due in part to a “fractionation effect”, in which the separation of the peptide mixtures into subfractions (i.e., E and FT fractions) yields more identiﬁcations by presenting simpler mixtures for analysis. To further evaluate the contributions of