posted on 2018-04-17, 00:00authored byMinh T.
N. Nguyen, Gerta Shema, René P. Zahedi, Steven H. L. Verhelst
About 2% of the genome of human and
other organisms codes for proteases.
An important step toward deciphering the biological function of a
protease and designing inhibitors is the profiling of protease specificity.
In this work we present a novel, label-free, proteomics-based protease
specificity profiling method that only requires simple sample preparation
steps. It uses proteome-derived peptide libraries and enriches the
cleaved sequences using strong cation exchange chromatography (SCX)
material in a pipet tip. As a demonstration of the method’s
versatility, we successfully determined the specificity of GluC, caspase-3,
chymotrypsin, MMP-1 and cathepsin G from several hundreds to almost
2000 cleavage events per protease. Interestingly, we also found a
novel intrinsic preference of cathepsin G for Asn at the P1 subsite,
which we confirmed using synthetic peptides. Overall, this method
is straightforward and requires so far the lowest investment in material
and equipment for protease specificity profiling. Therefore, we think
it will be applicable in any biochemistry laboratory and promote an
increased understanding of protease specificity.