A new evaluation technique for the detection of impurities in purified proteins via CE with native UV‐LIF

Rodat, Audrey; Couderc, Bettina; Feurer, Bernard; Couderc, François

doi:10.1002/elps.200900369

Cited by 12 publications

(9 citation statements)

References 30 publications

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“…Among the commercial detection modes developed for CE, LIF detection is one of the most sensitive . Because only proteins containing aromatic amino acids (tryptophan, tyrosine, and phenylalanine) exhibit native fluorescence, labeling dyes are required to confer fluorescent properties on proteins . The fluorescent labels are normally chemically attached to the protein through amines or other reactive groups.…”

Section: Introductionmentioning

confidence: 99%

On‐line capillary electrophoresis derivatization method for high sensitivity analysis of ubiquitin in filtered cerebrospinal fluid

et al. 2013

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Electrophoretically mediated microanalysis was implemented for on-line fluorescence derivatization of ubiquitin (UBI), a potential biomarker of Alzheimer disease. Ubiquitin was labeled on its amino groups by the Fluoprobes® 488 N-hydroxysuccinimide. The pH of the BGE, the applied electric field, and the washing process of the capillary were optimized. The best derivatization yield was obtained at pH 9.5 under an electric field of ∼210 V/cm. The concentration and volume of the fluorescent dye as well as the sample medium and volume of injected UBI were also optimized. In order to improve further the LOD of UBI, electrophoretically mediated microanalysis was performed in a wider (100 μm id) and longer (110 cm) capillary. As expected, these capillary dimensions improved the analytical sensitivity of UBI when compared to the 50 μm id capillary. Under the optimized conditions, this analytical methodology allowed the analysis of free UBI in standard solution with a LOD of ∼15 nM. Finally, the on-line fluorescent derivatization of UBI was applied to the detection and the quantification of UBI in cerebrospinal fluid (CSF) samples. Due to the high salt concentration of biological samples, desalting of CSF was required prior to the CE analysis. Quantification of UBI in CSF either by a direct evaluation of peak areas or using the standard addition method was achieved.

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Section: Introductionmentioning

confidence: 99%

On‐line capillary electrophoresis derivatization method for high sensitivity analysis of ubiquitin in filtered cerebrospinal fluid

et al. 2013

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“…The emitted light is detected using a diode or photomultiplier tube. Recently Rodat et al described further improvements to the detection sensitivity in LIF detection [14,15]. Light-emitting diodes, although not laser sources, produce monochromatic light and have been used successfully for the detection of various biomolecules in CE [16,17].…”

Section: Instrumentation and Laser Sourcesmentioning

confidence: 99%

“…[11, [64][65][66][67] Allophycocyanins, phycocyanins, phycoerythrins 500-650 CZE [45,46,68] Green fluorescent protein 488 CZE [42] Protein impurities 266 CZE [15] …”

Section: Proteins and Peptidesmentioning

confidence: 99%

Bioanalytical Applications of Capillary Electrophoresis with Laser-Induced Native Fluorescence Detection

Tseng

Barrett³

2010

Bioanalysis

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In this article we describe recent developments and applications of capillary electrophoresis (CE) coupled with laser-induced native fluorescence (LINF) detection in the analysis of biological, pharmaceutical and environmental samples. Compared with traditional UV absorbance detection used in CE, the LINF technique can greatly improve the concentration sensitivity of CE without the need for derivatization; the only requirement being that the analyte must have native fluorescence. Instrumentation and laser sources used in CE-LINF are summarized and specific applications of CE-LINF to small-biomolecule analysis, profiling of human biofluids, detection of native fluorescent peptides and proteins, single-cell analysis and the use of online sample preconcentration methods are also reviewed in detail.

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“…More recently, a work on impurities in pharmaceutical recombinant protein has been reported by Rodat et al., studying IgG in CGE using SDS in the migration buffer (SDS‐CGE) . This technique is 80 times more sensitive than the SDS‐CGE/UV absorbance and in the same range than silver staining (LOD was 25 ng/mL (160 pM)).…”

Section: Applicationsmentioning

confidence: 99%

Capillary electrophoresis hyphenated with UV‐native‐laser induced fluorescence detection (CE/UV‐native‐LIF)

2016

Self Cite

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Native laser-induced fluorescence using UV lasers associated to CE offers now a large related literature, for now 30 years. The main works have been performed using very expensive Ar-ion lasers emitting at 257 and 275 nm. They are not affordable for routine analyses, but have numerous applications such as protein, catecholamine, and indolamine analysis. Some other lasers such as HeCd 325 nm have been used but only for few applications. Diode lasers, emitting at 266 nm, cheaper, are extensively used for the same topics, even if the obtained sensitivity is lower than the one observed using the costly UV-Ar-ion lasers. This review presents various CE or microchips applications and different UV lasers used for the excitation of native fluorescence. We showed that CE/Native UV laser induced fluorescence detection is very sensitive for detection as well as small aromatic biomolecules than proteins containing Trp and Tyr amino acids. Moreover, it is a simple way to analyze biomolecules without derivatization.

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A new evaluation technique for the detection of impurities in purified proteins via CE with native UV‐LIF

Cited by 12 publications

References 30 publications

On‐line capillary electrophoresis derivatization method for high sensitivity analysis of ubiquitin in filtered cerebrospinal fluid

On‐line capillary electrophoresis derivatization method for high sensitivity analysis of ubiquitin in filtered cerebrospinal fluid

Bioanalytical Applications of Capillary Electrophoresis with Laser-Induced Native Fluorescence Detection

Capillary electrophoresis hyphenated with UV‐native‐laser induced fluorescence detection (CE/UV‐native‐LIF)

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