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Toolbox Accelerating Glycomics (TAG): Glycan Annotation from MALDI-TOF MS Spectra and Mapping Expression Variation to Biosynthetic Pathways.

ABSTRACT: Glycans present extraordinary structural diversity commensurate with their involvement in numerous fundamental cellular processes including growth, differentiation, and morphogenesis. Unlike linear DNA and protein sequences, glycans have heterogeneous structures that differ in composition, branching, linkage, and anomericity. These differences pose a challenge to developing useful software for glycomic analysis. To overcome this problem, we developed the novel Toolbox Accelerating Glycomics (TAG) program. TAG consists of three units: 'TAG List' creates a glycan list that is used for database searching in TAG Expression; 'TAG Expression' automatically annotates and quantifies glycan signals and draws graphs; and 'TAG Pathway' maps the obtained expression information to biosynthetic pathways. Herein, we discuss the concepts, outline the TAG process, and demonstrate its potential using glycomic expression profile data from Chinese hamster ovary (CHO) cells and mutants lacking a functional Npc1 gene (Npc1 knockout (KO) CHO cells). TAG not only drastically reduced the amount of time and labor needed for glycomic analysis but also detected and quantified more glycans than manual analysis. Although this study was limited to the analysis of N-glycans and free oligosaccharides, the glycomic platform will be expanded to facilitate the analysis of O-glycans and glycans of glycosphingolipids.

SUBMITTER: Miura N

PROVIDER: S-EPMC7650810 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Toolbox Accelerating Glycomics (TAG): Glycan Annotation from MALDI-TOF MS Spectra and Mapping Expression Variation to Biosynthetic Pathways.

Miura Nobuaki N Hanamatsu Hisatoshi H Yokota Ikuko I Okada Kazue K Furukawa Jun-Ichi JI Shinohara Yasuro Y

Biomolecules 20200928 10

Glycans present extraordinary structural diversity commensurate with their involvement in numerous fundamental cellular processes including growth, differentiation, and morphogenesis. Unlike linear DNA and protein sequences, glycans have heterogeneous structures that differ in composition, branching, linkage, and anomericity. These differences pose a challenge to developing useful software for glycomic analysis. To overcome this problem, we developed the novel Toolbox Accelerating Glycomics (TAG ...[more]

PMID: 32998456

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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

Toolbox Accelerating Glycomics (TAG): Glycan Annotation from MALDI-TOF MS Spectra and Mapping Expression Variation to Biosynthetic Pathways.

Publications

Toolbox Accelerating Glycomics (TAG): Glycan Annotation from MALDI-TOF MS Spectra and Mapping Expression Variation to Biosynthetic Pathways.

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