In-solution sample preparation for liquid chromatography tandem mass spectrometry (LC-MS/MS)

MAP-rich tubulin solutions (0.2 mL in 20 mM TrisCl pH 8.5, 0.01% azide at 0.5 mg/mL) were diluted to 1 mL before the proteins were reduced with 10 mM dithiothreitol in a boiling water bath for 3 min, alkylated with 50 mM iodoacetamide, and dialyzed against 20 mM ammonium bicarbonate pH 8 to remove salts. The volume of the carbamidomethylated proteins was reduced from 1 mL to 0.05 mL in a vacuum centrifuge. Protein concentration, estimated from absorbance at 280 nm in a NanoDrop spectrophotometer (Thermo Fisher Scientific), indicated a recovery of about 65%. Protein at a concentration of about 1.3 μg/μL in 50 μL of 20 mM ammonium bicarbonate was digested with 1 μg trypsin at 37˚C for 16 h. Trypsin was inactivated by addition of formic acid to 0.1%. Particles that could plug the narrow tubing in the liquid chromatography system were removed by centrifuging the samples for 30 min at 14,000 x g in a microfuge. The top 15 μL were transferred to autosampler vials for liquid chromatography tandem mass spectrometry (LC-MS/MS). The protein concentration in the 15 μL supernatant ranged from 0.5 to 0.9 μg/μL.

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