Single-label fluorescent derivatization of peptides

Little, Michael J.; Paquette, Donald M.; Harvey, M.; Banks, Peter

doi:10.1016/s0003-2670(96)00488-6

Cited by 21 publications

(12 citation statements)

References 23 publications

(5 reference statements)

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“…Although they also react slowly to form secondary fluorescent products , their use in place of a fluorescent dye are attractive because it reduces the background signal by several orders of magnitude. Many reports describe also the use of fluorescent dyes such as FITC and rhodamine‐based dyes for LIF detection in the 400–600 nm wavelength range to get near nM LODs. These latter compounds are more reactive than FITC, especially when the dye is activated by the succinimidyl ester group which can react efficiently with unprotonated amines to form a stable amide bond .…”

Section: Introductionmentioning

confidence: 99%

Derivatization strategies for CE‐LIF analysis of biomarkers: Toward a clinical diagnostic of familial transthyretin amyloidosis

et al. 2014

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We report three derivatization strategies for CE analysis with LIF detection (CE-LIF) of two synthetic peptides mimicking the wild and mutated fragments of interest for the diagnosis of familial transthyretin amyloidosis. The precapillary derivatization of the peptides with three optical tags, 5-carboxytetramethylrhodamin succinimidyl ester (TAMRA-SE), naphtalene-2,3-dicarboxyaldehyde (NDA), and 3-(2-furoyl)quinoline-2-carboxyaldehyde (FQ) has been investigated by CE-LIF detection and MS. Results provide evidence that high reaction yields have been reached whereas the multitagging phenomenon has occurred for both NDA and TAMRA-SE labeling procedures. The derivatization and electrokinetic separation of a mixture of the two peptides of interest for the pathology diagnosis (22-aa peptides that differ only from one amino acid) were achieved using both approaches. The highest resolution with a value of 2.5 was obtained with TAMRA-SE labeled derivatives whereas NDA gave the best detection sensitivity (LOD of 2.5 μM). The validation of the developed methods showed a good linearity (R ≥ 0.997) between the peak area of the labeled derivatives and the peptide concentration for both NDA and FQ labeling procedures. The intraday RSDs of A and the migration times were less than 3.8 and 2.2%, respectively.

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Section: Introductionmentioning

confidence: 99%

Derivatization strategies for CE‐LIF analysis of biomarkers: Toward a clinical diagnostic of familial transthyretin amyloidosis

et al. 2014

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“…This primary amino reactive probe has been used for a single-label fluorescent derivatization of peptides via their primary a-amino groups at a derivatization pH lower than usual derivatization (pH < 9.0), which discriminates the derivatization of e-amino from a-amino groups [136]. Recombinant hirudin (r-hirudin), a polypeptide of 65 amino acids which is a thrombin inhibitor, was detected by CE-based immunoassay (CEIA) with LIF detection using FITC as derivatization reagent in order to check purity levels and to perform pharmacokinetics studies [137].…”

Section: Fitcmentioning

confidence: 99%

LIF detection of peptides and proteins in CE

2007

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CE- and microchip-based separations coupled with LIF are powerful tools for the separation, detection and determination of biomolecules. CE with certain configurations has the potential to detect a small number of molecules or even a single molecule, thanks to the high spatial coherence of the laser source which permits the excitation of very small sample volumes with high efficiency. This review article discusses the use of LIF detection for the analysis of peptides and proteins in CE. The most common laser sources, basic instrumentation, derivatization modes and set-ups are briefly presented and special attention is paid to the different fluorogenic agents used for pre-, on- and postcapillary derivatization of the functional groups of these compounds. A table summarizing major applications of these derivatization reactions to the analysis of peptides and proteins in CE-LIF and a bibliography with 184 references are provided which covers papers published to the end of 2005.

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“…As the laser used in this study has 488 nm wavelength, FITC can be used as a labeling agent [26,27]. It can be applied in aqueous solvents and reacts with primary and secondary amines at pH values around 9; it has also frequently been used for analysis in biological samples [28,29].…”

Section: Sample Pretreatmentmentioning

confidence: 99%

Determination of sertraline and N‐desmethylsertraline in human plasma by CE with LIF detection

et al. 2007

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A method has been developed for the analysis of the antidepressant drug sertraline together with its main metabolite N-desmethylsertraline (DMS) in human plasma. It is based on CE with LIF detection (lambda = 488 nm). A SPE procedure is employed for biological sample pretreatment, followed by a derivatization step with FITC; reboxetine was the internal standard. The effect of CD, acetone and N-methyl-D-glucamine (GLC) as constituents of the BGE for analyte separation was investigated. The final BGE consisted of 20 mM carbonate buffer, pH 9.0, with 2.5 mM heptakis(2,6-di-O-methyl)-beta-CD, 50 mM GLC and 20% v/v acetone. With 30 kV applied voltage, the electrophoretic run is completed in 7.5 min. Linearity was observed in the plasma concentration range from 3.0 to 500 ng/mL for sertraline and 4.0 to 500 ng/mL for DMS. Extraction yield was >97.1%, precision - expressed as RSD% - was <3.7, accuracy (recovery) was >95.6%. Due to its sensitivity and selectivity, the method was suited for the analysis of plasma samples from patients undergoing therapy with sertraline.

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Single-label fluorescent derivatization of peptides

Cited by 21 publications

References 23 publications

Derivatization strategies for CE‐LIF analysis of biomarkers: Toward a clinical diagnostic of familial transthyretin amyloidosis

Derivatization strategies for CE‐LIF analysis of biomarkers: Toward a clinical diagnostic of familial transthyretin amyloidosis

LIF detection of peptides and proteins in CE

Determination of sertraline and N‐desmethylsertraline in human plasma by CE with LIF detection

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