Peptide codes for multiple protease activity assay via high-resolution mass spectrometric quantitation.

RATIONALE: Proteases, which are involved in a number of biological processes, play important roles in human health. Alterations in protease activities have been associated with many diseases. Thus, effective methods for multiple protease assay are very essential andmay help to discover more about their functions in different physiological processes. Here, we proposed a strategy of peptide codes for label-free analysis of multiple protease activities using high-resolution mass spectrometry (HRMS). METHODS: The peptide codes were designed to contain a coding region and the substrate of the protease, respectively. Upon the cleavage of target proteases, the coding regions could be rapidly identified and directly quantitated by accurate m/z extractions without internal standard. Therefore, the coding region could be used as the unique “Protease ID” for the identification of the corresponding protease, and the amount of the cleavage product was used for protease activity analysis. RESULTS: The method was validated by using trypsin and chymotrypsin as model proteases, with the designed “Trypsin ID” and “Chymotrypsin ID” occurring at m/z 761.270 and 711.243, respectively. The peak area of “Protease ID” was proportional to trypsin and chymotrypsin concentration in the range from 0.01 to 10 nM and 10 to 2000 nM, respectively. The proposedmethod could also be used for screening of protease inhibitors, whichmight be of great importance for drug discovery. CONCLUSIONS: This method provided a label-free and accurate quantitative platform for convenient identification and activity analysis of multiple proteases, which could potentially be applied in clinical diagnosis. Copyright © 2016 John Wiley & Sons, Ltd.