title: Simple, automated, high resolution mass spectrometry method to determine the disulfide bond and glycosylation patterns of a complex protein: subgroup A avian sarcoma and leukosis virus envelope glycoprotein.
abstract: Enveloped viruses must fuse the viral and cellular membranes to enter the cell. Understanding how viral fusion proteins mediate entry will provide valuable information for antiviral intervention to combat associated disease. The avian sarcoma and leukosis virus envelope glycoproteins, trimers composed of surface (SU) and transmembrane heterodimers, break the fusion process into several steps. First, interactions between SU and a cell surface receptor at neutral pH trigger an initial conformational change in the viral glycoprotein trimer followed by exposure to low pH enabling additional conformational changes to complete the fusion of the viral and cellular membranes. Here, we describe the structural characterization of the extracellular region of the subgroup A avian sarcoma and leukosis viruses envelope glycoproteins, SUATM129 produced in chicken DF-1 cells. We developed a simple, automated method for acquiring high resolution mass spectrometry data using electron capture dissociation conditions that preferentially cleave the disulfide bond more readily than the peptide backbone amide bonds that enabled the identification of disulfide-linked peptides. Seven of nine disulfide bonds were definitively assigned; the remaining two bonds were assigned to an adjacent pair of cysteine residues. The first cysteine of surface and the last cysteine of the transmembrane form a disulfide bond linking the heterodimer. The surface glycoprotein contains a free cysteine at residue 38 previously reported to be critical for virus entry. Eleven of 13 possible SUATM129 N-linked glycosylation sites were modified with carbohydrate. This study demonstrates the utility of this simple yet powerful method for assigning disulfide bonds in a complex glycoprotein.
authors: Department of Molecular Medicine, the Mayo Clinic, Rochester, Minnesota 55905, USA.
authors: Gennett M
authors: GM
authors: Pike
authors: Benjamin J
authors: BJ
authors: Madden
authors: Deborah C
authors: DC
authors: Melder
authors: M Cristine
authors: MC
authors: Charlesworth
authors: Mark J
authors: MJ
authors: Federspiel
mesh_terms: D001355:Alpharetrovirus / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002645:Chickens; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D013058:Mass Spectrometry / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D006023:Glycoproteins; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins