Glycoform analysis of recombinant and human immunodeficiency virus envelope protein gp120 via higher energy collisional dissociation and spectral-aligning strategy

Weiming Yang; Punit Shah; Shadi Toghi Eshghi; Shuang Yang; Shisheng Sun; Minghui Ao; Abigail Rubin; J Brooks Jackson; Hui Zhang

doi:10.1021/ac500876p

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Glycoform analysis of recombinant and human immunodeficiency virus envelope protein gp120 via higher energy collisional dissociation and spectral-aligning strategy

Journal article

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Peer reviewed

Glycoform analysis of recombinant and human immunodeficiency virus envelope protein gp120 via higher energy collisional dissociation and spectral-aligning strategy

Weiming Yang, Punit Shah, Shadi Toghi Eshghi, Shuang Yang, Shisheng Sun, Minghui Ao, Abigail Rubin, J Brooks Jackson and Hui Zhang

Analytical chemistry (Washington), Vol.86(14), pp.6959-6967

07/15/2014

DOI: 10.1021/ac500876p

PMCID: PMC4215848

PMID: 24941220

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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.

HIV Envelope Protein gp120 - genetics

Recombinant Proteins - metabolism

Amino Acid Sequence

Recombinant Proteins - analysis

Humans

Glycopeptides - chemistry

Molecular Sequence Data

Recombinant Proteins - chemistry

Ions

Virion - chemistry

Glycosylation

Recombinant Proteins - genetics

HIV Envelope Protein gp120 - metabolism

Glycopeptides - analysis

HEK293 Cells

HIV Envelope Protein gp120 - analysis

HIV Envelope Protein gp120 - chemistry

Carbohydrate Conformation

Tandem Mass Spectrometry - methods

Details

Title: Subtitle: Glycoform analysis of recombinant and human immunodeficiency virus envelope protein gp120 via higher energy collisional dissociation and spectral-aligning strategy
Creators: Weiming Yang - Department of Pathology, School of Medicine, Johns Hopkins University , 1550 Orleans Street , Baltimore, Maryland 21205, United States
Punit Shah
Shadi Toghi Eshghi
Shuang Yang
Shisheng Sun
Minghui Ao
Abigail Rubin
J Brooks Jackson
Hui Zhang
Resource Type: Journal article
Publication Details: Analytical chemistry (Washington), Vol.86(14), pp.6959-6967
DOI: 10.1021/ac500876p
PMID: 24941220
PMCID: PMC4215848
NLM abbreviation: Anal Chem
ISSN: 0003-2700
eISSN: 1520-6882
Publisher: United States
Grant note: P01HL107153 / NHLBI NIH HHS U01 AI069482 / NIAID NIH HHS 1U01 AI69482-01 / NIAID NIH HHS P01 HL107153 / NHLBI NIH HHS
Language: English
Date published: 07/15/2014
Academic Unit: Pathology; VPMA - Administration
Record Identifier: 9984047637302771

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