Monitoring proteolytic processing events by quantitative mass spectrometry

Abstract

Protease activity plays a key role in a wide variety of biological processes including gene expression, protein turnover and development. misregulation of these proteins has been associated with many cancer types such as prostate, breast, and skin cancer. thus, the identification of protease substrates will provide key information to understand proteolysis-related pathologies. Areas covered: Proteomics-based methods to investigate proteolysis activity, focusing on substrate identification, protease specificity and their applications in systems biology are reviewed. Their quantification strategies, challenges and pitfalls are underlined and the biological implications of protease malfunction are highlighted. Expert commentary: Dysregulated protease activity is a hallmark for some disease pathologies such as cancer. Current biochemical approaches are low throughput and some are limited by the amount of sample required to obtain reliable results. Mass spectrometry based proteomics provides a suitable platform to investigate protease activity, providing information about substrate specificity and mapping cleavage sites.

Keywords: Proteolysis; isotope labeling; mass spectrometry; protease; protease activity; proteomics; substrate recognition.

Figure 1

Protease nomenclature system. The protease cleavage site occurs between amino acid p1 and p1′. The new N terminus will be located at p1′, which is also known as the neo terminus [5].

Figure 2

Diagram of Terminal Amine Isotopic Labeling of Substrate (TAILS) workflow. In general, after the sample is exposed to the protease, primary amines are chemically protected. After trypsin digestion, a new unlabeled N-terminus is generated, facilitating their binding to the polyglycerol matrix. These peptides are captured after ultracentrifugation [24].

Figure 3

Schematic workflow of Proteomics Identification of Protease Cleavage Sites (PICS). After trypsin digestion, amines are derivatized by reductive methylation and cysteines are blocked with iodoacetamide. The peptide library is then incubated with the protease. Neo peptides are biotinylated and recovered using streptavidin [–65].

Figure 4

COFRADIC approach. For in vivo studies, WT cells and cells where the protease is knocked out (KO) are analyzed. After the whole proteome is extracted, proteins are alkylated. All primary amine are acetylated prior to trypsin digestion. Neo-N-terminal peptides are further identified after multiple chromatographic steps.

Figure 5

Comparison between SPECS and glyco-enrichment method. While SPECS is only applicable to the study of sheddase proteases, glycol-enrichment can also capture peptides derived from intramembrane protease activity.