Immunopurification of isopeptides from a tryptic digest of MAP-rich tubulin

MAP-rich tubulin (100 μg) in 0.2 mL of 20 mM TrisCl pH 8.5, 0.01% azide (w/v) was treated with 10 mM dithiothreitol for 3 min in a boiling water bath, followed by alkylation of sulfhydryl groups with 50 mM iodoacetamide for 30 min in the dark. The sample was diluted with water to 0.5 mL. The 0.5 mL sample was injected into a hydrated Slide-A-Lyzer cassette 7,000 MWCO Pierce 66373. After 48 hours of dialysis against 2 x 4 L of 20 mM ammonium bicarbonate at 4˚C, the desalted MAP-rich tubulin was recovered in 0.45 mL of 20 mM ammonium bicarbonate. The carbamidomethylated MAP-rich tubulin was digested with 2 μg trypsin overnight at 37˚C. Trypsin was heat inactivated in a boiling water bath for 5 min.

The 450 μL of carbamidomethylated, desalted tryptic peptides were added to 3 mg Dynabeads-Protein G-81D1C2 and rotated overnight at room temperature. The beads were washed 3 times with phosphate buffered saline, and 2 times with water. Bound peptides were released with 50 μl of 1% (v/v) formic acid pH 2, followed by a second extraction with 50 μl of 1% formic acid. The extracted peptides were dried in a vacuum centrifuge. The dry peptides were dissolved in 20 μL water. The sample was centrifuged at 15,300xg for 20 min before the top 10 μL were transferred to an autosampler vial for mass spectrometry analysis.