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.
Highlights
Cell surfaces are coated with a variety of intricately arranged glycoconjugates such as glycoproteins and glycolipids, and glycosylation is thought to be essential for maintaining homeostasis in mammalian cells [1,2]
We developed Toolbox Accelerating Glycomics (TAG) software that provides a modifiable and expandable glycan list, automatically annotates and quantifies the glycan signals on MALDI-TOF mass spectrometry (MS) spectra, quantifies and sorts the results, and visualizes variation in glycan expression based on glycan biosynthetic pathways
To support the discrimination of structural isomers, TAG can provide a list of possible structure candidates with the same composition, which assists the design of further experiments such as MS/MS and exoglycosidase digestion
Summary
Cell surfaces are coated with a variety of intricately arranged glycoconjugates such as glycoproteins and glycolipids, and glycosylation is thought to be essential for maintaining homeostasis in mammalian cells [1,2] As omics approaches such as transcriptomics and proteomics have contributed enormously to our understanding of various biological processes in the postgenomic era, rapid and precise analysis of cellular glycomics is attracting a lot of attention. Glycans are empirically annotated from raw mass data, and quantitative analysis calculations are often carried out manually using spreadsheet software such as Microsoft Excel. Such manual approaches are too time-consuming and labor-intensive for large-scale glycomic analysis. Lack of a comprehensive glycan list and obtaining a non-standard resultant format owing to manual analyses can cause glycomics to be difficult
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