Enzyme degradation, high performance liquid chromatography and liquid secondary ion mass spectrometry in the analysis of glycoproteins

Smith, Kevin D.; Harbin, Anne-Marie; Carruthers, Robert A.; Lawson, A. M.; Hounsell, Elizabeth F.

doi:10.1002/bmc.1130040612

Cited by 17 publications

(2 citation statements)

References 50 publications

(10 reference statements)

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“…Bovine fetuin is a well-characterized glycoprotein consisting of 341 amino acids [11]. Partial mass mapping (using fast-atom bombardment) of its tryptic digest has been reported [21]. It contains three N-linked glycosylation sites whose carbohydrate structures and site heterogeneity has been studied extensively by NMR [12][13][14][15].…”

Section: Instrumentationmentioning

confidence: 99%

Characterization of protein iv-glycosylation by reversed-phase microbore liquid chromatography / electrospray mass spectrometry, complementary mobile phases, and sequential exoglycosidase digestion

Medzihradszky

Maltby

Hall

et al. 1994

J. Am. Soc. Mass Spectrom.

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A strategy for the identification of the site occupancy and glycoform heterogeneity, including sialylation occurring at specific sites of N-linked giycoproteins is presented using the asparagine-linked glycosylation on bovine fetuin for illustration. This is achieved by microbore high-performance liquid chromatography/electrospray ionization mass analysis (LC/ESIMS) of the tryptic glycopeptide mixtures with an acetonitrile-based mobile phase followed by sequential steps of residue (and linkage) specific glycoform degradation and LC/ESIMS analysis at each stage. In addition, chromatographic separation of the site-specific glycoforms of tryptic glycopeptides is accomplished by the use of an alternative, mass spectrometrically compatible mobile phase-water/ethanol/propanol/formic acid. By employing this nontraditional mobile phase for characterizing the complete tryptic digest, and using highly specific exoglycosidases in combination with LC/ESIMS analysis, a previously uncharacterized carbohydrate (a disialo biantennary complex oligosaccharide) was identified as a novel structure at Asn(81) of bovine fetuin. (J Am Sot Mass Spectrom 1994, 5, 350-358).

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Section: Instrumentationmentioning

confidence: 99%

Characterization of protein iv-glycosylation by reversed-phase microbore liquid chromatography / electrospray mass spectrometry, complementary mobile phases, and sequential exoglycosidase digestion

Medzihradszky

Maltby

Hall

et al. 1994

J. Am. Soc. Mass Spectrom.

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“…Further analysis utilized reversed phase HPLC (Smith ef al., 1990) to confirm that HepG2 cells released fully sialylated AGP before and after cytokine stimulation. Reference to Fig.…”

Section: Discussionmentioning

confidence: 99%

Chromatographic investigation of the glycosylation pattern of alpha‐1‐acid glycoprotein secreted by the HEPG2 cell line; a putative model for inflammation?

Elliott

Watson

et al. 1995

Biomedical Chromatography

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In certain pathophysiological conditions, such as rheumatoid arthritis, there are alterations in the glycosylation pattern of the acute phase protein, alpha-1-acid glycoprotein (AGP). These changes are likely to be functionally significant, however, verification of the latter role requires a system which reflects in vivo glycosylation changes in AGP and also produces sufficient quantities of the protein for further study. The human hepatoma cell line HepG2 is documented as displaying a shift in the glycosylation pattern of glycoproteins from normal state to acute phase after stimulation with inflammatory mediators. We have isolated AGP from the culture medium of HepG2 cells both before and after stimulation with a cytokine preparation and analysed the glycosylation pattern of each preparation, after enzymatic release, by high pH anion-exchange chromatography. Before stimulation, the glycosylated population was similar to a profile of AGP isolated from normal plasma; however, cytokine stimulation resulted in a shift to a profile which was consistent with that of AGP from a rheumatoid arthritis sufferer. Thus a HepG2 cell culture system is capable of being a crude model of the changes in glycosylation of acute phase proteins although it has a tendency to produce oligosaccharide chains which are not fully sialylated.

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General Strategies for the Characterization of Carbohydrates from Recombinant Glycoprotein Therapeutics

Gerwig¹,

Damm²

1998

Bioseparation and Bioprocessing

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Enzyme degradation, high performance liquid chromatography and liquid secondary ion mass spectrometry in the analysis of glycoproteins

Cited by 17 publications

References 50 publications

Characterization of protein iv-glycosylation by reversed-phase microbore liquid chromatography / electrospray mass spectrometry, complementary mobile phases, and sequential exoglycosidase digestion

Characterization of protein iv-glycosylation by reversed-phase microbore liquid chromatography / electrospray mass spectrometry, complementary mobile phases, and sequential exoglycosidase digestion

Chromatographic investigation of the glycosylation pattern of alpha‐1‐acid glycoprotein secreted by the HEPG2 cell line; a putative model for inflammation?

General Strategies for the Characterization of Carbohydrates from Recombinant Glycoprotein Therapeutics

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