Characterization of 9-Aminopyrene-1,4,6-trisulfonate Derivatized Sugars by Capillary Electrophoresis with Laser-Induced Fluorescence Detection

Evangelista, Ramon A.; Liu, Ming-Sun; Chen, Fu-Tai A.

doi:10.1021/ac00109a051

Cited by 202 publications

(159 citation statements)

References 16 publications

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“…35 As we report on the separation of IgG glycans, this is less crucial, as sialylation is quite low, representing only about up to 5% of the total glycan pool from each species. The detection limit of APTS-labelled glycans by CE-LIF is estimated to be 0.4 nM using a 3 mW 488 nm argon-ion laser, 36 which is slightly less sensitive than HILIC-UPLC; however, the injection volume of the latter is on average 3 orders of magnitude higher than in CE. CE-LIF allows for the separation of all IgG glycans in 8 min based on the conditions applied in this paper with the overall separation time just over 20 min.…”

Section: Q2mentioning

confidence: 99%

Comparison of separation techniques for the elucidation of IgG N-glycans pooled from healthy mammalian species

Adamczyk

Tharmalingam-Jaikaran

Schomberg

et al. 2014

Carbohydrate Research

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a b s t r a c tThe IgG N-glycome provides sufficient complexity and information content to serve as an excellent source for biomarker discovery in mammalian health. Since oligosaccharides play a significant role in many biological processes it is very important to understand their structure. The glycosylation is cell type specific as well as highly variable depending on the species producing the IgG. We evaluated the variation of N-linked glycosylation of human, bovine, ovine, equine, canine and feline IgG using three orthogonal glycan separation techniques: hydrophilic interaction liquid chromatography (HILIC)-UPLC, reversed phase (RP)-UPLC and capillary electrophoresis with laser induced fluorescence detection (CE-LIF). The separation of the glycans by these high resolution methods yielded different profiles due to diverse chemistries. However, the % abundance of structures obtained by CE-LIF and HILIC-UPLC were similar, whereas the analysis by RP-UPLC was difficult to compare as the structures were separated by classes of glycans (highly mannosylated, fucosylated, bisected, fucosylated and bisected) resulting in the co-elution of many structures. The IgGs from various species were selected due to the complexity and variation in their N-glycan composition thereby highlighting the complementarity of these separation techniques.

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Section: Q2mentioning

confidence: 99%

Comparison of separation techniques for the elucidation of IgG N-glycans pooled from healthy mammalian species

Adamczyk

Tharmalingam-Jaikaran

Schomberg

et al. 2014

Carbohydrate Research

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“…The results show that a partially opened triple-helix conformation was formed after treatment with NaOH and the corresponding biological activities correlated with the degree of strand opening. Preparation of Laminarin-AP Derivative-The AP probe, designed to specifically label the reducing end of glucans, was prepared according to a modification of the procedure of Evanglista et al (21) Specifically, 100 mg of laminarin (0.005 mmol) was mixed with 2.6 mg (0.011 mmol) of AP (in 0.5 ml Me 2 SO), 2 ml of 15% acetic acid, 17.6 mg (0.28 mmol) of sodium cyanoborohydride, and 42 mg (0.31 mmol) of ammonium sulfate (NH 4 ) 2 SO 4 . The 5-ml glass reaction vial was heated in a water bath at 75°C for 1 h, then dialyzed with double distilled water for 2 days.…”

mentioning

confidence: 99%

Observation of a Partially Opened Triple-helix Conformation in 1→3-β-Glucan by Fluorescence Resonance Energy Transfer Spectroscopy

Young¹,

Dong²,

Jacobs³

2000

Journal of Biological Chemistry

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This study used fluorescence resonance energy transfer (FRET) spectroscopy as an indirect method to investigate the effect of NaOH treatment on the conformation of a triple-helix (133)-␤-D-glucan and then evaluated the effect of conformation on biological activity. Previous studies have suggested that treatment of the triple-helix glucans with NaOH produces single-helix conformers. FRET spectra of the triple-helix glucan, laminarin, doubly labeled with 1-aminopyrene as donor probe and fluorescein-5-isothiocyanate as acceptor probe attached at the reducing end, showed that a partially opened triplehelix conformer was formed on treatment with NaOH. Increasing degrees of strand opening was associated with increasing concentrations of NaOH. Based on these observations we propose that a partially opened triplehelix rather than a single helix, is formed by treating the triple-helix glucans with NaOH. After neutralizing the NaOH, changes in FRET indicated that the partially opened conformer gradually reverts to the triple-helix over 8 days. Laminarian was stabilized at different degrees of partial opening and its biological activity examined using the Limulus amebocyte lysate assay and nitric oxide production by alveolar macrophage. Both Limulus amebocyte lysate activity and nitric oxide production were related to the degree of opening of the triple-helix. Partially open conformers were more biologically active than the intact triple-helix.(133)-␤-D-Glucans have been shown to systemically enhance the immune system, resulting in antitumor, antibacterial, and wound healing activities (1). Differences in these activities are thought to be related to three factors: molecular conformation, the degree of branching, and the molecular weight (M r ) (2). Studies have shown that high M r (100,000 -200,000 g/mol) glucans with a degree of branching of 0.20 -0.33 are most active (2), however, data describing the effects of molecular conformation on biological activity are less clear. This is in part due to the lack of adequate methodology to characterize the tertiary structure of glucans.Most spectroscopic techniques only provide data about the secondary structure of glucans. Methods such as solid state 13 C NMR spectroscopy (3) or multi-angle laser-light scattering (4) have had some success in relating the tertiary structure of glucans to their conformation based biological activity, however, their use is limited by availability, cost, and efficacy for evaluating glucan structure in solution. A direct observation technique, such as x-ray crystallography would assist in understanding glucan structure and its biological relevance; however, it is difficult to crystallize glucans and only a limited number of these studies have been done (5-7).Fluorescence resonance energy transfer (FRET) 1 is a phenomenon of non-radioactive energy transfer over relatively long distances that has been used to characterize the spatial relationship of donor-and acceptor-labeled molecules in biological systems (8). If a system contains two fluorophores an...

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“…Several detection systems are utilised in carbohydrate analysis and these include light scattering detection systems, which are used mainly with size exclusion chromatography, integrated pulsed electrochemical detection (IPED) used frequently with HPAEC. CE is employed with ultraviolet spectrophotometers or laser induced fluorescence detection (Evangelista et al, 1995). The modes of detection used with CE require that the carbohydrates should be derivatised before analysis (Rassi and Smith, 1995).…”

Section: Introductionmentioning

confidence: 99%

Profiling of carbohydrate polymers in biotechnology using microdialysis sampling, high performance anion exchange chromatography with integrated pulsed electrochemical detection/mass spectrometry

Okatch¹,

Torto²

2003

Afr. J. Biotechnol.

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The analysis of carbohydrate polymers is very demanding and challenging because of the similar physical and chemical properties they possess. Enzymatic hydrolysis is employed to cleave the polymers. The use of enzymes in analytical chemistry requires an analytical system that has on-line capability, is fast, reproducible, robust, and offers sensitive detection. More importantly the system employed should offer in-situ sampling and sample clean-up so as to reduce the sample handling steps and the analysis time. One such system consists of microdialysis sampling, high performance anion exchange chromatography with integrated pulsed electrochemical detection/mass spectrometry. The on-line system offers sampling, sample clean-up, separation and detection of saccharides as a way of profiling carbohydrate polymers. The wealth of information attained include carbohydrate polymer sequencing, glycosidic linkages, anomericity and result in the unequivocal characterisation of the polymers. This review focuses on the applications of a total on-line system for the profiling of carbohydrate hydrolysates. The above-mentioned analytical system has been employed to carbohydrate polymers and hydrolysates from starch, lignocellulosic material and legumes.

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Characterization of 9-Aminopyrene-1,4,6-trisulfonate Derivatized Sugars by Capillary Electrophoresis with Laser-Induced Fluorescence Detection

Cited by 202 publications

References 16 publications

Comparison of separation techniques for the elucidation of IgG N-glycans pooled from healthy mammalian species

Comparison of separation techniques for the elucidation of IgG N-glycans pooled from healthy mammalian species

Observation of a Partially Opened Triple-helix Conformation in 1→3-β-Glucan by Fluorescence Resonance Energy Transfer Spectroscopy

Profiling of carbohydrate polymers in biotechnology using microdialysis sampling, high performance anion exchange chromatography with integrated pulsed electrochemical detection/mass spectrometry

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