title: Global site-specific N-glycosylation analysis of HIV envelope glycoprotein.
abstract: HIV-1 envelope glycoprotein (Env) is the sole target for broadly neutralizing antibodies (bnAbs) and the focus for design of an antibody-based HIV vaccine. The Env trimer is covered by ∼90N-linked glycans, which shield the underlying protein from immune surveillance. bNAbs to HIV develop during infection, with many showing dependence on glycans for binding to Env. The ability to routinely assess the glycan type at each glycosylation site may facilitate design of improved vaccine candidates. Here we present a general mass spectrometry-based proteomics strategy that uses specific endoglycosidases to introduce mass signatures that distinguish peptide glycosites that are unoccupied or occupied by high-mannose/hybrid or complex-type glycans. The method yields >95% sequence coverage for Env, provides semi-quantitative analysis of the glycosylation status at each glycosite. We find that most glycosites in recombinant Env trimers are fully occupied by glycans, varying in the proportion of high-mannose/hybrid and complex-type glycans.
authors.affiliation: Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: Liwei
authors.initials: L
authors.lastname: Cao
authors.affiliation: Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: Jolene K
authors.initials: JK
authors.lastname: Diedrich
authors.affiliation: Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California 92037, USA.|Department of the IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: Daniel W
authors.initials: DW
authors.lastname: Kulp
authors.affiliation: Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California 92037, USA.|Department of the IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: Matthias
authors.identifier: 0000-0001-9212-4307
authors.initials: M
authors.lastname: Pauthner
authors.affiliation: Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: Lin
authors.initials: L
authors.lastname: He
authors.affiliation: Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: Sung-Kyu Robin
authors.initials: SR
authors.lastname: Park
authors.affiliation: Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California 92037, USA.|Department of the IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: Devin
authors.initials: D
authors.lastname: Sok
authors.affiliation: Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: Ching Yao
authors.initials: CY
authors.lastname: Su
authors.affiliation: Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: Claire M
authors.initials: CM
authors.lastname: Delahunty
authors.affiliation: Department of the IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: Sergey
authors.initials: S
authors.lastname: Menis
authors.affiliation: Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: Raiees
authors.initials: R
authors.lastname: Andrabi
authors.affiliation: Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: Javier
authors.initials: J
authors.lastname: Guenaga
authors.affiliation: Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: Erik
authors.initials: E
authors.lastname: Georgeson
authors.affiliation: Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Kubitz
authors.affiliation: Department of the IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Adachi
authors.affiliation: Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California 92037, USA.|Department of the IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: Dennis R
authors.initials: DR
authors.lastname: Burton
authors.affiliation: Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California 92037, USA.|Department of the IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: William R
authors.initials: WR
authors.lastname: Schief
authors.affiliation: Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: John R
authors.identifier: 0000-0001-5267-1672
authors.initials: JR
authors.lastname: Yates
authors.affiliation: Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.|Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California 92037, USA.|Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: James C
authors.initials: JC
authors.lastname: Paulson
mesh_terms: D000939:Epitopes / Q000737:chemistry; D006031:Glycosylation; D015699:HIV Envelope Protein gp120 / Q000737:chemistry / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D013058:Mass Spectrometry; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry; D055503:Protein Multimerization; D015203:Reproducibility of Results
chemical_list: D000939:Epitopes; D015699:HIV Envelope Protein gp120; D010455:Peptides; D011134:Polysaccharides