research articles Scheme 1. Synthesis of IBBa Lin et al. a See “Experimental Procedures” for detailed description. Reagents: (a) TBDMSCl, Et3N, THF, 86%; (b) 2-phenyl-1,3-dithiane, n-butyllithium, THF, 91%; (c) methyl bromoacetate, TBAF, THF, 93%; (d) 2,2′-(Ethylenedioxy)bisethylamine, MeOH, 75%; (e) biotin, CDI, DMF, 56%; (f) HgClO4, CH3CN/H2O, 69%; (g) iodoacetic acid, DCC, DMAP, 81%. contains a thiol-reactive iodoacetamido group, a cleavable benzoin group and biotin (Scheme 2A). We ﬁrst studied the capture and release reactions of IBB using the model Cys-containing peptide Ac-TpepC (Scheme 2B). Reaction of Ac-TpepC (0.25 mM) and DTT (5 mM) with IBB (50 mM) in 100 mM sodium phosphate, pH 7.0 buffer containing 33.3% TFE completely converted the peptide (peak I) to the Ac-TpepC-IBB conjugate (peak II) after 20 min at room temperature (Figure 1B). LC-MS analyses of Ac-TpepC-IBB indicated that the conjugate was stable at pH 4.5, but increas- ingly unstable at higher pH, yielding the hydrolysis product S-carboxymethyl-Ac-TpepC (peak V), as indicated in Figure 2C-F. However, the Ac-TpepC-IBB conjugate was very stable in 50 mM sodium acetate, pH 4.5 for up to 16 h at room temperature (Figure 2G). Although the plotted data are from total ion chromatograms, MS/MS analyses conﬁrmed the structures of Ac-TpepC (Figure S1, Supporting Information), its IBB conjugate (Figure S2, Supporting Information) and the hydrolysis product S-carboxymethyl-Ac-TpepC (Ac-TpepC-IAA, Figure S3, Supporting Information). The sensitivity of the thiol- IBB conjugate to hydrolysis in mild base is consistent with the properties of similar benzoin esters.37 Cys-Peptide Enrichment. Our approach to evaluating cap- ture and release of Cys-peptides in complex mixtures is illustrated in Scheme 2C. Treatment of a tryptic peptide digest with IBB/TFE at pH 7.0 results in alkylation of Cys-peptides to form the biotin-tagged Cys-peptide conjugates, which are applied to a streptavidin sepharose column at pH 4.5. The ﬂow through (FT) fraction includes non-Cys-peptides, which are not 5464 Journal of Proteome Research Vol. 9, No. 10, 2010 biotinylated. Nonbiotinylated peptides that nonspeciﬁcally bind to the column are removed by washing with pH 4.5 sodium acetate buffer and by additional washes with 2 M NaCl. The captured Cys-peptides are then eluted (E) by hydrolysis with ammonium bicarbonate. A global peptide mixture (G) is generated as a reference for Cys-peptide enrichment by incubating the IBB-treated peptide mixture at pH 8 before applying to the streptavidin column. Neither the Cys-peptides nor the non-Cys-peptides are retained on the streptavidin column; this control corrects for any nonspeciﬁc effects of the IBB labeling step or irreversible, nonspeciﬁc peptide binding to the streptavidin column. For each experiment, RKO cells or yeast lysates were subjected to denaturation, reduction, and tryptic digestion. Two aliquots of each sample then were alkylated with IBB. One IBB- labeled aliquot was applied to a streptavidin column to generate the FT and E fractions described above, whereas the other was ﬁrst hydrolyzed with ammonium bicarbonate before applying to the streptavidin column. The ﬂow-through from this sample is the G fraction, which serves as a reference for evaluating enrichment of Cys-peptides. Equal amounts of peptides from the FT and E fractions from the ﬁrst sample and from the G fraction from the second sample were then analyzed. In a preliminary test of the speciﬁcity and reproducibility of this IBB Cys-peptide enrichment method, three replicate samples of FT, E and G fractions prepared from yeast and from RKO cell lysates were analyzed by reverse phase LC-MS/MS (no IEF). For the replicate E fractions from RKO cells, the three