Proteolytic Post-translational Modification of Proteins: Proteomic Tools and Methodology

Abstract

Proteolytic processing is a ubiquitous and irreversible post-translational modification involving limited and highly specific hydrolysis of peptide and isopeptide bonds of a protein by a protease. Cleavage generates shorter protein chains displaying neo-N and -C termini, often with new or modified biological activities. Within the past decade, degradomics and terminomics have emerged as significant proteomics subfields dedicated to characterizing proteolysis products as well as natural protein N and C termini. Here we provide an overview of contemporary proteomics-based methods, including specific quantitation, data analysis, and curation considerations, and highlight exciting new and emerging applications within these fields enabling in vivo analysis of proteolytic events. Proteolysis involves the breakdown of proteins into smaller polypeptides or amino acids through the hydrolysis of peptide bonds by a protease. This represents a remarkably significant, but often underappreciated, post-translational modification (PTM) 1 in that is it irreversible yet also ubiquitous. Consequently, the functional sequence of a protein can very rarely be predicted from its transcript, as proteolysis products form new (neo-) N and C termini. These cleavage events, or proteolytic processing events, can result in activation, inactivation, completely altered protein function, and even excision of "neo-proteins" with growth factor activity from an extracellular matrix parent molecule, and they regulate a vast array of biological processes (1). These include DNA replication, cell cycle progression, cell proliferation, and cell death, as well as pathological processes such as inflammation, cancer, arthritis, and cardiovascular disease. For example, in protein synthesis and maturation, precise selective removal of the N-terminal methionine and the signal peptide is essential for correct protein maturation and secretion. In some proteins, scission of the chain forms a molecule with four termini when linked by disulfide bridges. Through the removal of signal, nuclear, and mitochondrial localization sequences and ectodomain shedding, proteases regulate protein localization, and in viral infection, via cleavage of pre-and pro-domains and polyprotein processing, inactive proteins are converted into their active form(s), are inactivated, or change receptor-binding affinity. Thus, proteolysis is involved in much more than the mere degradation and turnover of proteins, important though these processes are in homeostasis.Proteases exist in all orders of life and constitute one of the largest enzyme families in humans (2), and more than 30 drugs targeting these enzymes are currently approved for clinical use (3). However, in order to fully comprehend the cellular function(s) of a given protease, one must have knowledge of the proteins processed by that protease, as well as the functions of these substrates and specific processing events. This is currently far from the case, as half of all human proteases have no known substrates (4). Degradomic...

Protein Termini and Their Modifications Revealed by Positional Proteomics

“…68,72-74 Given the higher reactivity of primary amines over carboxyl groups, positional proteomics initially focused on identifying N-terminal peptides, with C-terminomics approaches slow to develop. Over the last decade, several laboratories have developed various strategies to isolate and characterize protein termini by positive or negative enrichment of N-or C-termini from highly complex protein mixtures (for recent reviews see references 75,76 ). All these strategies follow a general bottom-up proteomics approach where 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 chemical labeling occurs at the protein level before the sample is proteolytically digested, thus unambiguously preserving terminal information.…”

A review on mechanisms and commercial aspects of food preservation and processing

“…The entire phenomena require the presence of miscellaneous protease enzymes [36]. Different specialized proteases play a key role in various regulatory processes.…”

Time-resolved Analysis of the Matrix Metalloproteinase 10 Substrate Degradome

“…S4), suggesting cooperative processing of ADAMTSL1 by both MMP10 and MMP2. DISCUSSION Recent advances in positional proteomics have enabled the reliable identification of protease substrates in complex biological samples (5). Evolving these powerful technologies further, we present a workflow for the time-resolved monitoring of protein termini by exploiting the multiplex capabilities of the recently introduced iTRAQ-TAILS analysis platform (15,21).…”

“…substrate degradomes, in complex and active proteomes (5,6). A common principle of these mass spectrometry-based methods is the enrichment and monitoring of N-terminal peptides (protein neo-N termini) that are newly generated by a test protease (7).…”