Iodoacetamide

Surface biotinylation was performed as described elsewhere (Gottardi et al., 1995 (link)). In brief, cells were rinsed in PBS without calcium or magnesium (PBS⁻) and labeled at 4°C for 30 min with 0.5 mg/ml sulfo-NHS-SS biotin (Pierce) in PBS⁻. The reaction was quenched with 20 mM glycine in PBS⁻. To assess the degradation kinetics of biotinylatable (surface) proteins, cells were placed at 37°C to induce endocytosis (and subsequent degradation) for various time points, washed with PBS⁻, and lysed with 1% Triton buffer described earlier. Biotinylated proteins were then enriched by affinity purification with NeutrAvidin beads (Pierce). To assess endocytosis rates, labeled cells were returned to 37°C for various time points but were then placed back on ice. Remaining biotin on the cell surface was stripped with MesNA (Sigma-Aldrich) in 50 mM Tris, pH 8.6, 100 mM NaCl, and 2.5 mM CaCl₂ followed by an alkylation step with 5 mg/ml iodoacetamide (Bio-Rad) in PBS⁻. Cells were washed with PBS⁻ before lysis and enrichment as described. One plate for each condition was not placed at 37°C and served as a control for total labeling efficiency, and an additional plate was not placed at 37°C but was subjected to the stripping and alkylation steps as a control for stripping efficiency.

Cy2, Cy3, and Cy5 were purchased from GE Healthcare. dimethylformamide was purchased from Aldrich. DTT, urea, agarose, glycerol, bromphenol blue, CHAPS, mineral oil, acrylamide, Bis, Tris base, glycine, SDS, iodoacetamide, ammonium persulfate, TEMED, Immobiline DryStrip gels (24 cm, pH 3–10), and Bio-Lyte solutions (pH 3–10) were purchased from Bio-Rad. Thiourea was purchased from Fluka (Buchs, Switzerland). Protease inhibitor mixture was purchased from Roche Applied Science. ACN and methanol were purchased from Fisher. TFA was purchased from Merck. Trypsin (sequencing grade) was purchased from Promega (Madison, WI). All buffers were prepared with Milli-Q water (Millipore, Bedford, MA).