The dynamic range of protein expression: A challenge for proteomic research

Corthals, Garry L.; Wasinger, Valerie C.; Hochstrasser, Denis F.; Sanchez, Jean‐Charles

doi:10.1002/(sici)1522-2683(20000401)21:6<1104::aid-elps1104>3.0.co;2-c

Cited by 558 publications

(259 citation statements)

References 95 publications

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“…The absolute mobility is related to the electrophoretic mobility, ω i by (2) The mass conservation law for each species can be expressed as follows:…”

Section: Nonlinear Numerical Simulationmentioning

confidence: 99%

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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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“…The absolute mobility is related to the electrophoretic mobility, ω i by (2) The mass conservation law for each species can be expressed as follows:…”

Section: Nonlinear Numerical Simulationmentioning

confidence: 99%

“…The need for fast, high-throughput, reproducible, and automated analytical separation techniques have slowed the advances of proteomic research and characterization [1][2][3]. Analytical-scale, biological separations are most commonly accomplished with the use of LC, CE, or SDS-PAGE.…”

Section: Introductionmentioning

confidence: 99%

Characterization of voltage degradation in dynamic field gradient focusing

Burke

Ivory

2008

Electrophoresis

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“…The use of capillary LC separation in conjunction with FTICR for analysis of complex proteolytic digests (i.e., a mammalian proteome) poses a major challenge for accurate mass measurements since protein concentrations of interest in, for example, yeast vary by Ͼ10,000-fold [40,41]. Therefore, LC peak intensities and the concentrations of eluted peptide mixtures span several orders of magnitude and result in large changes in ion populations in the ICR cell, causing cyclotron frequency shifts and greater mass measurement errors.…”

mentioning

confidence: 99%

An automated high performance capillary liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometer for high-throughput proteomics

Belov

Anderson

Wingerd

et al. 2004

J. Am. Soc. Mass Spectrom.

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We describe a fully automated high performance liquid chromatography 9.4 tesla Fourier transform ion resonance cyclotron (FTICR) mass spectrometer system designed for proteomics research. A synergistic suite of ion introduction and manipulation technologies were developed and integrated as a high-performance front-end to a commercial Bruker Daltonics FTICR instrument. The developments incorporated included a dual-ESI-emitter ion source; a dualchannel electrodynamic ion funnel; tandem quadrupoles for collisional cooling and focusing, ion selection, and ion accumulation, and served to significantly improve the sensitivity, dynamic range, and mass measurement accuracy of the mass spectrometer. In addition, a novel technique for accumulating ions in the ICR cell was developed that improved both resolution and mass measurement accuracy. A new calibration methodology is also described where calibrant ions are introduced and controlled via a separate channel of the dual-channel ion funnel, allowing calibrant species to be introduced to sample spectra on a real-time basis, if needed. We also report on overall instrument automation developments that facilitate high-throughput and unattended operation. These included an automated version of the previously reported very high resolution, high pressure reversed phase gradient capillary liquid chromatography (LC) system as the separations component. A commercial autosampler was integrated to facilitate 24 h/day operation. Unattended operation of the instrument revealed exceptional overall performance: Reproducibility (1-5% deviation in uncorrected elution times), repeatability (Ͻ20% deviation in detected abundances for more abundant peptides from the same aliquot analyzed a few weeks apart), and robustness (high-throughput operation for 5 months without significant downtime). When combined with modulated-ionenergy gated trapping, the dynamic calibration of FTICR mass spectra provided decreased mass measurement errors for peptide identifications in conjunction with high resolution capillary LC separations over a dynamic range of peptide peak intensities for each spectrum of 10 3 , and Ͼ10 5 for peptide abundances in the overall separation. (J Am Soc Mass Spectrom 2004, 15, 212-232)

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“…However, the concentrations of individual proteins in cells can span five orders of magnitude while the total number of different proteins expressed number in the thousands [17]. Therefore, any realistic and reliable representation of protein expression will only be possible if more powerful analytical techniques are developed.…”

Section: Resultsmentioning

confidence: 99%

On-line strong cation exchange μ-HPLC-ESI-MS/MS for protein identification and process optimization

Bihan

Duewel

Figeys

2003

J. Am. Soc. Mass Spectrom.

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We have developed an on-line strong cation exchange (SCX)-ESI-MS/MS platform for the rapid identification of proteins contained in mixtures. This platform consists of a SCX precolumn followed by a nanoflow SCX column on-line with an electrospray ion trap mass spectrometer. We also used this platform to study the dynamics of peptide separation/ extraction by SCX, in particular to understand the parameters affecting the performance of SCX in multidimensional chromatography. For example, we have demonstrated that the buffer typically used for tryptic digestion of protein mixtures can have a detrimental effect on the chromatographic behaviour of peptides during SCX separations, thereby affecting certain peptide quantitation approaches that rely on reproducible peptide fractionation. We have also demonstrated that band broadening results when a step (discontinuous) gradient approach is used to displace peptides from the SCX precolumn, reducing the separation power of SCX in multidimensional chromatography. In contrast, excellent chromatographic peak shapes are observed when a defined (continuous) gradient is used. Finally, using a tryptic digest of a protein extract derived from human K562 cells, we observed that larger molecular weight peptides are identified using this on-line SCX approach compared to the more conventional reverse phase (RP) LC/MS approach. Both methods used in tandem complement each other and can lead to a greater number of peptide identifications from a given sample.

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The dynamic range of protein expression: A challenge for proteomic research

Cited by 558 publications

References 95 publications

Characterization of voltage degradation in dynamic field gradient focusing

Characterization of voltage degradation in dynamic field gradient focusing

An automated high performance capillary liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometer for high-throughput proteomics

On-line strong cation exchange μ-HPLC-ESI-MS/MS for protein identification and process optimization

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