Utility of N-peracetylation of proteins for their structure determination by mass spectrometry.

Acetylation of the animo groups (N-terminus and lysine) of proteins before enzymatic or chemical cleavage was explored as an approach to provide additional information in the course of the determination of amino acid sequences. The major advantage is the ability to differentiate glutamine from lysine, because only the latter is acetylated and thus increases in mass by 42 Da. Horse heart cytochrome c could be fully N-actetylated and even on prolonged digestion with chymotrypsin underwent very little tryptic cleavage, in contrast to the native protein where this side reaction is extensive. Sperm whale myoglobin is more difficult to acetylate, but even at 40%-50% average acetylation, all 19 lysines could be identified unambiguously. A proteolytic digest of acetylated protein is thus a useful component of strategies for the determination of the primary structure of proteins by tandem mass spectrometry.