Lys-C/Arg-C, a More Specific and Efficient Digestion Approach for Proteomics Studies

Nowadays, bottom-up approaches are
predominantly adopted in proteomics
studies, which necessitate a proteolysis step prior to MS analysis.
Trypsin is often the best protease in choice due to its high specificity
and MS-favored proteolytic products. A lot of efforts have been made
to develop a superior digestion approach but hardly succeed, especially
in large-scale proteomics studies. Herein, we report a new tandem
digestion using Lys-C and Arg-C, termed Lys-C/Arg-C, which has been
proven to be more specific and efficient than trypsin digestion. Reanalysis
of our previous data (Anal. Chem. 2018, 90 (3), 1554–1559) revealed that both Lys-C
and Arg-C are trypsin-like proteases and perform better when considered
as trypsin. In particular, for Arg-C, the identification capacity
is increased to 2.6 times and even comparable with trypsin. The good
complementarity, high digestion efficiency, and high specificity of
Lys-C and Arg-C prompt the Lys-C/Arg-C digestion. We systematically
evaluated Lys-C/Arg-C digestion using qualitative and quantitative
proteomics approaches and confirmed its superior performance in digestion
specificity, efficiency, and identification capacity to the currently
widely used trypsin and Lys-C/trypsin digestions. As a result, we
concluded that the Lys-C/Arg-C digestion approach would be the choice
of next-generation digestion approach in both qualitative and quantitative
proteomics studies. Data are available via ProteomeXchange with identifier
PXD009797.