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Nat Protoc. 2018 Jun;13(6):1196-1212. doi: 10.1038/nprot.2018.024. Epub 2018 May 3.

Global site-specific analysis of glycoprotein N-glycan processing.

Author information

1
Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California, USA.
2
Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery and IAVI Neutralizing Antibody Center, Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, California, USA.
3
Department of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA.
4
Ragon Institute of MGH, MIT and Harvard, Cambridge, Massachusetts, USA.

Abstract

N-glycans contribute to the folding, stability and functions of the proteins they decorate. They are produced by transfer of the glycan precursor to the sequon Asn-X-Thr/Ser, followed by enzymatic trimming to a high-mannose-type core and sequential addition of monosaccharides to generate complex-type and hybrid glycans. This process, mediated by the concerted action of multiple enzymes, produces a mixture of related glycoforms at each glycosite, making analysis of glycosylation difficult. To address this analytical challenge, we developed a robust semiquantitative mass spectrometry (MS)-based method that determines the degree of glycan occupancy at each glycosite and the proportion of N-glycans processed from high-mannose type to complex type. It is applicable to virtually any glycoprotein, and a complete analysis can be conducted with 30 μg of protein. Here, we provide a detailed description of the method that includes procedures for (i) proteolytic digestion of glycoprotein(s) with specific and nonspecific proteases; (ii) denaturation of proteases by heating; (iii) sequential treatment of the glycopeptide mixture with two endoglycosidases, Endo H and PNGase F, to create unique mass signatures for the three glycosylation states; (iv) LC-MS/MS analysis; and (v) data analysis for identification and quantitation of peptides for the three glycosylation states. Full coverage of site-specific glycosylation of glycoproteins is achieved, with up to thousands of high-confidence spectra hits for each glycosite. The protocol can be performed by an experienced technician or student/postdoc with basic skills for proteomics experiments and takes ∼7 d to complete.

PMID:
29725121
PMCID:
PMC5941933
[Available on 2019-06-01]
DOI:
10.1038/nprot.2018.024

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