Proteomics electrophoretic techniques, protein

Although protocols have been constantly improved in recent years (Molloy 2000 Olsson et al. 2002 Luche et al. 2003), hydrophobic membrane proteins, especially those with multiple transmembrane domains (TMD) are generally underrepresented in 2-DE based proteome studies (Santoni et al. 2000). To account for these inherent limitations alternative electrophoretic techniques have to be applied. 

Automation has been inttoduced into the analysis of peptides and proteins, especially in the study of proteomes. Exttacts of proteins from cells are separated using two dimensional gel techniques, usually a chromatographic and an electrophoretic separation. Protein spots are identified and cut out. After tryptic digestion the mixture of peptide fragments is analysed by mass spectrometry, usually either by MALDI-ToF or by nanoelectrospray analysis. The pattern of masses obtained from the peptides is then compared with a database showing the patterns obtained from many proteins. Matches are con ared and identifications are proposed. Several manufacturers offer complete systems which carry out this analysis automatically. 

Wittig I. and Schagger H. 2009. Native electrophoretic techniques to identify protein-protein interactions, Proteomics, 23 5214-5223. 

Electrophoretic techniques are used for nonvolatile compounds, which are permanently or temporarily charged, such as proteins or organic salts. Electrophoretic techniques have an increasing importance in biomedical fields, such as proteomics. 

The dynamic range of protein expression represents a main obstacle since abundant proteins are seldom of interest and others such as transcription factors are only present in a few copies. There is no detector that is able to visualize all proteins at the same time so that prefractionation and the investigation of subproteomes is required. In fact, pre-MS sample preparation techniques exploiting electrophoretic, chromatographic, or chemical properties of the analyte are often the bottleneck of proteomics. 

Mass spectrometry provides a wealth of information for proteomics research, enzymology, and protein chemistry in general. The techniques require only miniscule amounts of sample, so they can be readily applied to the small amounts of protein that can be extracted from a two-dimensional electrophoretic gel. The accurately measured molecular mass of a protein is one of the critical parameters in its identification. Once the mass of a protein is accurately known, mass spectrometry is a convenient and accurate method for detecting changes in mass due to the presence of bound cofactors, bound metal ions, covalent modifications, and so on. 

The use and development of high-resolving separation techniques as well as highly accurate mass spectrometers is nowadays essential to solve the proteome complexity. Currently, more than a single electrophoretic or chromatographic step is used to separate the thousands of proteins found in a biological sample. This separation step is followed by analysis of the isolated proteins (or peptides) by mass spectrometry (MS) via the so-called soft ionization techniques, such as electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) combined with the everyday more powerful mass spectrometers. Two fundamental analytical strategies can be employed the bottom-up and the top-down approach. 

Debye layer on the analyte backbone, and the frictional force from the surrounding fluid. Therefore, it is not a trivial matter to determine or calculate the electrophoretic mobility of a given protein/peptide a priori from the sequence information. Also, electrophoretic mobilities of many proteins among a given proteome can be similar. Therefore, the CZE is not an ideal technique for separating a very complex protein mixture for sample preparation purposes. 

Control of the proteome is obviously of primary importance, since control at the cellular level is exerted via the activities of enzymes either via catalysis of individual steps in pathways, performing transport functions, or acting as regulatory proteins. The proteome is amenable to analysis via extractive techniques followed by chromatographic or electrophoretic separation and analysis, usually by mass spectrometry (MS) (Gavin et al, 2002 Picotti et al., 2013 Washburn, Worters, Yates, 2001). These methods have been particularly useful for gene annotation. 

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