Characterization of pathotype-specific epitopes of newcastle disease virus fusion glycoproteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and post-source decay sequencing.
Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) was used to characterize the F2 polypeptide of the fusion (F) protein of an avirulent isolate (VRI 82-6409) of Newcastle disease virus (NDV) that was previously identified by immunochemical screening as having a variant cleavage activation sequence in its fusion protein precursor (F0). The major glycoform of the intact F2 polypeptide of the VRI 82-6409 isolate was 89 Da smaller than the F2 polypeptide of the avirulent V4 isolate of the Queensland strain of NDV. Analysis of AspN protease digests of the F2 polypeptides by MALDI/TOF-MS, with and without high-performance liquid chromatographic (HPLC) separation, showed this mass difference to be due to a combination of differences in the extents of glycosylation and an amino acid difference in the AspN peptides derived from the C-termini of the F2 polypeptides. Accuracies achieved in analysis of the AspN peptides allowed the identification of this amino acid difference as glutamic acid in the VRI 82-6409 isolate compared with glycine in the V4 isolate. Analysis of fragments formed by post-source decay (PSD) of ions of the C-terminal AspN peptides localized the difference to the C-terminal residues of the respective F2 polypeptides. The present study demonstrated that MALDI/TOF-MS is a highly effective technique for the characterization of NDV variants identified by immunochemical screening of pathotype-specific epitopes at the C-termini of their F2 polypeptides.
Lopaticki
S
S
Biomolecular Research Institute, Parkville, Victoria, Australia.
Morrow
C J
CJ
Gorman
J J
JJ
D000595:Amino Acid Sequence; D000941:Antigens / Q000737:chemistry* / Q000276:immunology; D010450:Endopeptidases / Q000737:chemistry; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology; D006023:Glycoproteins / Q000737:chemistry* / Q000276:immunology; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009522:Newcastle disease virus / Q000737:chemistry* / Q000276:immunology / Q000472:pathogenicity; D010446:Peptide Fragments / Q000032:analysis; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014760:Viral Fusion Proteins / Q000737:chemistry* / Q000276:immunology
D000941:Antigens; D000939:Epitopes; D006023:Glycoproteins; D010446:Peptide Fragments; D010455:Peptides; D014760:Viral Fusion Proteins; D010450:Endopeptidases