Cleavable Biotin Reagent for Cys-Peptide Enrichment Cys-Peptide Enrichment. Streptavidin sepharose beads were prewashed three times with 50 mM sodium acetate buffer, pH 4.5 and then diluted with this buffer to achieve a 50:50 (v/v) bead/buffer slurry. Tryptic peptides corresponding to 1.5 mg yeast or RKO cell protein were redissolved in 100 µ L of 100 mM sodium phosphate buffer, pH 7.0. After reducing with 10 mM dithiothreitol for 5 min, the sample was treated with 50 µ L IBB (200 mM in TFE) in the dark at room temperature for 20 min to label cysteinyl peptides. The mixture was then added to 15 mL streptavidin sepharose bead/buffer slurry and incu- bated for 30 min in the dark at room temperature with gentle mixing. The beads were then washed sequentially with 50 mM sodium acetate buffer, pH 4.5 (2 × 7.5 mL), 50 mM sodium acetate buffer, pH 4.5 containing 2 M NaCl (7.5 mL), and 50 mM ammonium bicarbonate, pH 8.0 (2 × 7.5 mL, quick). To release carboxymethyl-cysteine containing peptides, the beads were extracted twice (5 h and overnight) with 7.5 mL of 50 mM ammonium bicarbonate, pH 8, at room temperature with gentle mixing. The combined extract was lyophilized, resus- pended in 1 mL of deionized water, desalted on a SEP-Pak vac 1 cc (100 mg) C-18 cartridge (Waters Corp., Milford, MA) and then evaporated to dryness in vacuo. IEF Fractionation of Peptides. Tryptic peptides (100 µ g) were resuspended in 500 µ L of 6 M urea and loaded in an IPGphor rehydration tray (GE Healthcare, Piscataway, NJ). Immobilized pH gradient strips (24 cm, pH 3.5-4.5) were placed over the samples and allowed to rehydrate overnight at ambient temperature. The loaded strips were focused at 21 °C on an Ettan IPGPhor-3 IEF system (GE Healthcare, Piscataway, NJ) using an initial step at 300 V for 900 Vh, then gradient to 1000 V for 3900 Vh, then gradient to 8000 V for 13500 Vh, then step to 8000 V for 93700 Vh. The strips were then cut into 10 (2.4 cm) pieces and placed in separate wells of a 96-well Falcon ﬂat bottom polystyrene enzyme-linked immunosorbent assay plate. Peptides were eluted from the strips with 200 µ L of 0.1% formic acid (FA) for 15 min, followed by 200 µ L of 50% ACN/ 0.1% FA for 15 min, then 200 µ L of 100% ACN/0.1% FA for 15 min. The combined solutions of extracted peptides were evaporated in vacuo, then resuspended in 1 mL of 0.1% triﬂuoroacetic acid and desalted over a 96-well C18 Oasis hydrophilic-lipophilic balance plate (30 µ m particle size, 10 mg packing) (Waters Corp., Milford, MA). The combined peptide solutions were evaporated in vacuo, redissolved in 100 µ L of 0.1% FA for LC-MS/MS analysis. LC-MS/MS Analysis of Peptide Mixtures. Reverse phase LC of peptide mixtures was done with an Eksigent nanoLC and autosampler (Dublin, CA). Peptides were separated on a packed capillary tip (Polymicro Technologies, 100 µ m × 11 cm) with Jupiter C18 resin (5 µ m, 300 Å, Phenomenex) using an in-line solid-phase extraction column (100 µ m × 6 cm) packed with the same C18 resin (using a frit generated with liquid silicate Kasil 129) similar to that previously described.30 LC was done at ambient temperature at a ﬂow rate of 0.6 µ L min-1 using mobile phases of 0.1% (v/v) formic acid in water (solvent A) and 0.1% (v/v) formic acid in acetonitrile (solvent B). A 95 min gradient was performed with a 15 min washing period (100% A for the ﬁrst 10 min followed by a gradient to 98% A at 15 min) to allow for solid-phase extraction and removal of any residual salts. Following the wash period, the gradient was increased to 25% B by 50 min, followed by an increase to 90% B by 65 min and held for 9 min before returning to the initial conditions. Peptides were analyzed using a Thermo LTQ- Orbitrap hybrid mass spectrometer (Thermo Fisher, San Jose, research articles CA). A full scan obtained for eluting peptides in the range of 400-2000 amu was collected on the Orbitrap at a resolution of 60 000, followed by ﬁve data-dependent MS/MS scans on the LTQ with a minimum threshold of 1000 set to trigger the MS/MS spectra. In data-dependent MS/MS experiments, dy- namic exclusion of previously analyzed precursors was set at 60 s with repeat of 1 and a repeat duration of 1. Centroided MS/MS spectra were recorded on the LTQ-Orbitrap using an isolation width of 2 m/z, an activation time of 30 ms, an activation q of 0.250 and 30% normalized collision energy using 1 microscan with a max ion time of 100 ms for each MS/MS scan and 1 microscan with a max ion time of 500 ms for each full MS scan. Data Analysis. Tandem mass spectra stored as centroided peak lists from mass spectra .RAW ﬁles were read and transcod- ed to mzData v1.05 ﬁles with the in-house developed “Scan- Sifter” software. Only MS/MS scans were written to the mzData ﬁles; MS scans were excluded. If 90% of the intensity of a tandem mass spectrum appeared at a lower m/z than that of the precursor ion, a single precursor charge was assumed; otherwise, the spectrum was processed under both double and triple precursor charge assumptions. Tandem mass spectra were assigned to peptides from the IPI Human database version 3.37 (69249 sequences) for RKO or the Saccharomyces Genome database (SGD, 6839 sequences) for yeast by the MyriMatch algorithm, version 1.2.11.31 The sequence database was doubled to contain each sequence in both normal and reversed orienta- tions, enabling false discovery rate estimation. MyriMatch was conﬁgured to expect all cysteines to bear carboxymethyl modiﬁcations (+58.00548 Da) and to allow for the possibility of oxidation on methionines (+15.99492 Da) and cyclization of N-terminal glutamine (-17.02655 Da). Candidate peptides were required to have tryptic cleavages or protein termini at both ends, though any number of missed cleavages was permitted. A precursor error was allowed range up to 0.1 m/z in either direction, but fragment ions were required to match within 0.5 m/z. The IDPicker algorithm v2.132 ﬁltered the identiﬁcations for each LC-MS/MS run to include the largest set for which a 1% identiﬁcation false discovery rate could be maintained. Because many identiﬁcations were done from captured Cys-peptides, only one peptide sequence that met the above criteria was required to identify a protein. Indistinguish- able proteins were recognized and grouped, and parsimony rules were applied to generate a minimal list of proteins that explained all of the peptides that passed our entry criteria. False discovery rates (FDR) were computed by the formula: FDR ) (2 × reverse)/(forward + reverse).33 The algorithm reported the number of spectra and number of distinct sequences observed for each protein and protein group in each sample set. Statistical Analysis. Log2-transformed spectral count ratios were compared using a Kruskal-Wallis one-way ANOVA test for multiple comparisons and 95% conﬁdence intervals were determined. Results Synthesis of IBB and its Reaction with a Model Thiol Peptide. We set out to prepare a thiol-reactive biotinylating reagent containing a benzoin ester, as these structures are liable to photodissociation.26,34,35 The synthesis of IBB (Scheme 1) was based on the preparation of methyl ester 4,36 followed by aminolysis to form 5 and subsequent biotinylation to give 6. Subsequent 1,3-dithian deprotection to form 7 was followed by iodoacetylation with iodoacetic acid to afford IBB (8), which Journal of Proteome Research Vol. 9, No. 10, 2010 5463