Monitoring proteolytic processing events by quantitative mass spectrometry

Abstract

Introduction 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 (MS) based proteomics provides a suitable platform to investigate protease activity, providing information about substrate specificity and mapping cleavage sites.

Thyroid Disorders Change the Pattern of Response of Angiotensinase Activities in the Hypothalamus-Pituitary-Adrenal Axis of Male Rats

“…The functional status of the endogenous peptidergic substrates is reflecting the proteolytic activity of the enzyme offering an accurate perspective for the possible functional changes of those enzymes under selective experimental conditions ( 5 ). We studied the activity of several aminopeptidases, called angiotensinases, in the hypothalamic-pituitary-adrenal axis as a reflection of the functional status of certain angiotensin peptides involved in blood pressure control.…”

“…However, it should be taken into account that these enzymatic activities may also reflect the hydrolysis of other peptides such as cholecystokinin by AspAP and GluAP ( 8 ) or enkephalins by AlaAP ( 9 ) (Figure 1 ). Therefore, the activity of these enzymes reflects the functional status of their respective endogenous substrates as well as of the derived peptides ( 5 ).…”

The Proteasome Activators Blm10/PA200 Enhance the Proteasomal Degradation of N-Terminal Huntingtin

“…To describe cleavage specificity, we used the Berger and Schechter nomenclature [ 61 ]. Proteolytic cleavage depends on substrate recognition by the protease [ 62 , 63 ]. According to Berger and Schechter, substrate residues around the protease-binding pocket are denoted as P4-P3-P2-P1↓P1′-P2′-P3′-P4′.…”

Approaches to identify and characterize microProteins and their potential uses in biotechnology

“…The MS analysis will therefore yield results based on small peptides which serve as surrogates to determine the precursor peptides. Although more widely used, this method is disadvantageous because it does not preserve information of PTMs and can result in difficulties in distinguishing isoforms and homologs [ 30 , 35 , 36 ]. Irrespective of what method is used to identify cleavage products, the resulting list of cleavage products can be assessed with the MiPFinder to determine if they qualify as potential microProteins.…”