Iodoacetamide

Sample preparation for LC-MS/MS analysis was performed as described previously [80 (link)]. Briefly, 150 µg of total protein from each sample was separated on a 12.5% SDS-PAGE gel (Lonza, Basel, Switzerland). The gel was stained with Coomassie brilliant blue and each of the protein lanes was cut into six bands and destained with 25 mM ammonium bicarbonate in 50% acetonitrile. The gel pieces were washed with acetonitrile, vacuum dried and rehydrated in reducing solution (10 mM DTT; Fluka Biochemica, Steinheim, Germany), 25 mM NH₄HCO₃. After 45-min incubation at 56 °C, solution was exchanged to alkylating solution (55 mM iodoacetamide; Amersham Biosciences, Little Chalfont, UK), 25 mM NH₄HCO₃ and samples were incubated in the dark at room temperature for 30 min. The gel pieces were washed with acetonitrile and vacuum dried before rehydrating in 80 µL of trypsinization buffer (25 mM NH₄HCO₃) containing 1 µg of sequencing-grade modified porcine trypsine (Promega, Madison, WI, USA) per sample. After overnight incubation at 37 °C, the trypsin solution was collected and remaining peptides were extracted from the gel pieces using extraction solution (50% acetonitrile, 5% formic acid; JT Baker, Center Valley, PA, USA). Trypsin solution was added to extraction solution and concentrated by vacuum drying to a final volume of about 20 µL.

Proteins were separated on a 10% SDS gel and then subjected to silver staining. Protein bands were excised, destained, and subjected to in-gel digestion with trypsin, as previously described [22 (link), 23 (link)]. Briefly, the protein band was destained with 1% potassium ferricyanide and 1.6% sodium thiosulfate (Sigma-Aldrich). Then, the proteins were reduced with 25 mM NH₄HCO₃ (Sigma-Aldrich) containing 10 mM dithiothreitol (Biosynth AG, Switzerland) at 60°C for 30 min and alkylated with 25 mM NH₄HCO₃ containing 55 mM iodoacetamide (Amersham Biosciences, Buckinghamshire, UK) at room temperature for 30 min. After reduction and alkylation, the proteins were digested with sequencing-grade modified porcine trypsin (20 μg/mL; Promega, Madison, WI, USA) at 37°C for 16 h.

Trifluoroacetic acid (TFA), α-cyano-4-hydroxycinnamic acid (CCA), acetonitrile (ACN) and dithiothreitol (DTT) were obtained from Sigma (St. Louis, MO, USA). Immobiline drystrips, ammonium persulfate, urea, agarose, glycerol, bromophenol blue, iodoacetamide (IAA), silver nitrate, 3-[(3-cholamidopropyl)- dimethylammonio]-1-propane (CHAPS), and N, N, N’, N’-tetramethylethylenediamine (TEMED) were from Amersham Pharmacia Biotech (Uppsala, Sweden). Acrylamide, Bis, Tris, glycine, SDS and SDS-PAGE protein standards were from Bio-Rad (Hercules, CA).