Protein purification by Off-Gel electrophoresis

Ros, Alexandra; Faupel, Michel; Mees, Hervé; Oostrum, Jan van; Ferrigno, Rosaria; Reymond, Frédéric; Michel, Philippe; Rossier, Joël S.; Girault, Hubert H.

doi:10.1002/1615-9861(200202)2:2<151::aid-prot151>3.0.co;2-9

Cited by 117 publications

(64 citation statements)

References 16 publications

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“…16 We have introduced a new concept named OFFGEL IEF with the first aim to purify proteins. 17 The technique was later successfully used for the isoelectric fractionation of Escherichia coli proteins, proving to be a promising tool for proteomic applications. 18 Besides these general efforts to develop IEF for protein fractionation, IEF has also been used for peptide separation in a shotgun approach, where proteins are first proteolized, and the resulting peptides mixture separated and analyzed by tandem mass spectrometry.…”

Section: Introductionmentioning

confidence: 99%

Modeling the Isoelectric Focusing of Peptides in an OFFGEL Multicompartment Cell

et al. 2007

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In proteomic analysis of complex samples at the peptide level (termed shotgun proteomics), an effective prefractionation is crucial to decrease the complexity of the peptide mixture for further analysis. In this perspective, the high-resolving power of the IEF fractionation step is a determining parameter, in order to obtain well-defined fractions and correct information on peptide isoelectric points, to provide an additional filter for protein identification. Here, we explore the resolving power of OFFGEL IEF as a prefractionation tool to separate peptides. By modeling the peak width evolution versus the peptide charge gradient at pI, we demonstrate that for the three proteomes considered in silico (Deinococcus radiodurans, Saccharomyces cerevisiae, and Homo sapiens), 90% of the peptides should be correctly focused and recovered in two wells at most. This result strongly suggests OFFGEL to be used as a powerful fractionation tool in shotgun proteomics. The influence of the height and shape of the compartments is also investigated, to give the optimal cell dimensions for an enhanced peptide recovery and fast focusing time.

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Section: Introductionmentioning

confidence: 99%

Modeling the Isoelectric Focusing of Peptides in an OFFGEL Multicompartment Cell

et al. 2007

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“…When an electric field is applied along the IPG gel, the current lines pass through the flow chamber inducing the migration of the ions [25]. Figure 7a illustrates the concentration isovalues of the positive and negative ions after 60 s when the concentrations of salt and supporting electrolyte are identical (c 1,2 /c 3,4 = 1).…”

Section: Numerical Simulationmentioning

confidence: 99%

“…In Off-Gel  electrophoresis, the biological sample to be purified is filled in a flowchamber [25,26] or in static multiwells [27], which are positioned on top of an IPG gel. The pH at the bottom of the chamber is chosen to be close to the pI of the proteins to be isolated.…”

Section: Introductionmentioning

confidence: 99%

Salt removal during Off-Gel? electrophoresis of protein samples

et al. 2005

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The Off-Gel  technology was recently described for protein fractionation in a solution placed on top of an immobilized pH gradient gel. In addition, this process was found to remove salts from the biological samples to analyze. This desalting effect is studied experimentally in a conductometric prototype cell. A simplified analytical model is developed to understand this process and a good agreement is found with the conductivity measurements. To illustrate the desalting of a biological sample, a 1 mg?mL 21 solution of b-lactoglobulin A in 0.1 M NaCl is subjected to electrophoresis in a single compartment Off-Gel  cell. The analysis of the resulting sample by ESI-MS demonstrates the effective removal of salt. A finite element diffusion-migration model is also used to illustrate how the nonuniformity of the electric field in the cell, associated with the salt migration, can slow down the desalting process.

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“…Among the techniques, Off-Gel IEF electrophoresis (OGE) is widely used to fractionate proteins and peptides by their isoelectric points (pI), and to recover the purified compounds directly in solution [7][8][9][10]. Fractioned proteins or peptides are buffered by the immobilized pH gradient (IPG) gel used for separation, and can be further analyzed by the standard LC-MS/MS workflow.…”

Section: Introductionmentioning

confidence: 99%

Porous silica enhanced proteolysis during Off-Gel separation for efficient protein identification

Gan

Qiao

et al. 2015

Talanta

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a b s t r a c tAn advanced approach is developed in this work for simultaneous on-line separation and digestion of proteins by combining the Off-Gel isoelectric focusing (IEF) and enzymatic nanoreactor enhanced proteolysis. The nanoreactor was prepared by preloading trypsin in amino-functionalized macroporous silica, and then directly added into Off-Gel wells. With the nanoreactor loaded Off-Gel device, effective digestion of proteins happened during IEF electrophoresis to generate directly fractionated tryptic peptides, which not only accelerated the experimental flow but also avoided sample loss, leading to a more comprehensive protein identification from complex biological samples. A successful identification of 3592 proteins was achieved from Hela cell line by using the approach followed with LC-MS/MS analysis, while only 1877 proteins were identified from the same sample when using standard in-solution proteolysis followed with Off-Gel electrophoresis and then LC-MS/MS analysis. Therefore, we have demonstrated that the approach can greatly simplify high-throughput proteomics and significantly improve protein identification.

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Protein purification by Off-Gel electrophoresis

Cited by 117 publications

References 16 publications

Modeling the Isoelectric Focusing of Peptides in an OFFGEL Multicompartment Cell

Modeling the Isoelectric Focusing of Peptides in an OFFGEL Multicompartment Cell

Salt removal during Off-Gel? electrophoresis of protein samples

Porous silica enhanced proteolysis during Off-Gel separation for efficient protein identification

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