Cleavable Biotin Reagent for Cys-Peptide Enrichment research articles Scheme 2. (A) Chemical Structure of Base-Cleavable Cysteine-Reactive Probe (IBB),a (B) Modiﬁcation of Model Peptide (Ac-TpepC) by IBB and Hydrolysis of the Conjugate, and (C) Experimental Design for Evaluation of IBB Labeling and Streptavidin Capture of Cys Peptidesb a Consists of three functional elements: a thiol-reactive iodoacetamido group, a mild base-cleavable benzoin linker and biotin afﬁnity tag. b FT denotes non-Cys-peptides not retained on streptavidin. E denotes S-carboxymethyl Cys-peptides released by hydrolysis of IBB. G denotes complete mixture (“global fraction”) generated by hydrolysis of IBB-treated lysate before application to streptavidin. IBB labeling of cysteinyl thiols is done at pH 7, whereas streptavidin capture is done at pH 4.5. Elution/hydrolysis of IBB Cys-peptide conjugates is done at pH 8. analyses yielded 1932 conﬁdent peptide identiﬁcations, of which 91% were sequences containing at least one Cys residue (Table 1). Individual values for the three replicates were 1367 identiﬁcations (92% Cys-peptides), 1428 identiﬁcations (93% Cys-peptides and 1432 identiﬁcations (91% Cys-peptides). Analysis of the FT fractions yielded similarly consistent data across the three replicates, with 3,664 conﬁdent peptide identiﬁcations, of which 98.6% were non-Cys-peptides. The average percentage of identiﬁed Cys-peptides in the G fractions was 6.7%. Analyses of the yeast fraction yielded similar results. These data indicated that the IBB Cys-peptide enrichment method displayed high speciﬁcity, efﬁciency and repeatability. Global Impact of Cys-Peptide Enrichment Using IBB in Proteome Analyses of Yeast and RKO Cells. For studies of the effect of IBB Cys-peptide enrichment, the E and G fractions were resolved into 10 fractions by IEF prior to LC-MS/MS. A narrow 3.5-4.5 pI range separation was chosen based on previous studies showing that a majority of proteins are represented by tryptic peptides in this range.28,38,39 All 30 IEF fractions then were analyzed by LC-MS/MS. Compared to the reverse phase LC-MS/MS analyses, these multidimensional analyses yielded a 5-fold increase peptide identiﬁcations in RKO cells and a 3-4 fold increase in yeast (Table 1). For global quantitative assessment of the impact of Cys- peptide enrichment on protein detection, we used spectral count data as a measure of protein abundance.40-42 In addi- tion, our choice of yeast as a model proteome for these studies was based on the availability of abundance-level annotation of most yeast proteins from the quantitative tandem afﬁnity puriﬁcation tag (TAP tag) studies of Ghaemmaghami et al.43 This characteristic of the yeast proteome was utilized in our recent work as part of the CPTAC network, which demonstrated a high correlation between spectral count data and yeast protein copy number.27 Protein cysteine content should be major determinant of the impact of IBB Cys-peptide capture on global protein identiﬁca- tions. We chose to benchmark our analyses on the ratio of cysteinyl tryptic peptides to total tryptic peptides (Cys-peptide fraction), which was calculated through in silico tryptic diges- tion of the entries in the IPI and SGD proteome databases. The digestion criteria used were: (i) allowable peptide length was 5-40 amino acids; (ii) K-X and R-X comprise the only cleavage Journal of Proteome Research Vol. 9, No. 10, 2010 5465