Journal article

S-pyridylethylation of intact polyacrylamide gels and in situ digestion of electrophoretically separated proteins: A rapid mass spectrometric method for identifying cysteine-containing peptides

RL Moritz, JS Eddes, GE Reid, RJ Simpson



In-gel proteolytic digestion of acrylamide-gel separated proteins is a method widely used for generating peptide fragments for the purpose of identifying proteins by Edman degratation, tandem mass spectrometry, and peptide-mass fingerprinting. However, it is well recognised for disulfide-bonded proteins electrophoresed under reducing conditions that if no precautions are taken to minimise disulfide bond formation during protein digestion or peptide isolation, complex peptide maps can result. Here, we describe an improved method for in-gel protein digestion. It consists of first reducing and S-pyridylethylating Coomassie Brilliant Blue R-250-stained proteins immobilised in the whole gel slab ..

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University of Melbourne Researchers