Icat reagent

Viable cells were incubated with 1 mM sodium periodate for 10 min to oxidize glycoproteins [66 (link)]. Oxidized glycoproteins were conjugated to hydrazide resin (Bio-Rad, Hercules, CA) at 4 °C overnight [67 (link)]. After washing sequentially with: 2 M NaCl, 2 % SDS, 200 mM propanolamine (0.1 M NaAcetate, pH 5.5), 40 % ethanol and 80 % ethanol; bound proteins were reduced with dithiothreitol, and alkylated with ICAT™ reagent (Life Technologies/Thermo Fisher Scientific, Applied Biosystems, Framingham, MA). Alkylated proteins were digested with trypsin and cysteine-containing peptides were captured using an avidin column (Life Technologies/Thermo Fisher Scientific). In addition to the cysteine-containing peptide fraction, peptides bound to the resin were also collected and analyzed. Release of peptides was achieved through PNGase-F digestion (New England BioLabs, Ipswich, MA.). While we found some overlap between the proteins identified in the two fractions, analysis of both the cysteine -containing fraction and the resin-bound fraction resulted in complementary coverage of the cell surface protein population.

Samples were lyophilized, reconstituted with deionized H₂O, and dialyzed against 0.6 M Guanidine HCl, 10 mM Tris buffer, pH 8. Proteins were reduced with Tris (2-carboxyethyl) phosphine and alkylated with ICAT™ reagent (Life Technologies/Thermo Fisher Scientific). Following dialysis (0.1 M NH₄Acetate), alkylated proteins were digested with trypsin. Cysteine-containing peptides were purified using an avidin column (Life Technologies/ Thermo Fisher Scientific).