title: Glycan profiles of the 27 N-glycosylation sites of the HIV envelope protein CN54gp140.
abstract: Hope rests on the envelope proteins of human immunodeficiency virus (HIV) as protective vaccines and thus their antibody binding sites are of prime interest. 2G12 and other human antibodies bind to a cluster of oligomannose N-glycans. Owing to the extreme number and density of N-glycosylation sites gp160 and its recombinant form gp140 represent challenging tasks for site-specific glycosylation analysis. We have conducted a glycosylation analysis of CN54gp140 by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) using an ion trap as well as a Q-TOF instrument and standard software for glycopeptide identification. First, a deglycosylated sample of the protease digest served to locate the elution positions of peptides covering all of the 27 potential N-glycosylation sites. Then, the assignments of the similarly eluting glycopeptides were verified by collision-induced decay MS/MS experiments with elevated fragmentation energy. The acquisition of site-specific glycan profiles was facilitated by the use of buffered eluent, which rounds up all glycoforms of a peptide into one peak. Calculation of the molecular mass drawn on the weighted averages of the glycans at each site led to the actual mass of gp140 of approximately 120 kDa.
authors.affiliation: Department of Chemistry, University of Natural Resource and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
authors.forename: Martin
authors.initials: M
authors.lastname: Pabst
authors.forename: Martina
authors.initials: M
authors.lastname: Chang
authors.forename: Johannes
authors.initials: J
authors.lastname: Stadlmann
authors.forename: Friedrich
authors.initials: F
authors.lastname: Altmann
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002240:Carbohydrate Sequence; D006224:Cricetinae; D006020:Glycopeptides / Q000737:chemistry / Q000235:genetics; D006031:Glycosylation; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D054299:env Gene Products, Human Immunodeficiency Virus / Q000737:chemistry* / Q000235:genetics
chemical_list: D006020:Glycopeptides; D011994:Recombinant Proteins; D054299:env Gene Products, Human Immunodeficiency Virus; C097729:gp140 envelope protein, Human immunodeficiency virus 1