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.
Highlights
The “32-kDa” protein 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
We demonstrate here that the “32-kDa” protein associated with high plains virus (HPV) can be characterized by time-of-flight mass spectrometry (TOFMS), following HPV isolation in pure culture by “vascular puncture inoculation” [9, 10, 7], a novel mechanical means of transmission
Previous attempts to characterize the HPV agent have been hindered by the need to transmit the infectious agent with the wheat curl mite vector, which naturally transmits wheat streak mosaic virus along with HPV
Summary
HPV Isolates—The HPV isolates to be characterized were obtained from symptomatic maize (Zea mays L.) collected in Kansas in 1996 (KS96) and Idaho in 1997 (ID97). Generation of HPV Protein for Analysis by TOFMS—HPV-specific protein for mass spectrometric (MS) analysis was obtained after electrophoretic separation (SDS-PAGE), staining with Coomassie alanine; BLAST, basic local alignment search tool; CID, collisioninduced dissociation; ELISA, enzyme-linked immunosorbent assay; MALDI, matrix-assisted laser desorption ionization; TOFMS, time-offlight mass spectrometry; MS/MS, tandem mass spectrometry; aa, amino acid. Protein or peptide sequences were determined by manual interpretation of the MS and MS/MS measurements and identified by data base searching with the computer programs MS-Tag, BLAST, or FASTA
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