Direct in-gel profiling of protease specificity (DIPPS)

Determination of protease specificity is of crucial importance for understanding protease function. We have developed the first gel-based label-free proteomic approach (DIPPS) that enables quick and reliable determination of protease cleavage specificities under large variety of experimental conditions. The methodology is based on in-gel digestion of the gel-separated proteome with the studied protease, enrichment of cleaved peptides by gel extraction and subsequent mass spectrometry analysis combined with a length-limited unspecific database search. We applied the methodology to profile ten proteases ranging from highly specific (trypsin, endoproteinase GluC, caspase-7 and legumain) to broadly specific (matrix-metalloproteinase-3, thermolysin and cathepsins K, L, S and V). Using DIPPS, we were able to perform specificity profiling of thermolysin at its optimal temperature of 75C, which confirmed the applicability of the method to extreme experimental conditions. Moreover, DIPPS enabled the first global specificity profiling of legumain at pH as low as 4.0, which revealed a pH-dependent change in the specificity of this protease, further supporting its broad applicability.