Global site-specific analysis of glycoprotein N-glycan processing

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.

Human Influenza Virus Hemagglutinins Contain Conserved Oligomannose N-Linked Glycans Allowing Potent Neutralization by Lectins

“…interference of the biosynthetic processing pathway, our observations were unexpected since HA is substantially less glycosylated than the surface glycoproteins of numerous other viruses. With up to thirty N-linked glycans per monomer, both HIV gp120 and MERS CoV S-2P are well-characterized examples that comprise numerous oligomannose sites arising due to close packing of their many structurally proximal glycosites (Cao et al, 2017(Cao et al, , 2018. By contrast, HA glycans are far fewer and appear sparsely arranged (Figure 4), suggesting that in this case, presence of oligomannose glycans is not due to steric restrictions imposed by neighboring glycan chains.…”

“…This proteomics-based method for site-specific analysis of N-linked glycan processing uses endoglycosidases to introduce mass signatures that contain either no glycan, minimally processed high-mannose or hybrid-type, or more extensively processed complex-type N-glycans. This allows a semi-quantitative assessment of the proportion of each glycoform present at each glycosite (Cao et al, 2017(Cao et al, , 2018.…”

“…Energy-minimized modeling of glycosylated crystal structures for all six representative HAs reveals conservation of both the positions and types of glycans at individual glycosylation sites, particularly within the group 1 and 2 HA phylogenetic classes (Air, 1981;Nobusawa et al, 1991). Comparison of the glycosylated HAs suggests overall location on HA, together with local structural features surrounding the glycosites to be the major determinants of oligomannose glycoforms, rather than dense packing of multiple sites, as observed in other viruses, such as the human immunodeficiency virus (HIV) (Cao et al, 2017(Cao et al, , 2018. Structural mapping of high-mannose glycosites in the HA of human H3N2 viruses reveals the presence of a small high-mannose patch, in close proximity to the RBS at the top of H3, accounting for the effectiveness of GNL neutralization.…”

Haploid genetic screens identify SPRING/C12ORF49 as a determinant of SREBP signaling and cholesterol metabolism

“…5c). To interrogate the nature of the glycan chain present on SPRING we used Endoglycosidase-H (Endo-H), which removes glycans from ER-and cis/medial Golgi-resident proteins, before they acquire complex modifications 31 . We observed that unlike LDLR, SPRING glycosylation remained Endo-H-sensitive (Supplementary Fig.…”

Glycosylation of viral surface proteins probed by mass spectrometry

“…introduce unique mass signatures for (potential) sites of glycosylation that carry no glycan, high-mannose/hybrid type glycans, and complex glycans [75 ]. These strategies have arisen from the need for a HIV-1 vaccine that utilizes HIV-1 Env, which contains 75-90 N-glycans [76].…”