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.

Fig. 1

MALDI-TOF MS profiles of the permethylated N-linked glycans from human neutrophils. Major peaks are annotated with the relevant carbohydrate structure shown in symbol form, according to the glycan nomenclature adopted by the CFG (http://www.functionalglycomics.org/). Neutrophil Sample 1 (US) is displayed in panels (a), (b) and (c). Neutrophil Sample 2 (UK) is displayed in panels (d), (e) and (f). For complete annotation of the spectra see Table 1. All molecular ions are present in sodiated form ([M +Na]⁺)

Fig. 2

MALDI-TOF/TOF mass spectrum of the [M +Na]⁺ molecular ion m/z 3766.9 (composition Fuc₂Hex₈HexNAc₉, Panel (a)) and m/z 3141.0 (compositions NeuAc₂Fuc₂Hex₅HexNAc₄ and Fuc₁Hex₇HexNAc_6, Panel (b)). Both spectra are derived from the permethylated N-glycans released with PNGase F from neutrophil Sample 2 (UK) (Fig. 1d and e). Assignments of the fragment ions are labelled

Fig. 3

MALDI-TOF profile of permethylated N-glycans after neuraminidase digestion. Panel (a) shows the lower mass region and panel (b) shows the higher mass region. All molecular ions are present in sodiated form ([M +Na]⁺)

Fig. 4

MALDI-TOF/TOF mass spectrum of the [M +Na]⁺ molecular ion m/z 3765 (composition Fuc₂Hex₈HexNAc₉), derived from the neuraminidase treated permethylated N-glycans from neutrophil Sample 2 (UK)

Fig. 5

MALDI-TOF MS profiles of the permethylated O-linked glycans from human neutrophils. Major peaks are annotated with the relevant carbohydrate structure shown in symbol form, according to the glycan nomenclature adopted by the CFG (http://www.functionalglycomics.org/). Neutrophil Sample 1 (US) is displayed in panel a), while neutrophil Sample 2 (UK) is displayed in panel (b)

Fig. 6

MALDI-TOF/TOF mass spectrum of the [M +Na]⁺ molecular ion m/z 1518.0 derived from the permethylated O-glycans of neutrophil Sample 2 (UK). Assignments of the fragment ions are labelled