Two-dimensional gel electrophoresis in bacterial proteomics

Abstract

Two-dimensional gel electrophoresis (2-DE) is a gel-based technique widely used for analyzing the protein composition of biological samples. It is capable of resolving complex mixtures containing more than a thousand protein components into individual protein spots through the coupling of two orthogonal biophysical separation techniques: isoelectric focusing (first dimension) and polyacrylamide gel electrophoresis (second dimension). 2-DE is ideally suited for analyzing the entire expressed protein complement of a bacterial cell: its proteome. Its relative simplicity and good reproducibility have led to 2-DE being widely used for exploring proteomics within a wide range of environmental and medically-relevant bacteria. Here we give a broad overview of the basic principles and historical development of gel-based proteomics, and how this powerful approach can be applied for studying bacterial biology and physiology. We highlight specific 2-DE applications that can be used to analyze when, where and how much proteins are expressed. The links between proteomics, genomics and mass spectrometry are discussed. We explore how proteomics involving tandem mass spectrometry can be used to analyze (post-translational) protein modifications or to identify proteins of unknown origin by de novo peptide sequencing. The use of proteome fractionation techniques and non-gel-based proteomic approaches are also discussed. We highlight how the analysis of proteins secreted by bacterial cells (secretomes or exoproteomes) can be used to study infection processes or the immune response. This review is aimed at non-specialists who wish to gain a concise, comprehensive and contemporary overview of the nature and applications of bacterial proteomics.

Bacterial Electron Transfer Chains Primed by Proteomics

“…This methodology has been used for whole-cell proteomics as well as for the proteomic analyses of subcellular fractions, such as the cytoplasmic proteins, the protein complement associated with the cytoplasmic membrane, with the cell surface outer membrane in Gram-negative bacteria or the cell envelope of Gram-positive species. All these aspects have been described in numerous publications and have been covered in reviews in which the merits and future perspectives of 2DE-MS are critically evaluated (eg, Brewis & Brennan, 2010;Chevalier, 2010;Cordwell, 2004;Curreem, Watt, Lau, & Woo, 2012;G€ org, Weiss, & Dunn, 2004;Issaq & Veenstra, 2008;Mathy & Sluse, 2008;Rabilloud, Chevallet, Luche, & Lelong, 2008a. Here, we would like to illustrate the opportunities and intrinsic shortcomings of 2DE-MS with our work on Methanothermobacter thermautotrophicus strain ΔH (Farhoud, 2011).…”

“…However, like in each protein staining method the practical applicability depends on two factors, detection limit and the dynamic range in protein amounts, expressed as log values, giving a linear response. Conventional staining dyes such as Coomassie Brilliant blue (CBB), colloidal Coomassie and silver nitrate have detection limits of 50, 8-16 and 0.5-4 ng of protein, respectively (Chevalier, 2010;Curreem et al, 2012). (Note that only pico-or femtomoles of proteins are needed for MS analysis.)…”

Isoelectric Point Separations of Peptides and Proteins

“…The use of immobilized pH gradients in strip format is now the standard procedure for carrying out 2DE [18,19]. Applications of 2DE span a broad range of disciplines and have been used for exploring the bacterial proteome [20], mapping protein phosphorylation sites [21,22] and expanding from two-dimensions. A report of a third dimension of electrophoresis has been used to eliminate co-migration challenges [23].…”

Global analysis of bacterial membrane proteins and their modifications

“…This technique has the capability to resolve thousands of proteins, and is very advantageous when studying certain protein features like isoforms. Numerous 2D-PAGE studies have assisted in advancing the identification and knowledge of bacterial membrane proteins as well as the proteomes of many bacterial species in general (Curreem et al, 2012;Li et al, 2007;Peng et al, 2005;Yun et al, 2008); However, this technique faces inherent problems: it is relatively time consuming and it is difficult to identify certain protein classes (e.g. proteins of extreme sizes or PI values).…”