Glycomic analysis of human mast cells, eosinophils and basophils

Abstract

In allergic diseases such as asthma, eosinophils, basophils and mast cells, through release of preformed and newly generated mediators, granule proteins and cytokines, are recognized as key effector cells. While their surface protein phenotypes, mediator release profiles, ontogeny, cell trafficking and genomes have been generally explored and compared, there has yet to be any thorough analysis and comparison of their glycomes. Such studies are critical to understand the contribution of carbohydrates to the induction and regulation of allergic inflammatory responses and are now possible using improved technologies for detecting and characterizing cell-derived glycans. We thus report here the application of high-sensitivity mass spectrometric-based glycomics methodologies to the analysis of N-linked glycans derived from isolated populations of human mast cells, eosinophils and basophils. The samples were subjected to matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) screening analyses and MALDI-TOF/TOF sequencing studies. Results reveal substantive quantities of terminal N-acetylglucosamine containing structures in both the eosinophil and the basophil samples, whereas mast cells display greater relative quantities of sialylated terminal epitopes. For the first time, we characterize the cell surface glycan structures of principal allergic effector cells, which by interaction with glycan-binding proteins (e.g. lectins) have the possibility to dictate cellular functions, and might thus have important implications for the pathogenesis of inflammatory and allergic diseases.

Fig. 1.

MALDI-TOF MS profiles of the permethylated N-linked glycans from human basophils (A), eosinophils (B) and mast cells (C). Major peaks are annotated with their proposed carbohydrate structure, according to the symbolic nomenclature adopted by the CFG (Varki et al. 2009). For complete annotations of the spectra, see Table I. All molecular ions are present in singly charged sodiated form ([M + Na]⁺).

Fig. 2.

MALDI-TOF/TOF MS/MS fragmentation spectrum of the [M + Na]⁺ molecular ion observed at m/z 1835.9 from the permethylated N-glycan pool derived from the human basophil cell sample (Figure 1A). The peak is by far the most abundant ion in the spectrum and corresponds to a monosaccharide composition of Fuc₁Hex₃HexNAc₄. Assignments of the product ions are indicated.

Fig. 3.

MALDI-TOF MS profile of the permethylated N-linked glycans from human LAD2 mast cells. Major peaks are annotated with their proposed carbohydrate structure, according to the symbolic nomenclature adopted by the CFG (Varki et al. 2009). For complete annotations of the spectra, see Table I. All molecular ions are present in singly charged sodiated form ([M + Na]⁺).

Fig. 4.

MALDI-TOF MS profile of the permethylated O-linked glycans from human LAD2 mast cells. Peaks are annotated with their proposed carbohydrate structure, according to the symbolic nomenclature adopted by the CFG (Varki et al. 2009). For complete annotations of the spectrum, see Table II. All molecular ions are present in a singly charged sodiated form ([M + Na]⁺).

Fig. 5.

MALDI-TOF/TOF MS/MS fragmentation spectrum of the [M + Na]⁺ molecular ion observed at m/z 1618.6 from the permethylated O-glycan derived from the human LAD2 mast cells (Figure 4). The peak corresponds to a monosaccharide composition of NeuAc₃Hex₁HexNAc₁. Assignments of the product ions are indicated.