Activity Profiling of Aminopeptidases in Cell Lysates Using a Fluorogenic Substrate Library

Aminopeptidases are exopeptidases that process peptide bonds at the N-terminus of protein substrates, and they are involved in controlling several metabolic pathways. Due to their involvement in diseases such as cancer or rheumatoid arthritis, their presence can also be used as a predictive biomarker. Here, we used a library of fluorogenic substrates containing natural and unnatural amino acids to reliably measure the aminopeptidase N (APN) activity in cell lysates obtained from human, pig and rat kidneys. We compared our results to the substrate specificity profile of isolated APN. Our data strongly support the observation that fluorogenic substrates can be successfully used to identify aminopeptidases and to measure their activity in cell lysates. Moreover, in contrast to assays using single substrates, which can result in overlapping specificity due to cleavage by several aminopeptidases, our library fingerprint can provide information about single enzymes.