Abstract
Time-of-flight mass spectrometry (TOFMS) has been applied to determine the complete coat protein amino acid sequences of a number of distinct brome mosaic virus (BMV) isolates. Ionization was carried out by both electrospray ionization and matrix-assisted laser desorption/ionization (MALDI). After determining overall coat protein masses, the proteins were digested with trypsin or Lys-C proteinases, and the digestion products were analyzed in a MALDI QqTOF mass spectrometer. The N terminus of the coat protein was found to be acetylated in each BMV isolate analyzed. In one isolate (BMV-Valverde), the amino acid sequence was identical to that predicted from the cDNA sequence of the "type" isolate, but deviations from the predicted amino acid sequence were observed for all the other isolates analyzed. When isolates were propagated in different host taxa, modified coat protein sequences were observed in some cases, along with the original sequence. Sequencing by TOFMS may therefore provide a basis for monitoring the effects of host passaging on a virus at the molecular level. Such TOFMS-based analyses assess the complete profiles of coat protein sequences actually present in infected tissues. They are therefore not subject to the selection biases inherent in deducing such sequences from reverse-transcribed viral RNA and cloning the resulting cDNA.
Highlights
Time-of-flight mass spectrometry (TOFMS) has been applied to determine the complete coat protein amino acid sequences of a number of distinct brome mosaic virus (BMV) isolates
In one isolate (BMV-Valverde), the amino acid sequence was identical to that predicted from the cDNA sequence of the “type” isolate, but deviations from the predicted amino acid sequence were observed for all the other isolates analyzed
As an illustration of how these methods can be applied to closely related virus isolates, we describe here TOFMS measurements on a group of geographic isolates of brome mosaic virus (BMV)
Summary
With the development of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), as well as new types of mass analyzers, mass spectrometry (MS) affords a rapid and efficient approach for obtaining more detailed information about virus coat protein sequences in infected tissue [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22]. (a) Propagation and maintenance are easier than for human and animal viruses, allowing faster experiment cycles This is important in experiments designed to investigate the link between altered virulence achieved by serial passage through alternative hosts and specific changes in viral coat protein amino acid sequence (see the example below). “Cruelty to plants” has not yet captured the attention of placard-waving demonstrators. (d) Most plant viruses are considerably less complex than most human, animal, and
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