title: Glycoform analysis of recombinant and human immunodeficiency virus envelope protein gp120 via higher energy collisional dissociation and spectral-aligning strategy.
abstract: Envelope protein gp120 of human immunodeficiency virus (HIV) is armored with a dense glycan shield, which plays critical roles in envelope folding, immune-evasion, infectivity, and immunogenicity. Site-specific glycosylation profiling of recombinant gp120 is very challenging. Therefore, glycoproteomic analysis of native viral gp120 is still formidable to date. This challenge promoted us to employ a Q-Exactive mass spectrometer to identify low abundant glycopeptides from virion-associated gp120. To search the HCD-MS data for glycopeptides, a novel spectral-aligning strategy was developed. This strategy depends on the observation that glycopeptides and the corresponding deglycosylated peptides share very similar MS/MS pattern in terms of b- and y-ions that do not contain the site of glycosylation. Moreover, glycopeptides with an identical peptide backbone show nearly resembling spectra regardless of the attached glycan structures. For the recombinant gp120, this "copy-paste" spectral pattern of glycopeptides facilitated identification of 2224 spectra using only 18 spectral templates, and after precursor mass correction, 1268 (57%) spectra were assigned to 460 unique glycopeptides accommodating 19 N-linked and one O-linked glycosylation sites (glycosites). Strikingly, we were able to observe five N- and one O-linked glycosites in native gp120. We further revealed that except for Asn276 in the C2 region, glycans were processed to contain both high mannose and hybrid/complex glycans; an additional four N-linked glycosites were decorated with high mannose type. Core 1 O-linked glycan Gal1GalNAc1 was seen for the O-linked glycosite at Thr499. This direct observation of site-specific glycosylation of virion-derived gp120 has implications in HIV glycobiology and vaccine design.
authors.affiliation: Department of Pathology, School of Medicine, Johns Hopkins University , 1550 Orleans Street , Baltimore, Maryland 21205, United States.
authors.forename: Weiming
authors.initials: W
authors.lastname: Yang
authors.forename: Punit
authors.initials: P
authors.lastname: Shah
authors.forename: Shadi
authors.initials: S
authors.lastname: Toghi Eshghi
authors.forename: Shuang
authors.initials: S
authors.lastname: Yang
authors.forename: Shisheng
authors.initials: S
authors.lastname: Sun
authors.forename: Minghui
authors.initials: M
authors.lastname: Ao
authors.forename: Abigail
authors.initials: A
authors.lastname: Rubin
authors.forename: J Brooks
authors.initials: JB
authors.lastname: Jackson
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D002236:Carbohydrate Conformation; D006020:Glycopeptides / Q000032:analysis* / Q000737:chemistry; D006031:Glycosylation; D057809:HEK293 Cells; D015699:HIV Envelope Protein gp120 / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007477:Ions; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014771:Virion / Q000737:chemistry
chemical_list: D006020:Glycopeptides; D015699:HIV Envelope Protein gp120; D007477:Ions; D011994:Recombinant Proteins