Application of CysTRAQ high throughput screening The CysTRAQ technology was applied according to our recently published method [23]. Briefly, peptides were labeled with iTRAQ (isobaric tags for relative and absolute quantitation) reagents (AB Sciex, Foster City, CA) for 2 h. The MIAC- and HCA-negative pooled samples were labeled with 114 and 116 tags, while MIAC- and HCA-positive pooled samples were labeled with 115 and 117 tags. After the labeling step, the samples were diluted three-fold with water, combined, acidified using TFA (Sigma) to pH 1�C2 and incubated for 30 min at 37��C to hydrolyze both RapiGest as well as unreacted iTRAQ tags. The hydrophobic part of RapiGest was removed by centrifugation and the supernatant was desalted using an Oasis HLB 1cc (30 mg) SPE column (Waters), and vacuum dried.

Twenty-five milligrams of the Thiopropyl-Sepharose 6B thiol affinity resin powder (GE Healthcare, Uppsala, Sweden) was rehydrated in water and washed with coupling buffer (50 mM Tris, 1 mM EDTA, pH 7.5). Peptides were dissolved in 20 ��l of 5 mM dithiothreitol (Sigma) in coupling buffer and reduced for 1 h at 60��C. The sample was diluted to 100 ��l with coupling buffer and incubated with the slurry for 2 h at 37��C. Unbound non-cysteinyl peptides were captured using the Macro SpinColumn (Harvard Apparatus, Holliston, MA). The beads were then washed with 2.5 ml of each of the following solutions: washing buffer (50 mM Tris, 1 mM EDTA, pH 8.0); 2 M NaCl; 80% acetonitrile (ACN), 0.1% TFA; and washing buffer. Bound peptides (cysteinyl peptides) were released by incubation with 100 ��l of 50 mM dithiothreitol in washing buffer for 1 h at 60��C.

Both fractions were desalted on Oasis HLB 1cc (10 mg) Extraction Cartridge SPE columns and dried. The peptides were reduced with 5 mM tris(2-carboxyethyl) phosphine hydrochloride for 1 hour at 60��C, and cysteines were blocked with 10 mM methyl methanethiosulfonate (AB Sciex) for 10 min at RT. Basic pH reversed-phase peptide fractionation Desalted cysteinyl and non-cysteinyl peptide fractions were redissolved in 200 ��l of 20 mM ammonium formate (NH4FA). The fractionation was performed on the Alliance 2695 HPLC system. Non-cysteinyl peptides (100 ��l) and cysteinyl peptides (200 ��l) were injected onto a Gemini C18 150��2 mm column (Phenomenex, Torrance, CA) filled with 3 ��m, 110 ? particles. The peptides were separated by a linear gradient, from 5% ACN, 20 mM NH4FA to 55% ACN, 20 mM NH4FA in 62 min. The eluting peptides were collected between 20 and 60 min of separation resulting in 18 collected fractions per sample. Each fraction Anacetrapib was acidified with formic acid, and the samples were dried in vacuo. LC-MS/MS analysis Each basic pH fraction was redissolved in 40 ��l of 5% ACN, 0.