High-throughput immunoglobulin G N-glycan characterization using rapid resolution reverse-phase chromatography tandem mass spectrometry

Prater, Bradley D.; Connelly, Heather; Qin, Qiang; Cockrill, Steven L.

doi:10.1016/j.ab.2008.10.023

Cited by 65 publications

(68 citation statements)

References 28 publications

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“…The shift in AU values of digested glycan structures run on RP-UPLC is not predictable based on sequential removal of terminal sugar residues. Since the sugar structures are separated based on the hydrophobicity of the glycan and the fluorescent tag, 41 the removal of terminal sugars may have an unpredictable influence on the shift of glycans after digestion. With the HILIC method one can expect reproducible shifts in the range of 0.4 to 490 1.15 (and 0.15 to 0.2 for bisecting GlcNAcs), for exoglycosidase digested glycans depending on the sugars removed.…”

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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“…By combining the compositional information provided by MS analysis with biological knowledge of probable glycan structures, glycan structures may be inferred (37). If available, tandem MS can also be applied to further elucidate the glycan structure (2,14,26,(34)(35)(36)(38)(39)(40)(41)(42). Glycan compositional mass profiling is an attractive option for biomarker discovery due to the simplicity of the data analysis and its potential for rapid-and high-throughput applications (when done by MALDI-MS).…”

Section: Glycomics For Biomarker Discovery: Compositional and Structumentioning

confidence: 99%

“…Compositional mass profiling utilizes high-resolution mass spectrometry (MS) to rapidly separate and identify glycans based on accurate mass. Glycans may be chromatographically fractionated offline and spotted for MALDI (in applications where speed is desirable) (15,(26)(27)(28)(29)(30)(31)(32)(33); or, they may be separated online by methods such as reversed phase (RP) liquid chromatography (LC) immediately prior to introduction into the mass spectrometer via electrospray ionization (where greater sensitivity is necessary) (34)(35)(36). Compositional mass profiling describes the glycans present in a sample in relation to the number of hexoses (such as glucose, galactose, or mannose), N-acetylhexosamines, deoxyhexoses (typically fucose), and sialic acids (such as N-acetylneuraminic acid or N-glycolylneuraminic acid) that each glycan is composed of.…”

Section: Glycomics For Biomarker Discovery: Compositional and Structumentioning

confidence: 99%

Glycoscience aids in biomarker discovery

Hua¹,

An²

2012

BMB Reports

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The glycome consists of all glycans (or carbohydrates) within a biological system, and modulates a wide range of important biological activities, from protein folding to cellular communications. The mining of the glycome for disease markers represents a new paradigm for biomarker discovery; however, this effort is severely complicated by the vast complexity and structural diversity of glycans. This review summarizes recent developments in analytical technology and methodology as applied to the fields of glycomics and glycoproteomics. Mass spectrometric strategies for glycan compositional profiling are described, as are potential refinements which allow structure-specific profiling. Analytical methods that can discern protein glycosylation at a specific site of modification are also discussed in detail.

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“…Fluorescence labeling using 2-aminobenzamide (2-AB) or less frequently 2-aminobenzoic acid (2-AA) followed by normal-phase high performance liquid chromatography (NP-HPLC) separation has become the most commonly used method because of the high sensitivity, high resolution, and good reproducibility [21,[27][28][29]. In addition, many of the derivatization reagents add hydrophobicity to the otherwise hydrophilic oligosaccharides and allow them to be analyzed by reversed-phase HPLC (RP-HPLC) [14][15][16][17][18].…”

mentioning

confidence: 99%

“…Released oligosaccharides can be analyzed by high performance anion-exchange chromatography with pulsedamperometric detection (HPAEC-PAD) [8], matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) [9][10][11], and a graphitized carbon column with mass spectrometry [12,13]. A variety of chemistry has been employed to derivatise released oligosaccharides either to introduce a chromophore or flurophore for detection by absorption or fluorescence [14][15][16][17][18][19][20][21][22][23], or to introduce a positive charged group to enhance mass spectrometry response [20,[24][25][26]. Fluorescence labeling using 2-aminobenzamide (2-AB) or less frequently 2-aminobenzoic acid (2-AA) followed by normal-phase high performance liquid chromatography (NP-HPLC) separation has become the most commonly used method because of the high sensitivity, high resolution, and good reproducibility [21,[27][28][29].…”

mentioning

confidence: 99%

Detection and Quantitation of Afucosylated N-Linked Oligosaccharides in Recombinant Monoclonal Antibodies Using Enzymatic Digestion and LC-MS

May

et al. 2012

J. Am. Soc. Mass Spectrom.

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The presence of N-linked oligosaccharides in the CH2 domain has a significant impact on the structure, stability, and biological functions of recombinant monoclonal antibodies. The impact is also highly dependent on the specific oligosaccharide structures. The absence of core-fucose has been demonstrated to result in increased binding affinity to Fcγ receptors and, thus, enhanced antibody-dependent cellular cytotoxicity (ADCC). Therefore, a method that can specifically determine the level of oligosaccharides without the core-fucose (afucosylation) is highly desired. In the current study, recombinant monoclonal antibodies and tryptic peptides from the antibodies were digested using endoglycosidases F2 and H, which cleaves the glycosidic bond between the two primary GlcNAc residues. As a result, various oligosaccharides of either complex type or high mannose type that are commonly observed for recombinant monoclonal antibodies are converted to either GlcNAc residue only or GlcNAc with the corefucose. The level of GlcNAc represents the sum of all afucosylated oligosaccharides, whereas the level of GlcNAc with the core-fucose represents the sum of all fucosylated oligosaccharides. LC-MS analysis of the enzymatically digested antibodies after reduction provided a quick estimate of the levels of afucosylation. An accurate determination of the level of afucosylation was obtained by LC-MS analysis of glycopeptides after trypsin digestion.

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High-throughput immunoglobulin G N-glycan characterization using rapid resolution reverse-phase chromatography tandem mass spectrometry

Cited by 65 publications

References 28 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

Glycoscience aids in biomarker discovery

Detection and Quantitation of Afucosylated N-Linked Oligosaccharides in Recombinant Monoclonal Antibodies Using Enzymatic Digestion and LC-MS

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