Profiling constitutive proteolytic events in vivo

Abstract

Most known organisms encode proteases that are crucial for constitutive proteolytic events. In the present paper, we describe a method to define these events in proteomes from Escherichia coli to humans. The method takes advantage of specific N-terminal biotinylation of protein samples, followed by affinity enrichment and conventional LC (liquid chromatography)-MS/MS (tandem mass spectrometry) analysis. The method is simple, uses conventional and easily obtainable reagents, and is applicable to most proteomics facilities. As proof of principle, we demonstrate profiles of proteolytic events that reveal exquisite in vivo specificity of methionine aminopeptidase in E. coli and unexpected processing of mitochondrial transit peptides in yeast, mouse and human samples. Taken together, our results demonstrate how to rapidly distinguish real proteolysis that occurs in vivo from the predictions based on in vitro experiments.

Finding the needles in the haystack: mapping constitutive proteolytic events in vivo

“…In the method described in this issue of the Biochemical Journal, Timmer et al [1] have developed a selective modification strategy that allows the amines on the side chains of lysine to be guanidinylated while leaving the free N-terminus of the protein intact ( Figure 1A). This allows the desired N-terminal amine to be labelled with an affinity tag for specific enrichment by affinity chromatography.…”

“…By comparison of the sequences of the isolated peptides with genome sequences, it is possible to determine which peptides represent native Ntermini and which have been trimmed by the action of a protease. The results from the study by Timmer et al [1] show that this method provides a relatively comprehensive list of the Nterminal peptides from a large number of proteins. Of particular note, the analysis of Escherichia coli, human, mouse and yeast cells provides a first comprehensive look at the specificity of methionine aminopeptidase, a protease that plays the important role of removing the initial N-terminal methionine found on most newly synthesized proteins.…”

“…Although the work presented by Timmer et al [1] provides a new method to approach the difficult task of globally mapping proteolytic events, a number of equally elegant methods have also been devised that serve to complement this approach. In particular, a number of research groups have made use of acylation to block free amino groups both on the N-terminal backbone nitrogen and also on lysine side chains [3,4].…”

“…These approaches have the advantage that they allow specific protein substrates to be linked to a defined protease of interest, but they generally do not allow assignment of the exact site of proteolysis. An obvious next step in the use of the tagging and enrichment strategy described by Timmer et al [1] would be to use isotopically labelled versions of the biotin tag that would allow direct quantitative monitoring of dynamic changes in proteolytic processing. Significantly, this method would allow the exact site of proteolysis to be mapped at the same time that the substrate is identified.…”

“…Such a difficult problem therefore requires development of technologies that will allow proteolytic events to be monitored on a systemwide level. In this issue of the Biochemical Journal, Timmer et al [1] describe an important new biochemical method for enrichment of the products of proteolysis. This work represents yet another step forward in our quest to understand protease function.…”