Quantitative Comparison of Global Carbohydrate Structures of Glycoproteins Using LC−MS and In-Source Fragmentation
| Autor: | Brian D. Kelley, James E. Vath, Wen Yu, Istvan Mazsaroff |
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| Rok vydání: | 1997 |
| Předmět: | |
| Zdroj: | Analytical Chemistry. 69:2517-2524 |
| ISSN: | 1520-6882 0003-2700 |
| DOI: | 10.1021/ac961116+ |
| Popis: | A comparative method for the quantitative analysis of the ratio of oxonium fragment (reporter) ions derived from sialic acid and N-acetylhexosamine residues on a large intact glycoprotein, the B domain of recombinant human factor VIII (rhFVIII), was developed. The method utilized liquid chromatography-electrospray ionization mass spectrometry (LC-ESI MS) on a single-quadrupole instrument. During development, systematic approaches such as full-matrix and simplex strategies were used for the optimization of the signal-to-noise ratio by controlling source temperature and cone voltage. The method was found to be precise (RSD = 0.84%), sensitive (capable of differentiating 1 sialic acid residue change among at least 29 sialic acids on a 103-kDa glycoprotein that is 38% carbohydrate), applicable to a wide range of loading (11.6-372 micrograms of FVIII), and accurate according to a comparison to matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. Combining the method with enzymatic removal of N-glycans, selective O-glycan analysis was also performed leading to differential fragment ion analysis ascribed to N- and O-glycans. Quantitative ESI in-source dissociation MS combined with LC can generally be used for glycoproteins, as one of the indicators, to compare the distribution of carbohydrate residues over N- and O-glycans, to investigate their isoforms, and compare batch-to-batch characteristics of biopharmaceuticals. |
| Databáze: | OpenAIRE |
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