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Home arrow Health arrow Analysis of Protein Post-Translational Modifications by Mass Spectrometry
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Introduction

Rebecca Pferdehirt, Florian Gnad and Jennie R. Lill

Proteomics and Biological Resources, Genentech Inc., South San Francisco, CA, USA

Post-translational Modification of Proteins

While the human proteome is encoded by approximately 20,000 genes [1, 2], the functional diversity of the proteome is orders of magnitude larger because of added complexities such as genomic recombination, alternative transcript splicing, or post-translational modifications (PTMs) [3, 4]. PTMs include the proteolytic processing of a protein or the covalent attachment of a chemical or proteinaceous moiety to a protein allowing greater structural and regulatory diversity. Importantly, PTMs allow for rapid modification of a protein in response to a stimulus, resulting in functional flexibility on a timescale that traditional transcription and translation responses could never accommodate. PTMs range from global modifications such as phosphorylation, methylation, ubiquitination, and glycosylation, which are found in all eukaryotic species in all organs, to more specific modifications such as crotonylation (thought to be spermatozoa specific) and hypusinylation (specific for EIF5a), which govern more tight regulation of associated proteins. Taken together, over 200 different types of PTMs have been described [5], resulting in an incredibly complex repertoire of modified proteins throughout the cell.

The addition and subtraction of PTMs are controlled by tight enzymatic regulation. For example, many proteins are covalently modified by the addition of a phosphate group onto tyrosine, serine, or threonine residues in a process called phosphorylation [6]. Phosphorylation is catalyzed by a diverse class of enzymes called kinases [7], whereas these phosphomoieties are removed by a second class of enzymes referred to as phosphatases. The tight regulation of kinases and phosphatases often creates “on/off” switches essential for regulation of sensitive signaling cascades. There are some exceptions to this rule however, and the hunt is still underway for the ever-elusive hypusine [8]

Analysis of Protein Post-Translational Modifications by Mass Spectrometry,

First Edition. Edited by John R. Griffiths and Richard D. Unwin.

© 2017 John Wiley & Sons, Inc. Published 2017 by John Wiley & Sons, Inc.

removing enzyme or putative enzymes responsible for the removal of protein arginine methylation. However, it is also possible that proteins bearing these PTMs are modulated or removed from the cell by other mechanisms of action. For example, proteolysis is rarely (if ever) reversible, and many proteins (e.g., blood clotting factors and digestive enzymes) are tightly governed by irreversible cleavage events where the active form is created after proteolysis of a proenzyme.

While PTMs such as phosphorylation and lysine acetylation exist in a binary “on/off” state, many other PTMs exhibit much more complex possible modification patterns. For example, lysine residues can be modified by covalent attachment of the small protein ubiquitin, either by addition of a single ubiquitin or by addition of ubiquitin polymers. In the latter case ubiquitin itself is used as the point of attachment for addition of subsequent ubiquitin monomers [9]. To add another layer of complexity, ubiquitin has seven lysines (K6, K11, K27, K29, K33, K48, and K63), each of which may be used as the point of polyubiquitin chain linkage, and each of which has a different functional consequence. For example, K63-linked chains are associated with lysosomal targeting, whereas K48-linked chains trigger substrate degradation by the proteasome. Thus, even within one type of PTM, multiple subtypes exist, further expanding the functional possibilities of protein modification.

In addition, many proteins are modified on multiple residues by different types of PTMs. A classic example is the PTM of histones. Histones are nuclear proteins that package and compact eukaryotic DNA into structural units called nucleosomes, which are the basic building blocks of chromatin and essential for regulation of gene expression. The C-termini of histones are composed of unstructured tails that protrude from nucleosomes and are heavily modified by methylation, acetylation, ubiquitylation, phosphorylation, SUMOylation, and other PTMs [10]. Overall, 26 modified residues on a single-core histone have been identified, and many of these residues can harbor multiple PTM types. In a generally accepted theory referred to as the “histone code,” the combination of PTMs on all histones comprising a single nucleosome or group of nucleosomes regulates fine-tuned expression of nearby genes.

As we begin to uncover the modified proteome, the importance of the interplay between multiple different PTMs has become increasingly apparent. One classic example is the involvement of both protein phosphorylation and ubiquitylation in the regulation of signaling networks [11]. Protein phosphorylation commonly promotes subsequent ubiquitylation, and the activities of ubiquitin ligases are also frequently regulated through phosphorylation. In a recent study by Ordureau et al., quantitative proteomic studies were employed to describe the PINK1 kinase-PARKIN UB ligase pathway and its disruption in Parkinson's disease [12]. The authors describe a feedforward mechanism where phosphorylation of PARKIN by PINK1 occurs upon mitochondrial damage, leading to ubiquitylation of mitochondria and mitochondrial proteins by PARKIN. These newly formed ubiquitin chains are then themselves phosphorylated by PINK1, which promotes association of phosphorylated PARKIN with polyubiquitin chains on the mitochondria, and ultimately results in signal amplification. This model exemplifies how intricate interactions between multiple different PTMs regulate protein localization, interactions, activity, and ultimately essential cellular processes.

Recent advances in mass spectrometry methods, instrumentation, and bioinformatics analyses have enabled the identification and quantification of proteome-wide PTMs. For example, it is now a common practice to identify ten thousand phosphorylation sites in a single phosphoproteome enrichment experiment [13]. In addition, precise quantitation allows a deeper understanding of the combinations and occupancy of PTMs within a given protein. Such MS-based PTM analyses have led to previously impossible discoveries, advancing our understanding of the role of PTMs in diverse biological processes.

 
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