Applications of Mass Spectrometry in Proteomics Izabela Sokolowska, A Armand G. Ngounou Wetie, 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 Characterisation of proteins and whole proteomes can provide a foundation to our understanding of physiological and pathological states and biological diseases or disorders. Constant development of more reliable and accurate mass spectrometry (MS) instruments and techniques has allowed for better identification and quantification of the thousands of proteins involved in basic physiological processes. Therefore, MS-based proteomics has been widely applied to the analysis of biological samples and has greatly contributed to our understanding of protein functions, interactions, and dynamics, advancing our knowledge of cellular processes as well as the physiology and pathology of the human body. This review will discuss current proteomic approaches for protein identification and characterisation, including post- translational modification (PTM) analysis and quantitative proteomics as well as investigation of protein–protein interactions (PPIs). Manuscript received: 27 March 2013. Manuscript accepted: 14 May 2013. Published online: 3 June 2013. Introduction Proteomics is the study of the proteome, which is the compre- hensive complement of proteins in a cell or organism at any given time. The proteome of an organism, or even of a single type of cells, is much more complex than its corresponding genome. This is primarily due to the changes that can be intro- duced via alternate splicing and post-translational modifications which affect virtually all proteins. The proteome differs from cell to cell and from time to time in its composition depending on the physiological or pathological state of cells or organisms. Understanding that proteins are the actual effectors of biological function has been an essential part of biochemistry for over a hundred years. [1] Due to proteomes’ complexity, their analysis is extremely challenging. Therefore, modern biochemical tech- nologies with improved separation and identification methods have been introduced. Mass spectrometry (MS) became the core of advanced methods in proteomics experiments. The integra- tion of MS with a variety of other analytical methods made it possible to examine virtually all types of samples derived from tissues, organs, and organisms. MS allowed for the identifica- tion and accurate quantification of thousands of proteins and peptides from complex biological samples. Of particular interest is the high potential for outcomes from proteomics experiments to improve the development of diag- nostics and therapeutic strategies in treating human diseases. [2,3] Understanding that proteins, their expression level, and inter- actions, influence the physiological function of our body as well as the development and progression of pathological conditions is a main force driving current biomedical investigations. Therefore, research aiding protein biomarker discovery is rapidly growing. Rapid and reliable identification and quantifi- cation of diagnostic and prognostic markers could improve the quality and length of many lives. [4,5] Putative biomarkers are not the only use of information gained from proteomic studies. Identification of interactions between proteins and their alter- nations can lead to development of new bio-therapeutics along with their targets and potentially to the understanding of the effects of existing therapies. Although the focus of our review is on MS and proteomics with biomedical applications, the same MS and proteomics approaches and techniques also apply to basic research in other animal systems as well as plants, yeast, bacteria, viruses, or various pathogens. Therefore, when we refer to biomedical applications, we in fact refer to all organ- isms. This review will provide an overview of current MS for qualitative identification and characterisation of proteins as well as its use in quantitative and structural proteomics. MS Principle and Instrumentation In proteomic approaches key parameters of MS-based experi- ments are sensitivity, resolution, dynamic range, and mass accuracy. Therefore, various elements need to be taken into consideration during a typical MS-based proteomics experiment (Fig. 1). Proteomics includes several steps that should be adapted according to a final experimental goal. First, proteins to be analysed are isolated from their source and subjected to biochemical fractionation using techniques such as affinity purification followed by 1D or 2D gel electrophoresis. Frac- tionated proteins are then subjected to enzymatic digestion (usually with trypsin) to create a mixture of peptides that may or CSIRO PUBLISHING Aust. J. Chem. 2013, 66, 721–733 http://dx.doi.org/10.1071/CH13137 Journal compilation Ó CSIRO 2013 www.publish.csiro.au/journals/ajc Review RESEARCH FRONT