Effectiveness and limitation of two-dimensional gel electrophoresis in bacterial membrane protein proteomics and perspectives

Bunai, Keigo; Yamane, Kunio

doi:10.1016/j.jchromb.2004.08.030

Cited by 97 publications

(85 citation statements)

References 40 publications

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“…In total, this proteomic profile probably represents the proteins most abundantly displayed on the cell envelope of B. longum NCIMB 8809, a human isolate with the capacity to produce an antimicrobial substance (O'Riordan & Fitzgerald, 1998). 2D electrophoresis-based workflows, the most popular techniques for the study of bacterial stress responses, do not provide information on hydrophobic proteins, mainly membrane proteins, due to technical difficulties in retaining their solubility during the isoelectrofocusing and limitations in reliable quantification (Bunai & Yamane, 2005;Speers & Wu, 2007). Some of these disadvantages have been overcome with new techniques based on improved solubilizing agents or isotopic labelling (Couté et al, 2007;Speers & Wu, 2007), expanding the possibilities for analysing bacterial membrane subproteomes.…”

Section: Discussionmentioning

confidence: 99%

The cell-envelope proteome of Bifidobacterium longum in an in vitro bile environment

et al. 2009

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Host-bacteria interactions are often mediated via surface-associated proteins. The identification of these proteins is an important goal of bacterial proteomics. To address how bile can influence the cell-envelope proteome of Bifidobacterium longum biotype longum NCIMB 8809, we analysed its membrane protein fraction using stable isotope labelling of amino acids in cell culture (SILAC). We were able to identify 141 proteins in the membrane fraction, including a large percentage of the theoretical transporters of this species. Moreover, the envelope-associated soluble fraction was analysed using different subfractionation techniques and differential in-gel fluorescence electrophoresis (DIGE). This approach identified 128 different proteins. Some of them were well-known cell wall proteins, but others were highly conserved cytoplasmic proteins probably displaying a 'moonlighting' function. We were able to identify 11 proteins in the membrane fraction and 6 proteins in the envelope-associated soluble fraction whose concentration varied in the presence of bile. Bile promoted changes in the levels of proteins with important biological functions, such as some ribosomal proteins and enolase. Also, oligopeptide-binding proteins were accumulated on the cell surface, which was reflected in a different tripeptide transport rate in the cells grown with bile. The data reported here will provide the first cell-envelope proteome map for B. longum, and may contribute to understanding the bile tolerance of these bacteria

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Section: Discussionmentioning

confidence: 99%

The cell-envelope proteome of Bifidobacterium longum in an in vitro bile environment

et al. 2009

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“…Results obtained by global solution-based, multidimensional shotgun investigations suggest that 16 O/ 18 O labeling is a reliable and powerful tool for comparative proteomics and offers significant advantages over the 2D-PAGE-based comparative proteomics by allowing unbiased proteome coverage [33] and high analytical throughput [34]. It is important to stress that relative changes in protein concentrations obtained by shotgun proteomics depict changes in protein abundances only at a given point in time.…”

Section: Introductionmentioning

confidence: 99%

18O Stable Isotope Labeling in MS-based Proteomics

Ye¹,

Luke²,

Andrésson³

et al. 2009

Briefings in Functional Genomics and Proteomics

113

126

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“…Subsequent studies on the E. coli membrane proteins have optimized this sample preparation method, enabling 394 membrane proteins to be identified (Fountoulakis and Gasser, 2003). Following this milestone in examining bacterial membrane proteins using 2-DE, many Gram-positive and Gram-negative bacterial membranes proteomes, such as those from Mycobacterium tuberculosis, Campylobacter jejuni, P. aeruginosa and S. aureus have been investigated (Nouwens et al, 2002;Bunai and Yamane, 2005;Gatlin et al, 2006;Poetsch and Wolters, 2008). Membrane proteins are often highly immunogenic, and have been investigated in search of potential vaccine targets.…”

Section: Membrane Proteinsmentioning

confidence: 99%

Two-dimensional gel electrophoresis in bacterial proteomics

et al. 2012

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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.

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Effectiveness and limitation of two-dimensional gel electrophoresis in bacterial membrane protein proteomics and perspectives

Cited by 97 publications

References 40 publications

The cell-envelope proteome of Bifidobacterium longum in an in vitro bile environment

The cell-envelope proteome of Bifidobacterium longum in an in vitro bile environment

18O Stable Isotope Labeling in MS-based Proteomics

Two-dimensional gel electrophoresis in bacterial proteomics

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