Multi-dimensional liquid phase based separations in proteomics

Wang, Hong; Hanash, Samir M.

doi:10.1016/s1570-0232(02)00335-5

Cited by 194 publications

(140 citation statements)

References 43 publications

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“…Analytical-scale, biological separations are most commonly accomplished with the use of LC, CE, or SDS-PAGE. Even state-of-the-art advancements, namely multidimensional LC [4] and 2-DE [5], have proven only partially successful in the deconvolution of complex mixtures [6][7][8].…”

Section: Introductionmentioning

confidence: 99%

Characterization of voltage degradation in dynamic field gradient focusing

Burke

Ivory

2008

Electrophoresis

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Dynamic field gradient focusing (DFGF) is an equilibrium gradient method that utilizes an electric field gradient to simultaneously separate and concentrate charged analytes based on their individual electrophoretic mobilities. This work describes the use of a 2-D nonlinear, numerical simulation to examine the impact of voltage loss from the electrodes to the separation channel, termed voltage degradation, and distortions in the electric field on the performance of DFGF. One of the design parameters that has a large impact on the degree of voltage degradation is the placement of the electrodes in relation to the separation channel. The simulation shows that a distance of about 3 mm from the electrodes to the separation channel gives the electric field profile with least amount of voltage degradation. The simulation was also used to describe the elution of focused protein peaks. The simulation shows that elution under constant electric field gradient gives better performance than elution through shallowing of the electric field. Qualitative agreement between the numerical simulation and experimental results is shown. The simulation also illustrates that the presence of a defocusing region at the cathodic end of the separation channel causes peak dispersion during elution. The numerical model is then used to design a system that does not suffer from a defocusing region. Peaks eluted under this design experienced no band broadening in our simulations. Preliminary experimental results using the redesigned chamber are shown.

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Section: Introductionmentioning

confidence: 99%

Characterization of voltage degradation in dynamic field gradient focusing

Burke

Ivory

2008

Electrophoresis

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“…This would result in less interference between peptides during the LC-MS/MS, thus leading to larger numbers of identified peptides. Very large increases in the number of identified peptides and proteins in complex samples were recently achieved by using multidimensional protein identification technology (MudPIT) combining strong cation exchange (SCX) with long RP gradients [3,6,11] (reviewed in [12][13][14]) and by performing repeated chromatographies with mass segmentation selection of peptides for fragmentation [3,6]. However, even with the increased number of steps employed for prefractionation, the number of identified proteins eventually reaches a plateau, since the concentration of some rare proteins is still below the needed threshold level of sensitivity for the mass spectrometers.…”

Section: The Hupo Plasma Proteome Project (Ppp) Goals and The Serum Amentioning

confidence: 99%

Evaluation of prefractionation methods as a preparatory step for multidimensional based chromatography of serum proteins

et al. 2005

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Prefractionations of proteins prior to their proteolysis, chromatography, and MS/MS analyses help reduce complexity and increase the yield of protein identifications. A number of methods were evaluated here for prefractionating serum samples distributed to the participating laboratories as part of the human Plasma Proteome Project. These methods include strong cation exchange (SCX) chromatography, slicing of SDS-PAGE gel bands, and liquid-phase IEF of the proteins. The fractionated proteins were trypsinized and the resulting peptides were resolved and analyzed by multidimensional protein identification technology coupled to IT MS/MS. The MS/ MS spectra were clustered, combined, and searched against the IPI protein databank using PepMiner. The identification results were evaluated for the efficacy of the different prefractionation methodologies to identify larger numbers of proteins at higher confidence and to achieve the best coverage of the proteins with the identified peptides. Prefractionation based on SCX resulted in the largest number of identified proteins, followed by gel slices and then the liquid-phase IEF. An important observation was that each of the methods revealed a set of unique proteins, some identified with high confidence. Therefore, for comprehensive identification of the serum proteins, several different prefractionation approaches should be used in parallel.

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“…This approach incorporates multidimensional highperformance liquid chromatography (HPLC), ESI-MS/ MS, and database searching algorithms. 6 Multidimensional HPLC was proposed more than a decade ago as a way to separate proteins and peptides, with the past few years having seen development of highly effective methods for peptide separation. An example is nanoflow capillary reverse-phase liquid chromatography (cRPLC)-ESI-MS/ MS, in which separation of complex digested protein mixtures by nanoflow cRPLC is followed immediately by online mass spectroscopic detection using automated acquisition modes that allow conventional MS and MS/ MS spectra to be collected in a data-dependent manner.…”

Section: Proteome Analysismentioning

confidence: 99%

Proteome analysis of prostate cancer

Kuruma

Egawa

Ohishi

et al. 2004

Prostate Cancer Prostatic Dis

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In this paper, we briefly review cancer proteomics in general, with particular attention to our proteome analyses of prostate cancer. Our efforts include development of new tools and novel approaches to discovering proteins potentially useful as cancer diagnostic and/or prognostic biomarkers or as therapeutic targets. To this end, we analyzed prostate cancer proteomes using twodimensional gel electrophoresis employing agarose gels for the initial isoelectric focusing step (agarose 2-DE), with mass spectrometry used for protein identification. Agarose 2-DE offers advantages over the more widely used immobilized pH gradient 2-DE for separating high molecular mass proteins (15-500 kDa), thereby increasing its power to detect changes in the cancer's high-molecular mass proteomes.

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Multi-dimensional liquid phase based separations in proteomics

Cited by 194 publications

References 43 publications

Characterization of voltage degradation in dynamic field gradient focusing

Characterization of voltage degradation in dynamic field gradient focusing

Evaluation of prefractionation methods as a preparatory step for multidimensional based chromatography of serum proteins

Proteome analysis of prostate cancer

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