Characterization of the Agent of “High Plains Disease”: Mass Spectrometry Determines the Sequence of the Disease-specific Protein
peer-reviewed · Journal of Biological Chemistry · 2004
| Date | 2004-01-01 |
| Type | peer-reviewed |
| Venue | Journal of Biological Chemistry |
| Publisher | American Society for Biochemistry and Molecular Biology |
| Contribution | downstream-application |
| DOI | 10.1074/jbc.M308506200 |
Abstract
The “32-kDa” protein specifically associated with high plains disease was characterized by time-of-flight mass spectrometry, after the agent had been isolated in pure culture by “vascular puncture inoculation,” a novel mechanical means of transmission. Two isolates from different geographic locations each consisted of a mixture of subpopulations that were highly homologous to an amino acid sequence derived from a nucleotide sequence (U60141) deposited in GenBank trade mark by the Nebraska group as “the probable N-protein of high plains virus.” However, the U60141 sequence was found to be incomplete; de novo sequencing of peptides produced by proteolytic digestions of the 32-kDa band from an SDS-PAGE separation showed that an additional 18 amino acid residues were present at the N terminus. BLAST (basic local alignment search tool) examination of the sequence showed no significant homology with any protein in the databases, indicating that the infectious agent of high plains disease is likely a member of a hitherto unclassified virus group.
Methods and tools
- High plains disease agent characterization: Downstream application of de novo peptide sequencing to an unidentified plant pathogen. The 32-kDa protein specific to high plains disease was sequenced by time-of-flight MS after the agent was isolated in pure culture by vascular puncture inoculation. De novo sequencing of peptides from proteolytic digests of the SDS-PAGE band corrected the public record: the GenBank nucleotide-derived sequence U60141, deposited as the probable N-protein of high plains virus, turned out to be incomplete, and 18 further residues were found at the N terminus. BLAST then returned no significant homology to any protein in the databases, indicating a hitherto unclassified virus group. A clean early demonstration of why de novo matters: the answer was unreachable by database search because the correct sequence was not in any database.