A strategy for the identification of site-specific glycosylation in glycoproteins using MALDI TOF MS |
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| Institution: | 1. School of Mechanical Engineering, Shenyang Ligong University, Shenyang, 110159, China;2. School of Mechanical Electronic and Control Engineering, Beijing Jiaotong University, Beijing, 100044, China;1. Department of Electronic Science, University of Calcutta, 92, A.P.C Road, Kolkata 700009, West Bengal, India;2. Molecular and Applied Mycology and Plant Pathology Laboratory, Department of Botany, University of Calcutta, 35, Ballygunge Circular Road, Kolkata 700019, West Bengal, India;3. Department of Polymer Science and Technology, University of Calcutta, 92, A.P.C Road, Kolkata 700009, West Bengal, India;1. School of Chemistry, The University of New South Wales, Sydney, NSW 2052, Australia;2. School of Pharmacy, King Khalid University, Abha, Saudi Arabia;3. Solid State & Elemental Analysis Unit, Mark Wainwright Analytical Centre, Division of Research, UNSW Australia, NSW 2052, Australia;1. Science and Liberal Art, Amnatcharoen Campus, Mahidol University, Amnatcharoen 37000, Thailand;2. Creative Chemistry and Innovation Research Unit, Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Mahasarakham University, Maha Sarakham 44150, Thailand;3. Department of Chemistry, and Center of Excellence for Innovation in Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand |
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| Abstract: | A strategy for investigation of site-specific glycosylation of glycoproteins has been developed, based on peptide mass fingerprinting using matrix assisted laser desorption ionisation time of flight mass spectrometry (MALDI TOF MS). The glycoprotein is subjected to sequential digestion with a protease and glycan-specific endoglycosidases or with the glycan-specific endoglycosidases followed by the protease. Peptides with characteristic masses are detected for sequences containing glycosylated asparagine residues. By using a panel of three proteases, chymotrypsin, protease V8 and trypsin, and endoglycosidases F3 and H and peptide N-glycanase F, it was possible to monitor the state of glycosylation of all putative N-glycosylation sites on three glycoproteins. It was deduced that all potential N-glycosylation sites in human serum transferrin (two) and α1-antitrypsin (three) were occupied by non-fucosylated, biantennary, disialylated, complex glycans. In contrast, only four (asparagines 19, 59, 146 and 270) out of the five potential sites were glycosylated in recombinant human β-glucosylceramidase, with the site nearest the C-terminal (asparagine 462) being unoccupied. The glycans at each site consisted of a mixture of non-fucosylated and core α1–6 fucosylated oligomannose glycans (Man3 GlcNAc2), derived from the enzymic truncation of complex glycans. |
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