ArgC-Like Digestion: Complementary or Alternative to Tryptic Digestion?

Enzymatic
digestion of complex protein samples is often performed
by use of multiple proteases to improve protein identification and
characterization. Combining trypsin with ArgC is one option to enhance
sequence coverage in bottom-up proteomics. However, the low selectivity
of this endoprotease derogates from the benefit of the combination.
Our approach here is to mimic ArgC digestion by chemically modifying
all lysine residues in proteins so that trypsin can only cleave C-terminal
to arginine. Four different amine modifications, dimethylation, acetylation,
propionylation, and carbethoxylation, were tested, and the protocols
were optimized. A nearly complete conversion of the primary amines
was achieved for all modifications. Tryptic digestion of Escherichia
coli lysate proteins after acylation of lysine residues shows
the most significant improvement compared with data received from
ArgC digest. After propionylation, 9216 unique peptides identified
1439 proteins, which, compared with a conventional tryptic digestion,
represents the identification of 150 additional proteins due to a
reasonable reduction of the sample complexity and higher fragmentation
efficiencies of the peptides. It is therefore concluded that the Arg-C
like digestion should no longer be regarded as a complementary approach
but forms a viable and superior alternative to the conventional trypsin
digestion.