The lectin-captured proteins, 100 mg, were digested with trypsin, using a procedure described previously . Proteins were first denatured with 6 m guanidine chloride in 0.1 m ammonium bicarbonate buffer, pH 8 and reduced by incubating with 5 mm DTTat 75°C for 1 h, and then alkylated for 2 h with 0.02 m iodoacetamide. The samples were solvent exchanged using 0.1 m ammonium bicarbonate buffer (pH 8) with a 10kDa Amicon filter (0.5 mL capacity; Millipore), and adjusted to the final protein concentration of 0.5 mg/mL. Then, 1 mg trypsin was added to each sample and incubated at ambient temperature overnight. For complete digestion, another aliquot of 1 mg trypsin was added, and the digestion was continued for a total of 24 h. Then, the peptides were separated on a C18 capillary column (in-house packed, 150 x 0.075 mm) using a Surveyor LC pump (Thermo Electron, San Jose, CA, USA). The flow rate was maintained at 300nL/min. The gradient was started at 5% ACN with 0.1% formic acid and a linear gradient to 40% ACN was achieved in 165 min, and then ramped to 60% ACN in 20 min and to 90% in next 10 min. Ten microliters of each sample containing 2 mg of protein was injected on the column from a Surveyor autosampler (Thermo Electron) using the full-loop injection mode. The resolved peptides were analyzed on an LCQ DECA XP IT mass spec trometer (Thermo Electron) with an ESI ion source. The temperature ofthe ion transfer tube was controlled at 185°C and the spray voltage was 2.0kV. The normalized collision energy was set at 35% for MS/MS. Data-dependent ion selection was monitored to select the most abundant five ions from an MS scan for MS/MS analysis. Dynamic exclusion was continued for a duration of 2 min.
Was this article helpful?