A multiple-capillary electrophoresis system for small-scale DNA sequencing and analysis

Zhang, Jianbin

doi:10.1093/nar/27.24.e36

Cited by 59 publications

(29 citation statements)

References 29 publications

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“…Our instruments are descendants from this class of instrument. [32] The second class of instruments employs a single separation capillary; these instruments are typically used for more general analytical applications. Commercial automated CZE systems tend to use 360 μm o.d.…”

Section: Resultsmentioning

confidence: 99%

Third-Generation Electrokinetically Pumped Sheath-Flow Nanospray Interface with Improved Stability and Sensitivity for Automated Capillary Zone Electrophoresis–Mass Spectrometry Analysis of Complex Proteome Digests

et al. 2015

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We have reported a set of electrokinetically pumped sheath flow nanoelectrospray interfaces to couple capillary zone electrophoresis with mass spectrometry. A separation capillary is threaded through a cross into a glass emitter. A side arm provides fluidic contact with a sheath buffer reservoir that is connected to a power-supply. The potential applied to the sheath buffer drives electro-osmosis in the emitter to pump the sheath fluid at nanoliter/minute rates. Our first generation interface placed a flat-tipped capillary in the emitter. Sensitivity was inversely related to orifice size and to the distance from the capillary tip to the emitter orifice. A second generation interface used a capillary with an etched tip that allowed the capillary exit to approach within a few hundred micrometers of the emitter orifice, resulting in a significant increase in sensitivity. In both the first and second-generation interfaces, the emitter diameter was typically 8-μm; these narrow orifices were susceptible to plugging and tended to have limited lifetime. We now report a third-generation interface that employs a larger diameter emitter orifice with very short distance between the capillary tip and the emitter orifice. This modified interface is much more robust and produces much longer lifetime than our earlier designs with no loss in sensitivity. We evaluated the third-generation interface for a 5,000-min (127 runs, 3.5 days) repetitive analysis of bovine serum albumin digest using an uncoated capillary. We observed a 10% relative standard deviation in peak area, an average of 160,000 theoretical plates, and very low carry-over (much less than 1%). We employed a linear-polyacrylamide (LPA) coated capillary for single-shot, bottom-up proteomic analysis of 300 ng of Xenopus laevis fertilized egg proteome digest, and identified 1,249 protein groups and 4,038 peptides in a 110 min separation using an LTQ-Orbitrap Velos mass spectrometer; peak capacity was ~330. The proteome dataset using this third generation interface based CZE-MS/MS is similar in size to that generated using a commercial ultra-performance liquid chromatographic analysis of the same sample with the same mass spectrometer and similar analysis time.

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Section: Resultsmentioning

confidence: 99%

Third-Generation Electrokinetically Pumped Sheath-Flow Nanospray Interface with Improved Stability and Sensitivity for Automated Capillary Zone Electrophoresis–Mass Spectrometry Analysis of Complex Proteome Digests

et al. 2015

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“…However, this separation is performed without operator intervention or supervision. We have demonstrated that a multiple capillary sheath-flow cuvette is an efficient detector in high throughput DNA sequencing (22)(23)(24). We plan to modify the two-dimensional electrophoresis instrument so that 96 samples can be analyzed in parallel.…”

Section: Discussionmentioning

confidence: 99%

Fully Automated Two-dimensional Capillary Electrophoresis for High Sensitivity Protein Analysis

Michels

Schoenherr

et al. 2002

Molecular & Cellular Proteomics

118

114

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We report a system for automated protein analysis. In the system, proteins are labeled with the fluorogenic reagent 3-(2-furoyl)quinoline-2-carboxaldehyde, which reacts with lysine residues and creates a highly fluorescent product. These labeled proteins are analyzed by submicellar capillary electrophoresis at pH 7.5 to perform a first dimension separation. Once the first components migrate from the capillary, a fraction is transferred to a second dimension capillary, where electrophoresis is performed at pH 11.1 to further separate the proteins. Laser-induced fluorescence is used as an ultrasensitive detector of the separated proteins. Successive fractions are transferred from the first dimension capillary to the second dimension capillary for further separation to generate, in serial fashion, a twodimensional electropherogram. The transfer of fractions is computer-controlled; there is no operator intervention once the sample has been injected. Zeptomoles of labeled proteins are detected, providing exquisite sensitivity. The resolution of complex samples into components requires sophisticated technology. Most separation techniques are capable of resolving, at most, several dozen components. Cal Giddings (1) recognized that the combination of two separation techniques is important in the resolution of complex mixtures. If the two separation techniques are based on unrelated characteristics of the sample, then the number of resolution elements is given by the product of the resolution elements of both separation steps. For example, isoelectric focusing and SDS-polyacrylamide gel electrophoresis can, individually, resolve ϳ50 components in a protein sample. Their combination, in two-dimensional electrophoresis, can resolve several thousand components (2).Unfortunately, classic two-dimensional electrophoresis requires manual manipulation of the sample. These manipulations, although reasonable for occasional use, are very tedious when performed in large-scale protein analysis projects. Furthermore, detection sensitivity is limited, and relatively large amounts of sample are required to detect weakly expressed components.Column-switching technology is an alternative means of performing two-dimensional separations and is commonly used in chromatographic resolution of complex samples. Most simply, a fraction containing the compound of interest is captured as it elutes from the first column and is transferred to another chromatographic column for additional resolution; fractionation is repeated with different chromatographic methods to achieve the desired purity.In multidimensional chromatography, also known as comprehensive chromatography, a second column is used to sequentially separate all fractions from the first column. These two-dimensional techniques are particularly useful when characterizing extremely complex samples. An early report used two successive chromatographic steps to purify peptides generated from the proteolytic digest of a human immunoglobulin (3).Jorgenson and others (4 -12) have developed elegant...

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“…Capillary array electrophorcsis has proven to be a powerful tool for DNA analysis (ZHANG et al, 1999). Automated instruments have been commercialized and used to sequence the vast majority of the human genome.…”

Section: Capillary Electrophoresismentioning

confidence: 99%

Protein Technologies: Proteome Analysis by Capillary Electrophoresis

Dovic̀hi

Michels

et al. 2002

Essentials of Genomics and Bioinformatics

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A multiple-capillary electrophoresis system for small-scale DNA sequencing and analysis

Cited by 59 publications

References 29 publications

Third-Generation Electrokinetically Pumped Sheath-Flow Nanospray Interface with Improved Stability and Sensitivity for Automated Capillary Zone Electrophoresis–Mass Spectrometry Analysis of Complex Proteome Digests

Third-Generation Electrokinetically Pumped Sheath-Flow Nanospray Interface with Improved Stability and Sensitivity for Automated Capillary Zone Electrophoresis–Mass Spectrometry Analysis of Complex Proteome Digests

Fully Automated Two-dimensional Capillary Electrophoresis for High Sensitivity Protein Analysis

Protein Technologies: Proteome Analysis by Capillary Electrophoresis

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