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Cells need to detect and react to changes in internal and external conditions. One method used to adjust to these changes is chemically modifying proteins. Conditional chemical changes are relayed from sensors to effectors via reversible post-translational modifications (PTMs) of proteins. PTMs play an important part in modifying the end product of expression, contribute to biological processes and diseased conditions, playing a key role in many cellular processes such as cellular differentiation (1), protein degradation, signaling and regulatory processes, regulation of gene expression, and protein-protein interactions (2,3).
PTMs can happen at any step of the protein lifespan. Many proteins are modified shortly after translation is completed to mediate proper folding or to direct the nascent protein to distinct cellular locations (such as the nucleus or membrane). Other modifications occur after folding and localization are completed to activate or inactivate catalytic activity. Proteins are also covalently linked to tags that target a protein for degradation. They are modified through a combination of post-translational cleavage and the addition of functional groups through a step-wise mechanism of protein maturation or activation.
PTMs occur at distinct amino acid side chains or peptide linkages and are most often mediated by enzymatic activity. Indeed, 5% of the proteome comprises enzymes that perform more than 200 types of PTMs (4). These enzymes include kinases, phosphatases, transferases, and ligases, which add or remove functional groups, proteins, lipids, or sugars to or from amino acid side chains, and proteases, which cleave peptide bonds to remove specific sequences or regulatory subunits. Many proteins can also modify themselves using autocatalytic domains, such as autokinase and autoprotolytic domains. PTMs can also be reversible based on the nature of the modification. As an example, phosphatases hydrolyze the phosphate group to remove it from the protein and reverse its biological activity (Figure 1).
Figure 1. Types of post-translational modifications (PTMs).
Protein phosphorylation (Figure 2) is the most commonly studied post-translational modification. It has been estimated that one-third of mammalian proteins may be phosphorylated, and this modification often plays a key role in modulating protein function. Recent developments in mass-spectrometry (MS) methods have enabled the identification of thousands of PTM sites. Consequently, novel enrichment strategies have uncovered the global cellular importance of several types of modifications (e.g., acetylation, ubiquitylation, O-GlNac, N-linked glycosylation). More than 200 diverse types of PTMs are currently known (5,6), ranging from small chemical modifications (e.g., phosphorylation and acetylation) to the addition of complete proteins (e.g., ubiquitylation, Figure 3).
Figure 2. WB result of phospho-Marcks antibody (10018-3-AP, 1:1500) with mouse J774 macrophage cells treated with PMA.
Figure 3. MDA-MB-453s cells were subjected to SDS PAGE followed by western blot with 10201-2-AP (ubiquitin antibody) at a dilution of 1:600.
The analysis of proteins and their PTMs is particularly important for the study of heart disease, cancer, neurodegenerative diseases, and diabetes (7). The main challenges in studying post-translationally modified proteins are the development of specific detection and purification methods. Fortunately, these technical obstacles are being overcome with a variety of new and refined proteomics technologies.