Proteomics emerges from the protein identification to protein functional elucidation, which depends to a large extent on the characterization of protein sequences. However, a large part of proteome sequences remains unannotated due to the limitation in proteolytic digestion by golden standard protease trypsin. Herein, we demonstrated that a cyanobacterial protease VAILase could specifically cleave at the short-chain aliphatic amino acids valine, alanine, leucine, isoleucine and threonine with cleavage specificity about 92% in total for proteomic analysis. The unique features of VAILase cleavage facilitate the characterization of most proteins and exhibit high complementarity to trypsin, and 22% of the covered sequences by VAILase are unique. VAILase can greatly improve the coverages of sequences with abundant aliphatic residues that are usually dark regions in conventional proteomic analysis, such as the transmembrane regions within anion exchanger 1 and photosystem II.