Determination of the disulfide bond arrangement of human respiratory syncytial virus attachment (G) protein by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
The attachment protein or G protein of the A2 strain of human respiratory syncytial virus (RSV) was digested with trypsin and the resultant peptides separated by reverse-phase high-performance liquid chromatography (HPLC). One tryptic peptide produced a mass by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) corresponding to residues 152-187 with the four Cys residues of the ectodomain (residues 173, 176, 182, and 186) in disulfide linkage and absence of glycosylation. Sub-digestion of this tryptic peptide with pepsin and thermolysin produced peptides consistent with disulfide bonds between Cys173 and Cys186 and between Cys176 and Cys182. Analysis of ions produced by post-source decay of a peptic peptide during MALDI-TOF-MS revealed fragmentation of peptide bonds with minimal fission of an inter-chain disulfide bond. Ions produced by this unprecedented MALDI-induced post-source fragmentation corroborated the existence of the disulfide arrangement deduced from mass analysis of proteolysis products. These findings indicate that the ectodomain of the G protein has a non-glycosylated subdomain containing a "cystine noose."
Gorman
J J
JJ
Biomolecular Research Institute, Parkville, Vic., Australia. jeff.gorman@bioresi.corn.au
Ferguson
B L
BL
Speelman
D
D
Mills
J
J
D000595:Amino Acid Sequence; D003553:Cystine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D015707:HN Protein; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D017421:Sequence Analysis / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014759:Viral Envelope Proteins; D014764:Viral Proteins / Q000737:chemistry*
D004220:Disulfides; D015707:HN Protein; D010446:Peptide Fragments; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C116204:attachment protein G; D003553:Cystine