Determination of the disulfide bond arrangement of human respiratory syncytial virus attachment (G) protein by matrix‐assisted laser desorption/ionization time‐of …

JJ Gorman, BL Ferguson, D Speelman… - Protein science, 1997 - Wiley Online Library
JJ Gorman, BL Ferguson, D Speelman, J Mills
Protein science, 1997Wiley Online Library
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 …
Abstract
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 Cys 173 and Cys 186 and between Cys 176 and Cys 182. 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.”
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