Mass spectrometric analysis of the N-glycoproteome in statin-treated liver cells with two lectin-independent chemical enrichment methods

• Published in: International journal of mass spectrometry Vol. 429; pp. 66 - 75
• Main Authors: Xiao, Haopeng, Hwang, Ju Eun, Wu, Ronghu
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.06.2018
• Subjects: Boronic acid-based enrichment; Click chemistry; Glycoproteomics; LC–MS/MS; Metabolic labeling; Glycoproteomics; Click chemistry; LC–MS/MS; Metabolic labeling; Boronic acid-based enrichment; LC-MS/MS
• ISSN: 1387-3806; 1873-2798
• DOI: 10.1016/j.ijms.2017.05.010

[Display omitted] •Two lectin-independent enrichment methods were comprehensively compared for global analysis of glycoproteins by MS.•The experimental results demonstrated that boronic acid-based method was more universal for glycopeptide enrichment.•The quantification results from the method combining metabolic labeling with click chemistry were more dynamic.•The site ratios were normalized to extract more information about protein expression and modification changes. Protein N-glycosylation is essential for mammalian cell survival and is well-known to be involved in many biological processes. Aberrant glycosylation is directly related to human disease including cancer and infectious diseases. Global analysis of protein N-glycosylation will allow a better understanding of protein functions and cellular activities. Mass spectrometry (MS)-based proteomics provides a unique opportunity to site-specifically characterize protein glycosylation on a large scale. Due to the complexity of biological samples, effective enrichment methods are critical prior to MS analysis. Here, we compared two lectin-independent methods to enrich glycopeptides for the global analysis of protein N-glycosylation by MS. The first boronic acid-based enrichment (BA) method benefits from the universal and reversible interactions between boronic acid and sugars; the other method utilizes metabolic labeling and click chemistry (MC) to incorporate a chemical handle into glycoproteins for future affinity enrichment. We comprehensively compared the performance of the two methods in the identification and quantification of glycoproteins in statin-treated liver cells. Based on the current results, the BA method is more universal in enriching glycopeptides, while with the MC method, cell surface glycoproteins were highly enriched, and the quantification results appear to be more dynamic because only the newly-synthesized glycoproteins were analyzed. In addition, we normalized the glycosylation site ratios by the corresponding parent protein ratios to reflect the real modification changes. In combination with MS-based proteomics, effective enrichment methods will vertically advance protein glycosylation research.