An analytical system for the characterization of highly heterogeneous mixtures of N-linked oligosaccharides

Briggs, John B.; Keck, Rodney G.; Ma, Stacey; Lau, Wendy; Jones, Andrew J. S.

doi:10.1016/j.ab.2009.03.006

Cited by 22 publications

(10 citation statements)

References 43 publications

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“…CE-LIF has proven to be a sensitive and high resolution separation method that is already broadly utilized in the analysis of complex carbohydrates, both by the biomedical as well as the biotechnology 90 industries. [24][25][26] CE separation is driven by an electric field mediated migration and is therefore based on charge to hydrodynamic volume ratio. Highly charged species migrate first.…”

Section: Introductionmentioning

confidence: 99%

“…27 The selection of the label can have a profound effect on both the sensitivity and resolving power of the method. 25 The ideal labelling agent for CE provides easy and fast glycan derivatization but also has high quantum yield, sufficient charge properties and an excitation wavelength that fits for commercially available la-100 sers. The most commonly used fluorophore which meets these criteria is 8-aminopyrene-1,3,6-trisulfonic acid (APTS).…”

Section: Introductionmentioning

confidence: 99%

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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: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

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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“…12, 33 IgGs are large glycoproteins with a single conserved N-glycosylation site at Asn297 on both heavy chains in the C H 2 domain of the Fc region. Analogous to the deglycosylation process of the other model glycoproteins, the IgG sample was thermally denatured in the presence of SDS, and the disulfide bonds were reduced prior to endoglycosidase digestion.…”

Section: Resultsmentioning

confidence: 99%

Rapid Release of N-Linked Glycans from Glycoproteins by Pressure-Cycling Technology

2010

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The standard, well-established sample preparation protocol to release N-linked glycans from glycoproteins for downstream analysis requires relatively long deglycosylation times (from several hours to overnight) and relatively high endoglycosidase concentration (1:250 -1:500 enzyme:substrate molar ratio). In this paper, we significantly improve this standard protocol by the use of pressure cycling technology (PCT) to increase the speed and decrease the relative amount of PNGase F during the release of N-linked glycans from denatured glycoproteins. With the application of pressure cycling from atmospheric to as high as 30 kPsi, >95% release of the asparagine linked glycans from bovine ribonuclease B, human transferrin and polyclonal human immunoglobulin was rapidly achieved in a few minutes using as low as 1:2500 enzyme:substrate molar ratio. The deglycosylation rate was first examined by SDS-PAGE at the protein level. The released glycans were then quantitated by capillary electrophoresis with laser induced fluorescence detection (CE-LIF). This new sample preparation protocol readily supports large scale glycan analysis of biopharmaceuticals with rapid deglycosylation times.

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“…The primary amine of the label reacts with the aldehyde group of the glycan to generate a Schiff base intermediate, which is then stabilized through reduction to form a secondary amine [55]. 2-aminobenzoic acid (2-AA), 2-aminobenzamide (2-AB), 2-aminopyridine (2-AP), and 5-amino-2-naphthalenesulfonic acid (ANSA) are some of the commonly utilized fluorescent labels [56–68]. …”

Section: Introductionmentioning

confidence: 99%

Electron detachment dissociation of fluorescently labeled sialylated oligosaccharides

Zhou

Håkansson

2011

Electrophoresis

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We explored the application of electron detachment dissociation (EDD) and infrared multiphoton dissociation (IRMPD) tandem mass spectrometry to fluorescently labeled sialylated oligosaccharides. Standard sialylated oligosaccharides and a sialylated N-linked glycan released from human transferrin were investigated. EDD yielded extensive glycosidic cleavages and cross-ring cleavages in all cases studied, consistently providing complementary structural information compared to IRMPD. Neutral losses and satellite ions such as C – 2H ions were also observed following EDD. In addition, we examined the influence of different fluorescent labels. The acidic label 2-aminobenzoic acid (2-AA) enhanced signal abundance in negative-ion mode. However, few cross-ring fragments were observed for 2-AA labeled oligosaccharides. The neutral label 2-aminobenzamide (2-AB) resulted in more cross-ring cleavages compared to 2-AA labeled species, but not as extensive fragmentation as for native oligosaccharides, likely resulting from altered negative charge locations from introduction of the fluorescent tag.

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An analytical system for the characterization of highly heterogeneous mixtures of N-linked oligosaccharides

Cited by 22 publications

References 43 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

Rapid Release of N-Linked Glycans from Glycoproteins by Pressure-Cycling Technology

Electron detachment dissociation of fluorescently labeled sialylated oligosaccharides

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