research articles proteins are reduced and digested without prior thiol alkylation and the peptides then are captured on the resin. After removal of noncovalently captured peptides, the Cys-peptides are released with a reducing agent. A variation on the thiol-disulﬁde capture strategy involves reversible modiﬁcation of cysteine residues using Ellman’s reagent and further isolation of these labeled Cys-peptides through combined fractional diagonal chromatography.17 More recently, p-hydroxymercuribenzoate based agarose beads were also reported to enrich Cys-peptides and further enhance the protein identiﬁcation through a mercury sulfhydryl interaction.18,19 Capture of cysteinyl peptides by covalent thiol alkylation with a biotinylating reagent is the basis of the isotope coded afﬁnity tag (ICAT) reagents,2 which employ an iodoacetamido elec- trophile that forms a stable thioether through SN2 substitution. These reagents are used to label thiols in reduced, intact proteins, which are then digested and the biotinylated Cys- peptides are enriched by chromatography on immobilized streptavidin. The ICAT reagents include stable isotope labels for relative quantitation (i.e., light and heavy forms). Recently, our research group has used biotin-tagged, thiol- reactive electrophiles as model probes to study the covalent modiﬁcation of proteins and the roles of protein alkylation in chemical toxicity and stress signaling.20-23 Through this work we also became familiar with two drawbacks of biotin-avidin capture. The minor drawback is that, for some labeled peptides, fragmentation of the adduct tag in collision-induced dissocia- tion complicates data analysis.24 The second, more signiﬁcant problem is the difﬁculty of recovering biotinylated peptides from avidin columns under mild conditions. Complete release of biotinylated peptides required extreme conditions (e.g., boiling SDS-PAGE loading buffer or acidic solvents), which frequently results in release of streptavidin protein from the resin. This problem had been addressed previously by the developers of second-generation versions of ICAT reagents, which incorporated a photocleavable linker6 or an acid- cleavable linker25 to release Cys-peptides from streptavidin. The latter is marketed as a “Cleavable ICAT” reagent (AB Sciex), which is a proprietary compound that incorporates light and heavy isotope tags, a biotin tag and the acid-cleavable linker and is used for comparative quantitation. The present study developed from our project funded by the National Cancer Institute Clinical Proteomic Technology As- sessment for Cancer (CPTAC) program, in which we proposed to quantitatively evaluate Cys-peptide capture via a global proteome analysis with current multidimensional LC-MS/MS technology. We also had recently described a photocleavable biotinyl linker for use in Click chemistry labeling of protein adducts formed by alkynyl electrophile probes.26 When we adapted this chemistry to synthesize a photocleavable, thiol- reactive biotinylating reagent, we learned that the linker was susceptible to mild base cleavage, thus enabling recovery of Cys-peptides under mild conditions compatible with our proteomics workﬂow. Our purpose was not to introduce a new reagent per se, but to characterize the impact of Cys-peptide enrichment on a global proteomic scale. Here we describe application of this reagent as a tool to characterize cysteine peptidomes and proteomes by multidimensional LC-MS/MS. We further employed a concentration-annotated yeast standard proteome described in a recent CPTAC study27 as a reference material to assess the enrichment and detection of Cys-peptides and proteins at known levels of abundance. Finally, we used these data to validate a spectral counting approach to assess 5462 Journal of Proteome Research Vol. 9, No. 10, 2010 Lin et al. the impact of Cys-peptide capture on proteomic inventory in a human cell line proteome. Experimental Procedures Materials. Streptavidin Sepharose High Performance was purchased from G.E. Healthcare Bioscience Corp (Pittsburgh, PA). Trypsin Gold was purchased from Promega (Madison, WI). The model Cys-containing peptide Ac-AVAGCAGAR (Ac-TpepC) was purchased from New England peptide (Gardner, MA). Synthesis of N-(2-(2-(2-(2-(3-(1-hydroxy-2-oxo-2-phenyleth- yl)phenoxy)acetamido)ethoxy)-ethoxy)ethyl)-5-(2-oxohexahy- dro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide (IBB) is de- scribed in the Supporting Information. All other chemical reagents were purchased from commercial sources and were used without further puriﬁcation. Model Peptide Studies. The cysteine containing peptide Ac- AVAGCAGAR (Ac-TpepC) was used as model peptide for initial studies if Cys-peptide capture. A sample of 40 nmol Ac-TpepC was mixed with 800 nmol dithiothreitol in 16 uL of 100 µ M sodium phosphate, pH 7 for 5 min at room temperature. To this solution was added 64 µ L of the same buffer and 40 µ L IBB (200 mM in triﬂuoroethanol (TFE)) and the solution was incubated in the dark for 20 min to form the S-alkyl-(IBB)-Ac- TpepC conjugate. Aliquots (8 µ L) were taken and diluted with (a) 92 µ L of 100 mM sodium phosphate buffer at either pH 5.5, 6.5, or 7.5 at 37 °C for 4 h; (b) with 92 µ L of 50 mM sodium acetate buffer, pH 4.5 at room temperature for 16 h; (c) with 92 µ L of 50 mM ammonium bicarbonate, pH 8.0 at room temperature for 2 h; or (d) with 92 µ L water. Samples (5 µ L) were subjected to chromatographic separa- tion on a 250 mm × 2.0 mm YMC ODS-AQ 5 µ m C18 column (Waters, Milford, MA) and eluted conjugates were analyzed with a Thermo LCQ DecaXP ion trap mass spectrometer (Thermo- Electron, San Jose, CA). The mobile phase consisted of 5% acetonitrile, 95% water, and 0.1% formic acid (solvent A) and 95% acetonitrile, 5% water, and 0.1% formic acid (solvent B). The ﬂow rate was 400 µ L min-1, and the gradient program was 100% solvent A from 0 to 5 min, 100 to 40% A by 20 min, 40 to 0% A by 22 min, then held at 0% A from 22 to 25 min, followed by a linear gradient to 100% A at 29 min, then held at 100% A from 29 to 34 min. Preparation of Yeast and Human Cell Tryptic Digests. A human colon adenocarcinoma cell line (RKO) was obtained from ATCC (Manassas, VA) and cultured in 175 mL cell culture ﬂasks in McCoy’s 5A media (Mediatech, Herndon, VA) supple- mented with 10% fetal bovine serum (Altas Biologicals, Fort Collins, CO) at 37 °C in 5% CO2. RKO cells were grown to approximately 90% conﬂuence, then harvested in 5 mL of 0.25% trypsin-EDTA, washed with PBS. A yeast protein extract was prepared at the National Institute of Standards and Technology for studies by the National Cancer Institute Clinical Proteomic Technology Assessment for Cancer (CPTAC) network, as de- scribed previously.27 Solubilization and tryptic digestion of proteins in the yeast and RKO cells was done by a modiﬁcation of the method of Wang et al.5 as we have described previously.28 The method employed triﬂuoroethanol to solubilize cell and tissue proteins prior to tryptic digestion. For the present study, the only modiﬁcation to the described method was that proteins were not treated with iodoacetamide following reduc- tion with dithiothreitol and before the digestion step. After digestion, the peptides were eluted from solid phase extraction columns in acetonitrile/water (4:1, v/v) and the solutions were evaporated to dryness in vacuo.