title: Simple approach to assign disulfide connectivity using extracted ion chromatograms of electron transfer dissociation spectra.
abstract: Increasing interest in production of protein-based pharmaceuticals (biotherapeutics) is accompanied by an increased need for verification of protein folding and correct disulfide bonding. Recombinant protein expression may produce aberrant disulfide bonds and could result in safety concerns or decreased efficacy. Thus, the thorough analysis of disulfide bonding is a necessity for protein therapeutics. The use of electron transfer dissociation (ETD) facilitates this analysis because disulfide bonds are preferentially cleaved when subjected to ETD. Here, we make use of this well-characterized reaction to assign disulfide bonding networks by coupling the use of extracted ion chromatograms (XICs) of cysteine-containing peptides with ETD analysis to produce an efficient assignment approach for disulfide bonding. This method can be used to assign a disulfide pattern in a de novo fashion, to detect disulfide shuffling, and to provide information on heterogeneity, when more than one disulfide bonding pattern is present. The method was applied for assigning the disulfide-bonding network of a recombinant monomer of the HIV envelope protein gp120. It was found that one region of the protein, the V1/V2 loops, had significant heterogeneity in the disulfide bonds.
authors.affiliation: Department of Chemistry, University of Kansas, Lawrence, Kansas 66045, USA.
authors.forename: Daniel F
authors.initials: DF
authors.lastname: Clark
authors.forename: Eden P
authors.initials: EP
authors.lastname: Go
authors.forename: Heather
authors.initials: H
authors.lastname: Desaire
mesh_terms: D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D004579:Electron Transport; D015699:HIV Envelope Protein gp120 / Q000737:chemistry*; D007477:Ions / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D053719:Tandem Mass Spectrometry
chemical_list: D004220:Disulfides; D015699:HIV Envelope Protein gp120; D007477:Ions; D010455:Peptides; D011994:Recombinant Proteins; D003545:Cysteine