The current state of two-dimensional electrophoresis with immobilized pH gradients

Görg, Angelika; Obermaier, Christian; Boguth, Günther; Harder, Alois; Scheibe, Burghardt; Wildgruber, Robert; Weiss, Walter

doi:10.1002/(sici)1522-2683(20000401)21:6<1037::aid-elps1037>3.0.co;2-v

Cited by 1,524 publications

(527 citation statements)

References 58 publications

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“…The traditional method of mapping the protein content of cells has been 2-D SDS-PAGE [35][36][37][38]. In this format the proteins are mapped according to pI in one dimension and electrophoresis, which is related to molecular weight, in the second dimension.…”

Section: Introductionmentioning

confidence: 99%

A protein molecular weight map of ES2 clear cell ovarian carcinoma cells using a two-dimensional liquid separations/mass mapping technique

Wang¹,

Kachman²,

Schwartz³

et al. 2002

Electrophoresis

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A molecular weight map of the protein content of ES2 human clear cell ovarian carcinoma cells has been produced using a two-dimensional (2-D) liquid separations/mass mapping technique. This method uses a 2-D liquid separation of proteins from whole cell lysates coupled on-line to an electrospray ionization-time of flight (ESI-TOF) mass spectrometer to map the accurate intact molecular weight (M(r)) of the protein content of the cells. The two separation dimensions involve the use of liquid isoelectric focusing as the first phase and nonporous silica reversed-phase high-performance liquid chromatography (HPLC) as the second phase of separation. The detection by ESI-TOF-MS provides an image of pI versus M(r) analogous to 2-D gel electrophoresis. Each protein is then identified based upon matrix-assisted laser desorption/ionization (MALDI)-TOF-MS peptide mapping and intact M(r) so that a standard map is produced against which other ovarian carcinoma cell lines can be compared. The accurate intact M(r) together with the pI fraction, and peptide map serve to tag the protein for future interlysate comparisons. An internal standard is also used to provide a means for quantitation for future interlysate studies. In the ES2 cell line under study it is shown that nearly 900 M(r) bands are detected over 17 pI fractions from pH 4 to 12 and a M(r) range up to 85 kDa and that around 290 of these bands can be identified using mass spectrometric based techniques. The protein M(r) is detected within an accuracy of 150 ppm and it is shown that many of the proteins in this human cancer sample are modified compared to the database. The protein M(r) map may serve as a highly reproducible standard Web-based method for comparing proteins from related human cell lines.

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Section: Introductionmentioning

confidence: 99%

A protein molecular weight map of ES2 clear cell ovarian carcinoma cells using a two-dimensional liquid separations/mass mapping technique

Wang¹,

Kachman²,

Schwartz³

et al. 2002

Electrophoresis

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“…The challenge of analysing a complicated proteome may be addressed by the use of very narrow-range IPGs (reviewed in [6][7][8]). These provide a means to 'zoom-in' on, and expand, crowded areas of broad-range/narrowrange IPG 2-D gels for better resolution and separation [9].…”

mentioning

confidence: 99%

“…Human heart (left ventricle) proteins were prepared from a tissue sample of unused transplant donor organ and subjected to 2-D PAGE essentially according to Görg et al [6] and Weekes et al [10]. Duplicate analytical loadings of broad-range (3-10NL; 100 g total protein), narrow-range (4-7L, 6-9L; 100 mg total protein) and very narrow-range IPGs (3.5-4.5L, 4-5L, 4.5-5.5L, 5-6L, 5.5-6.7L; 200 g total protein) were used.…”

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confidence: 99%

Zooming-in on the proteome: Very narrow-range immobilised pH gradients reveal more protein species and isoforms

Westbrook¹,

Yan²,

Wait³

et al. 2001

Electrophoresis

142

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Two-dimensional gel electrophoresis (2-DE) enables separation of complex mixtures of proteins on a single polyacrylamide gel according to isoelectric point, molecular weight, solubility, and relative abundance. For this reason, 2-DE together with mass spectrometry (MS) has become a key technology in proteome analysis. The introduction of immobilised pH gradients (IPGs) for isoelectric focusing of proteins affords improved reproducibility and permits full-scale proteome analyses to be undertaken. Whilst broad-range IPGs are useful for investigating simple proteomes (e.g. Mycoplasma genitalium) it is becoming clear that additional resolving power is needed for separating the more complex proteomes of eukaryotic organisms. The use of narrow-range and very narrow-range IPGs provides the means with which to dissect a complex proteome. We have compared very narrow-range IPGs (3.5-4.5L, 4-5L, 4.5-5.5L, 5-6L, and 5.5-6.7L) with broad- (3-10NL) and narrow-range IPGs (4-7L and 6-9L) for the visualisation of the human heart proteome. The superior ability of very narrow-range IPGs to separate different protein species and isoforms, compared with 3-10NL and 4-7L 2-D gels is demonstrated. The results are supported by MS identifications which further show that reduction of the number of comigrating protein species results in less ambiguous and more reliable database search results.

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“…Although 2-D PAGE has been greatly improved by advances in gel quality and refinement of separation methodologies [31], it is still problematic for separating large proteins (e.g., .100 000 Da under conditions when smaller proteins can also be observed), extremely acidic or basic proteins (pI ,3.5 or .9.5), and less soluble (e.g., membrane) proteins. Such proteins may, in total, account for as much as half of all proteins that may be expressed by an organism.…”

Section: Gel-based Separationmentioning

confidence: 99%

Proteomics based on high-efficiency capillary separations

2002

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Identifying and quantifying in a high throughput manner the proteins expressed by cells, tissues or an organism provides the basis for understanding the functions of its constituents at a "systems" level. As a result, proteome analysis has increasingly become the focus of significant interest and research over the past decade. This is especially true following the recent stunning achievements in genomics analyses. However, unlike the static genome, the complexities and dynamism of the proteome present significant analytical challenges and demand highly efficient separations and detection technologies. A number of recent technological advancements have been in direct response to these challenges. Currently, strategically mated combinations of sophisticated separations techniques and advanced mass spectrometric detection represent the best approach to addressing the intricacies of the proteome. Liquid-phase separations, often within capillaries, are increasingly recognized as the best separations technique for this approach. In combination on-line with mass spectrometry, liquid-phase separations provide the improved analytical sensitivity, sample throughput, and quantitation capabilities necessitated by the multifaceted problems within proteomics analyses. This review focuses primarily on current high-efficiency capillary separations techniques, including both capillary liquid chromatography and capillary electrophoresis, applied to the analysis of complex proteomic samples. We emphasize developments at our laboratory and illustrate technical advances that attempt to review the role of separations within the broader context of a state-of-the-art integrated proteomics effort.

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The current state of two-dimensional electrophoresis with immobilized pH gradients

Cited by 1,524 publications

References 58 publications

A protein molecular weight map of ES2 clear cell ovarian carcinoma cells using a two-dimensional liquid separations/mass mapping technique

A protein molecular weight map of ES2 clear cell ovarian carcinoma cells using a two-dimensional liquid separations/mass mapping technique

Zooming-in on the proteome: Very narrow-range immobilised pH gradients reveal more protein species and isoforms

Proteomics based on high-efficiency capillary separations

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