Capillary array isoelectric focusing with laser-induced fluorescence detection: milli-pH unit resolution and yoctomole mass detection limits in a 32-channel system

Dada, Oluwatosin O.; Ramsay, Lauren M.; Dickerson, Jane A.; Cermak, Nathan; Jiang, Rong; Zhu, Cuiru; Dovic̀hi, Norman J.

doi:10.1007/s00216-010-3595-x

Cited by 21 publications

(20 citation statements)

References 23 publications

(29 reference statements)

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“…Fluorescent labeling using neutral or anionic reagents disrupts both the zone electrophoresis and the isoelectric focusing profile of standards [42–44]. We employed the cationic fluorogenic reagent Chromeo P540 in this experiment; this reagent converts cationic lysine residues to cationic products, generating clean electrophoretic profiles [5, 17]. …”

Section: Resultsmentioning

confidence: 99%

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Capillary isoelectric focusing with pH 9.7 cathode for the analysis of gastric biopsies

et al. 2011

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Capillary isoelectric focusing tends to suffer from poor reproducibility, particularly for the analysis of complex protein samples from cellular or tissue homogenates. This poor reproducibility appears to be associated with erratic variations in electroosmotic flow. One cause of electroosmotic flow variation is degradation of the capillary coating caused by the extremely basic solution commonly used during mobilization and focusing; this degradation of the capillary coating can be reduced by employing a CAPS mobilization buffer at pH 9. Another cause of variation is protein adsorption to the capillary wall, which causes an increase in electroosmotic flow. The effects of protein adsorption can be reduced by use of surfactants in the buffer and by employing an extremely low sample loading. We report the use of CAPS mobilization buffer in combination with an ultrasensitive laser-induced fluorescence detector for the reproducible analysis of ~2 ng of protein from a Barrett’s esophagus biopsy.

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Section: Resultsmentioning

confidence: 99%

“…Among capillary electrophoresis separation methods, capillary isoelectric focusing (cIEF) provides the greatest resolving power, but has typically been the most challenging technique to perform [1–5]. In isoelectric focusing, ampholytes establish a pH gradient in the capillary and the analytes focus to their isoelectric points.…”

Section: Introductionmentioning

confidence: 99%

Capillary isoelectric focusing with pH 9.7 cathode for the analysis of gastric biopsies

et al. 2011

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“…This system is easy to be coupled with certain detection methods to achieve high-throughput analyses. For instance, Dada et al have introduced a detection system that can record fluorescence data from 32 channels simultaneously [17]. Up to now, the highest-throughput CAE system contained 768 lanes on a microfluidic chip [18].…”

Section: Introductionmentioning

confidence: 99%

A prototypic system of parallel electrophoresis in multiple capillaries coupled with microwell arrays

Ren

Dai

et al. 2011

Electrophoresis

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We present a microfluidic system that can be directly coupled with microwell array and perform parallel electrophoresis in multiple capillaries simultaneously. The system is based on an array of glass capillaries, fixed in a polydimethylsiloxane (PDMS) microfluidic scaffold, with one end open for interfacing with microwells. In this capillary array, every two adjacent capillaries act as a pair to be coupled with one microwell; samples in the microwells are introduced and separated by simply applying voltage between two electrodes that are placed at the other ends of capillaries; thus no complicated circuit design is required. We evaluate the performance of this system and perform multiple CE with direct sample introduction from microwell array. Also with this system, we demonstrate the analysis of cellular contents of cells lysed in a microwell array. Our results show that this prototypic system is a promising platform for high-throughput analysis of samples in microwell arrays.

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“…Compared to zone electrophoresis, the focusing properties of cIEF allow use of much larger injection volumes, which facilitates analysis of very dilute analyte [24–26]. …”

Section: Introductionmentioning

confidence: 99%

Capillary Isoelectric Focusing-Tandem Mass Spectrometry and Reversed-Phase Liquid Chromatography-Tandem Mass Spectrometry for Quantitative Proteomic Analysis of Differentiating PC12 Cells By Eight-Plex Isobaric Tags for Relative and Absolute Quantification

et al. 2013

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We report the application of capillary isoelectric focusing for quantitative analysis of a complex proteome. Biological duplicates were generated from PC12 cells at days 0, 3, 7, and 12 following treatment with nerve growth factor. These biological duplicates were digested with trypsin, labeled using eight-plex iTRAQ chemistry, and pooled. The pooled peptides were separated into 25 fractions using reversed-phase liquid chromatography (RPLC). Technical duplicates of each fraction were separated by capillary isoelectric focusing (cIEF) using a set of amino acids as ampholytes. The cIEF column was interfaced to an Orbitrap Velos mass spectrometer with an electrokinetically-pumped sheath-flow nanospray interface. This HPLC-cIEF-ESIMS/MS approach identified 835 protein groups and produced 2,329 unique peptides IDs. The biological duplicates were analyzed in parallel using conventional strong-cation exchange (SCX) – RPLC-ESIMS/MS. The iTRAQ peptides were first separated into eight fractions using SCX. Each fraction was then analyzed by RPLC-ESI-MS/MS. The SCX-RPLC approach generated 1,369 protein groups and 3,494 unique peptide IDs. For protein quantitation, 96 and 198 differentially expressed proteins were obtained with RPLC-cIEF and SCX-RPLC, respectively. The combined set identified 231 proteins. Protein expression changes measured by RPLC-cEIF and SCX-RPLC were highly correlated.

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Capillary array isoelectric focusing with laser-induced fluorescence detection: milli-pH unit resolution and yoctomole mass detection limits in a 32-channel system

Cited by 21 publications

References 23 publications

Capillary isoelectric focusing with pH 9.7 cathode for the analysis of gastric biopsies

Capillary isoelectric focusing with pH 9.7 cathode for the analysis of gastric biopsies

A prototypic system of parallel electrophoresis in multiple capillaries coupled with microwell arrays

Capillary Isoelectric Focusing-Tandem Mass Spectrometry and Reversed-Phase Liquid Chromatography-Tandem Mass Spectrometry for Quantitative Proteomic Analysis of Differentiating PC12 Cells By Eight-Plex Isobaric Tags for Relative and Absolute Quantification

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