Global site-specific analysis of glycoprotein N-glycan processing

Autor: Liwei Cao, Sung-Kyu Robin Park, Claire M. Delahunty, Dennis R. Burton, Yuanhui Ma, Jason S. McLellan, Matthias Pauthner, James C. Paulson, Jolene K. Diedrich, John R. Yates, Nianshuang Wang
Rok vydání: 2018
Předmět:
Zdroj: Nature Protocols. 13:1196-1212
ISSN: 1750-2799
1754-2189
DOI: 10.1038/nprot.2018.024
Popis: This protocol describes a semiquantitative glycoproteomics method using sequential treatment with endoglycosidases to create unique mass signatures to determine glycan occupancy and proportion of high-mannose and complex glycans at each glycosite. 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.
Databáze: OpenAIRE
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