Gel slices for analysis of KQ crosslinked peptides in MAP-rich tubulin

Peptides from an abundant protein can suppress mass spectrometry detection of peptides from less abundant proteins. In the case of MAP-rich tubulin, the abundant tubulin peptides can suppress detection of microtubule associated peptides. We overcame this problem by separating proteins on an SDS polyacrylamide gel, staining with Coomassie blue, cutting the visible bands from the gel, and analyzing tryptic peptides extracted from gel slices. Gel slices were taken from 5 SDS gels, ranging from 15 kDa to the top of the separating gel. Three separate samples were prepared. A total of 29 gel slices were analyzed. Proteins in each of the 29 gel slices were reduced with dithiothreitol, alkylated with iodoacetamide, and digested with trypsin. Peptides were extracted in preparation for LC-MS/MS using the method described by Peeples et al. [28].

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