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It is increasingly clear that distinct glycans also play pivotal roles in the virus's ability to shield and evade the host's immune system.\u00a0Recently, there has been a great advancement in structural identification and quantitation of viral glycosylation, especially spike proteins. Given the ongoing pandemic and the high demand for structure analysis of SARS\u2010CoV\u20102 densely glycosylated spike protein, mass spectrometry methodologies have been employed to accurately determine glycosylation patterns. There are still many challenges in the determination of site\u2010specific glycosylation of SARS\u2010CoV\u20102 viral spike protein. This is compounded by some conflicting results regarding glycan site occupancy and glycan structural characterization. These are probably due to differences in the expression systems, form of expressed spike glycoprotein, MS methodologies, and analysis software. In this review, we recap the glycosylation of spike protein and compare among various studies. Also, we describe the most recent advancements in glycosylation analysis in greater detail and we explain some misinterpretation of previously observed data in recent publications. Our study provides a comprehensive view of the spike protein glycosylation and highlights the importance of consistent glycosylation determination.<\/jats:p>","DOI":"10.1002\/pmic.202100322","type":"journal-article","created":{"date-parts":[[2022,6,14]],"date-time":"2022-06-14T14:19:31Z","timestamp":1655216371000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":26,"title":["Site\u2010specific glycosylation of SARS\u2010CoV\u20102: Big challenges in mass spectrometry analysis"],"prefix":"10.1002","volume":"22","author":[{"given":"Diana","family":"Campos","sequence":"first","affiliation":[{"name":"Max\u2010Planck\u2010Institut fuer Herz\u2010 und Lungenforschung  Bad Nauheim Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael","family":"Girgis","sequence":"additional","affiliation":[{"name":"Department of Bioengineering Volgenau School of Engineering and Computing George Mason University  Fairfax 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