Improved protease digestion conditions for membrane protein detection.

ABSTRACT: This work presents improved protease digestion conditions for membrane protein detection. The enzymatic digest of bacteriorhodopsin (BR), a model membrane protein with seven transmembrane domains (TMDs) was investigated. An initial in-gel digestion identified 17% BR sequence coverage, including part of the seventh TMD. To improve sequence coverage, BR digest was tested with different concentrations of RapiGest, methanol (MeOH) and SDS using either trypsin or chymotrypsin. Two improved conditions, 0.01% SDS or the combination of 10% MeOH and 0.01% RapiGest, were chosen. Trypsin digestions in both conditions achieved more than 40% BR sequence coverage compared to 0% using standard digestion conditions. Peptides detected from trypsin and chymotrypsin digestions in the same condition were comb

SUBMITTER: Min L 

PROVIDER: S-EPMC4739822 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature