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Structural characterisation of neutrophil glycans by ultra sensitive mass spectrometric glycomics methodology.

ABSTRACT: Neutrophils are the most abundant white blood cells in humans and play a vital role in several aspects of the immune response. Numerous reports have implicated neutrophil glycosylation as an important factor in mediating these interactions. We report here the application of high sensitivity glycomics methodologies, including matrix assisted laser desorption ionisation (MALDI-TOF) and MALDI-TOF/TOF analyses, to the structural analysis of N- and O-linked carbohydrates released from two samples of neutrophils, prepared by two separate and geographically remote laboratories. The data produced demonstrates that the cells display a diverse range of sialylated and fucosylated complex glycans, with a high level of similarity between the two preparations.

SUBMITTER: Babu P

PROVIDER: S-EPMC2791480 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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Structural characterisation of neutrophil glycans by ultra sensitive mass spectrometric glycomics methodology.

Babu Ponnusamy P North Simon J SJ Jang-Lee Jihye J Chalabi Sara S Mackerness Kathryn K Stowell Sean R SR Cummings Richard D RD Rankin Sara S Dell Anne A Haslam Stuart M SM

Glycoconjugate journal 20091101 8

Neutrophils are the most abundant white blood cells in humans and play a vital role in several aspects of the immune response. Numerous reports have implicated neutrophil glycosylation as an important factor in mediating these interactions. We report here the application of high sensitivity glycomics methodologies, including matrix assisted laser desorption ionisation (MALDI-TOF) and MALDI-TOF/TOF analyses, to the structural analysis of N- and O-linked carbohydrates released from two samples of ...[more]

PMID: 18587645

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Project description:Sensitive glycomics analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is of great importance but significantly hampered by their low ionization efficiency and labile sialic acid moieties. Chemical derivatization offers a viable way to improve both the ionization efficiency and analytical sensitivity of the glycans in MS analysis by altering their hydrophobicity or charge property. Here we employed Girard's reagent T (GT) for on-target derivatization (GTOD) of reducing glycan under mild acid condition to form stable hydrazones at room temperature, allowing rapid and sensitive identification of neutral and sialylated glycans in positive-ion mode as only permanently positive charged molecular ions without multiple ion adducts by MALDI-TOF-MS. The MS signal intensities of lactose, sialylated N-glycans derived from bovine fetuin and neutral N-glycans derived from RNaseB and ovalbumin were boosted by 7.44, 9.13, 12.96 and 13.47 folds on average (n?=?3), respectively. More importantly, after GTOD strategy, unwanted desialylation of sialylated glycans during MS was suppressed. The detection limit of the assay is desirable since the nanogram of N-glycans derived from 0.16??g ovalbumin could be detected. The assay demonstrated good stability (RSD?2.95%, within 10 days), reliable reproducibility (RSD?=?2.96%, n?=?7) and a desirable linear dynamic range from 78?nmol/mL to 10??mol/mL. The strategy has been successfully applied to MS analysis of reducing glycans from human milks, neutral and sialylated O-, N-glycans from glycoproteins, and reducing glycans derived from glycosphingolipids, presenting neater [M]+ signals which allow detection of more low-abundance glycans and assignation of Neu5Ac vs. Neu5Gc or fucose vs. hexose in glycans due to the absence of the ambiguous interpretation from multiple peaks (ion adducts [M+Na]+ and [M+K]+). Moreover, the GTOD assay prevents desialylation during MALDI-TOF-MS profiling and enables distinct linkage-specific characterization of terminal sialic acids of N-glycans derived from human serum protein when combines with an esterification.

Dataset Information

Structural characterisation of neutrophil glycans by ultra sensitive mass spectrometric glycomics methodology.

Publications

Structural characterisation of neutrophil glycans by ultra sensitive mass spectrometric glycomics methodology.

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