Two-Dimensional Electrophoresis of Membrane Proteins Using Immobilized pH Gradients

Molloy, Mark P.

doi:10.1006/abio.2000.4514

Cited by 300 publications

(147 citation statements)

References 57 publications

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“…Grand average hydropathy scores in several model organisms range from Ϫ2.2 (most hydrophilic) to 1.7 (most hydrophobic) (25). Despite progress (26 -28), only a few proteins with positive grand average hydropathy scores have been identified by 2D electrophoresis (29,30). PC3 cells otherwise lacking PSCA were stably transfected with the PSCA gene.…”

Section: Resultsmentioning

confidence: 99%

Selective Detection of Membrane Proteins Without Antibodies

Arnott¹,

Kishiyama²,

Luis³

et al. 2002

Molecular & Cellular Proteomics

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A method has been developed, called the mass western experiment in analogy to the Western blot, to detect the presence of specific proteins in complex mixtures without the need for antibodies. Proteins are identified with high sensitivity and selectivity, and their abundances are compared between samples. Membrane protein extracts were labeled with custom isotope-coded affinity tag reagents and digested, and the labeled peptides were analyzed by liquid chromatography-tandem mass spectrometry. Ions corresponding to anticipated tryptic peptides from the proteins of interest were continuously subjected to collision-induced dissociation in an ion trap mass spectrometer; heavy and light isotope-coded affinity tag-labeled peptides were simultaneously trapped and fragmented accomplishing identification and quantitation in a single mass spectrum. This application of ion trap selective reaction monitoring maximizes sensitivity, enabling analysis of peptides that would otherwise go undetected. The cell surface proteins prostate stem cell antigen (PSCA) and ErbB2 were detected in prostate and breast tumor cell lines in which they are expressed in known abundances spanning orders of magnitude. Molecular & Cellular Proteomics 1:148 -156, 2002.

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Section: Resultsmentioning

confidence: 99%

Selective Detection of Membrane Proteins Without Antibodies

Arnott¹,

Kishiyama²,

Luis³

et al. 2002

Molecular & Cellular Proteomics

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“…To optimize the solubilization of particular samples such as those containing larger and more hydrophobic proteins, variations in the concentration of chaotropes and reducing agents and utilization of zwitterionic detergents or reducing agents such as tributylphosphine (TBP) has been recommended [94][95][96].…”

Section: Sample Preparationmentioning

confidence: 99%

Two‐dimensional gel electrophoresis and mass spectrometry for biomarker discovery

Penque

2009

Proteomics Clinical Apps

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The proteome project, initiated in 1995, was made possible by 2-DE combined with MS. The project main objective was and remains the identification of all proteins expressed by a cell, tissue or organism in a given time and condition. Following this objective, the global profiling of proteins in health versus pathological state by the 2-DE/MS-based proteomic approach has contributed to the elucidation of the basic mechanisms of disease by discovering candidate disease biomarkers and disease targets for new drug development. This review will briefly summarize the historical evolution of 2-DE up to today, and review 2-DE/MS technology and its specific methods of study of immunoresponse (immunoproteomics), PTM of proteins, complex proteinprotein interactions (interactome), the proteome of cell membrane and intracellular proteome turnover in disease biomarker discovery. 2-DE historical evolutionGlobal profiling of proteins in healthy versus pathological states is a strategy to discover biomarkers for early diagnostics, disease progression, treatment response and novel targets for therapeutic drugs development. The idea of making an inventory of all the proteins in a cell, tissue or organism with normal or abnormal physiology, was (re)initiated in the middle of the 1990s with the proteome project [1]. Since then, many technologies to make the analysis of the proteome a reality have been united.The perfect technological 'marriage' that made the proteome project feasible was between high resolution 2-DE for separation of large numbers of proteins and MS for identification and characterization of the proteins displayed on this gel [2]. However, before this happy union occured, science and technology devoted to protein study had a long journey until 2-DE and MS combination was possible (Fig. 1). It started in the last century, in 1937, when Tiselius [3] introduced electrophoresis technique as a modification of Reiner's early concept (1927) [4] to analyse a complex mixture of proteins such as serum proteins. Only 20 years later, in 1959, electrophoresis on a gel support formed by crosslinked polymerization of Cyanogum 41, a commercial name for two organic monomers, acrylamide and the crosslinking agent, N,N1-methylenebisacrylamide, was shown to be useful and was named PAGE by Raymond and Weintraub [5]. The adaptation of polyacrylamide gel to IEF in a stable pH gradient, developed by Svensson in 1962 [6], was possible by the end of the 1960s [7][8][9]. About this time, the 2-D by combining IEF to separate undenatured proteins according to their charge and gradient SDS-PAGE for a second separation, according to their molecular mass, was for the first time introduced by Kenrick and Margolis (1970) [10]. Only in 1975, was IEF in denaturing conditions, as known today, used to follow the 2-D PAGE and it was described in three independent works [11][12][13]. The most powerful demonstraCorrespondence: Dr. Deborah Penque, Laboratório de Proteó-mica, Departamento de Genética, Instituto Nacional de Saúde, Dr. Ricardo Jorge, I.P., A...

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“…Moreover, quantitation of proteins on 2-DE gels is cumbersome and requires many replicate gels. In addition hydrophobic, extremely basic proteins and those with a very high or low molecular weight are often difficult to analyse using such approaches [6][7][8]. The application of chromatography in proteomic studies has been used increasingly over recent years [9,10].…”

Section: Introductionmentioning

confidence: 99%

Multidimensional liquid phase protein separations in conjunction with stable isotope labelling for quantitative proteomics

et al. 2007

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A method for quantitative protein profiling has been developed utilising multidimensional liquid phase protein separations in conjunction with stable isotope labelling. This approach combines the advantages of high throughput, automated, reproducible protein separations with accurate protein quantitation performed in the mass spectrometer. Escherichia coli cells were grown in the presence and absence of the DNA methylation inhibitor 5-Azacytidine on 14 N and 15 N enriched media. Protein separations were performed using ion exchange chromatography in the first dimension and RP capillary chromatography in the second dimension. UV absorbance measurements were used for the initial semiquantitative identification of differentially expressed proteins. Selected peaks from the mixed 15 N/ 14 N lysates were used for the accurate quantitation performed in the mass spectrometer using the ratios of the stable isotopes. Using this approach, a number of differentially expressed proteins have been identified. Moreover, this approach overcomes a number of caveats associated with multidimensional liquid phase protein separations, including the presence of multiple proteins present in a single chromatographic peak.

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Two-Dimensional Electrophoresis of Membrane Proteins Using Immobilized pH Gradients

Cited by 300 publications

References 57 publications

Selective Detection of Membrane Proteins Without Antibodies

Selective Detection of Membrane Proteins Without Antibodies

Two‐dimensional gel electrophoresis and mass spectrometry for biomarker discovery

Multidimensional liquid phase protein separations in conjunction with stable isotope labelling for quantitative proteomics

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