Identification of Post-Translational Modifications by Mass Spectrometry Armand G. Ngounou Wetie, A Izabela Sokolowska, A Alisa G. Woods, A,B and Costel C. Darie A,C A Biochemistry and Proteomics Group, Department of Chemistry and Biomolecular Science, Clarkson University, 8 Clarkson Avenue, Potsdam, NY, 13699-5810, USA. B Neuropsychology Clinic and Psychoeducation Services, SUNY Plattsburgh, Plattsburgh, NY, 12901, USA. C Corresponding author. Email: cdarie@clarkson.edu Proteins are the effector molecules of many cellular and biological processes and are thus very dynamic and flexible. Regulation of protein activity, structure, stability, and turnover is in part controlled by their post-translational modifications (PTMs). Common PTMs of proteins include phosphorylation, glycosylation, methylation, ubiquitination, acetylation, and oxidation. Understanding the biology of protein PTMs can help elucidate the mechanisms of many pathological conditions and provide opportunities for prevention, diagnostics, and treatment of these disorders. Prior to the era of proteomics, it was standard to use chemistry methods for the identification of protein modifications. With advancements in proteomic technologies, mass spectrometry has become the method of choice for the analysis of protein PTMs. In this brief review, we will highlight the biochemistry of PTMs with an emphasis on mass spectrometry. Manuscript received: 30 March 2013. Manuscript accepted: 17 May 2013. Published online: 1 July 2013. Introduction Chemistry of Proteins Proteins are biopolymers that are made up of amino acids and can therefore be viewed as chemical molecules. Post- translational modifications (PTMs) of proteins make use of the many functional groups present on a protein backbone with different side chains. We can distinguish between two types of PTMs. One occurs by the enzyme-catalysed covalent addition of an electrophilic group to the mostly nucleophile side chain residues of the protein. The other type occurs through covalent cleavage of peptide backbones. Of the 22 proteinogenic amino acids known, only leucine, isoleucine, valine, alanine, and phenylalanine have not been shown to be modifiable in vivo. [1] The amino acid residues of proteins are mostly nucleophilic with the cysteinic thiol group being one of the most used for the regioselective modification of proteins. Thiol groups of cysteines can undergo numerous reactions such as alkylation (with a-haloketones or Michael acceptors, e.g. maleimide deri- vatives). Another functional nucleophilic group used in amino acid residues is the e-amino group of lysine. There are many reactions based on the site-specific modification of primary amines in proteins. Reactions of lysines with esters (formation of amides), sulfonyl chlorides (building of sulfonamides), iso- cyanates (production of ureas), and isothiocyanates (thioureas) are common. [2] Furthermore, the functional side chains of arginine, histidine, aspartic acid, glutamic acid, and tyrosine can acts as nucleophiles as well. In general, the nucleophilicity order of amino acids follows the trend: R–S  . R–NH 2 .. R– COO  ¼ R–O  . However, this order depends on several other factors such as the polarisability of the nucleophile or the solvent used (protic or aprotic) or the conjugation of the nucleophile. For example, R–COO  (carboxylate) is less nucleophilic than R–O  (alkoxide) because of conjugation. Through chemistry, non-proteinogenic amino acids and other functional groups can be introduced into proteins. PTM Proteomics: Biological Implications The human genome encodes ,25000 genes, of which ,2% code for protein synthesis. However, due to alternative splicing mechanisms, ,100000 proteins are genetically encoded. Fur- thermore, by means of PTM Proteomics: Biological Implica- tions, this number increases to some 1–2 million protein entities. [3] PTMs can occur either enzymatically (phosphoryla- tion, glycosylation) or non-enzymatically (glycation, oxidation) (Fig. 1). [4] Protein PTMs expand the dimension and the repertoire of functions performed by these biomolecules. [5] Hundreds of unrelated PTMs exist that are known and characterised with regard to their involvement in physiological and pathological bioprocesses (Fig. 1). For this reason, it is of high importance to identify and characterise PTMs to be able to prevent and treat diseases. Most PTMs are reversible, but can also be spontaneous (no involvement of enzymes). Proteomics is designed to identify proteins, their sequence, and known modifications as well as their quantitation in a biological sample. [6–14] It is likely that due to challenges associated with analytical techniques and data analysis, many protein modifications are still to be determined. CSIRO PUBLISHING Aust. J. Chem. 2013, 66, 734–748 http://dx.doi.org/10.1071/CH13144 Journal compilation Ó CSIRO 2013 www.publish.csiro.au/journals/ajc Review RESEARCH FRONT