Determination of the Complete Amino Acid Sequence for the Coat
Protein of Brome Mosaic Virus by Time-of-Flight Mass Spectrometry
EVIDENCE FOR MUTATIONS ASSOCIATED WITH CHANGE OF PROPAGATION
HOST*
Yi-Min
She
,
Steve
Haber§,
Dallas L.
Seifers¶,
Alexander
Loboda
,
Igor
Chernushevich
,
Hélène
Perreault**,
Werner
Ens
, and
Kenneth G.
Standing

From the
Department of Physics & Astronomy,
University of Manitoba, Winnipeg, MB R3T 2N2, Canada, the
§ Cereal Research Centre, Agriculture & Agrifood Canada,
Winnipeg, MB R3T 2M9, Canada, the ¶ Agricultural Research Center,
Kansas State University, Hays, Kansas 67601-9228,
MDS Sciex,
Concord, ON L4K 4V8, Canada, and the ** Department of Chemistry,
University of Manitoba, Winnipeg, MB R3T 2N2, Canada
Received for publication, January 9, 2001, and in revised form, March 21, 2001
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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.
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INTRODUCTION |
Among the parameters characterizing a virus are the mass and the
amino acid sequence of the viral coat protein. Molecular masses have
traditionally been determined by gel electrophoresis; a method whereby
the accuracy and resolution are incapable of distinguishing proteins
that differ in mass by less than ~1%. The amino acid sequence
provides more definitive information, but most sequences currently in
the literature have been deduced from nucleic acid sequences of
specific cDNA clones. Thus they fail to take into account changes
that are not defined by the nucleic acid, such as post-translational
modifications. They also fail to provide a complete profile of the
viral coat protein population actually present in infected tissue.
With the development of electrospray ionization
(ESI)1 and matrix-assisted
laser desorption/ionization (MALDI), as well as new types of mass
analyzers, mass spectrometry (MS) now affords a rapid and efficient
approach for obtaining more detailed information about virus coat
protein sequences in infected tissue (1-22).
The simplest type of MS observation determines the mass of the coat
protein subunit. This can be carried out with an accuracy of a few
daltons (in a few tens of kilodaltons), which is usually sufficient to
distinguish coat proteins of different viruses or different isolates of
the same virus species (15). However, much more information can be
deduced by digesting the protein with specific endopeptidases and
analyzing the digestion products by MS (i.e. peptide
mapping). These analyses can often define the parts of the protein
sequences that agree with those predicted from nucleic acid data and
thus identify the regions where the amino acid sequence differs from prediction.
Still more detailed information can be obtained from tandem mass
spectrometry (MS/MS), in which a given proteolytic fragment ion is
selected by one mass analyzer and is forced to collide with the
molecules of a target gas. The resulting daughter ions are then
characterized by a second mass analyzer. This increases the analytical
power of MS and allows the rapid determination of amino acid sequences
from multiple protein specimens, even if they are closely related.
We have applied these approaches to investigate a number of plant
viruses. Such viruses are of considerable economic importance, but in
addition, the use of plant viruses as model systems for basic
investigations has several advantages. (a) Propagation and maintenance are easier than for human and animal viruses, allowing faster experiment cycles. This is particularly 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). (b) Compared with many human and animal viruses, plant
viruses pose little risk to humans. (c) There is little
difficulty in meeting requirements for health and care of hosts to be
infected, and experiments can accommodate high rates of host
destruction if necessary. "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 bacterial viruses, making it easier to generate well defined test
materials for experiments.
We have determined the ratio of m/z (~27,000)
for intact virions of brome mosaic virus (BMV) (15, 16), measured the
coat protein masses of more than 20 plant virus isolates by
time-of-flight mass spectrometry (TOFMS) (17), and subsequently
determined the complete amino acid sequences of several groups of ssRNA
plant viruses (18-22). These measurements have allowed us to identify variations among individual isolates. Analyses of such variations may
point to evolutionary relationships, whether the variations are caused
by post-translational modifications, deletions, point mutations, or
other mechanisms.
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). This virus is the
type member of the bromoviridae (23) and was one of the
first multipartite-genome ssRNA plant viruses to have its genome
completely sequenced (24-26). It is found in the Great Plains of North
America and has also been reported from Europe and western Asia (27).
In nature, BMV predominantly infects grasses and cereals and may cause
sporadic crop losses (28). It is unusual in its ability to also infect
an array of experimental broadleaf hosts (27).
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EXPERIMENTAL PROCEDURES |
Virus Isolates and Sample Preparation--
We first examined the
BMV isolates listed in Table I. BMV-P and
BMV-N were obtained from the Cereal Research Center collection of virus
isolates (28); isolates investigated subsequently, such as BMV-P* and
-N*, were derived from these in 1999 by repropagation in alternate
hosts. The other isolates listed (BMV1 to BMV4) were supplied to the
Manitoba TOFMS laboratory as coded samples from the collection of
Kansas State University; their subsequent identification (after the
analyses were carried out) is listed in the next column. Care was taken
to propagate and purify the BMV isolates separately and to eliminate
the possibilities of cross-contamination. Virus purification was
carried out according to published methods (28, 29).
After the removal of low molecular weight impurities (<5000 Da) by
centrifugal filtration, the purified coat protein was lyophilized to
determine its amount. A 1% (w/w, enzyme/protein) trypsin or endoproteinase Lys-C solution was added to the protein solution (1 µg/µl) in 25 mM ammonium bicarbonate, followed by
overnight enzymatic digestion at 37 C. The reaction was terminated by
freezing, and the products were stored at
20 °C for subsequent
MALDI and ESI analyses.
Mass Spectrometry--
Measurements of the undigested protein
masses were performed on MALDI/TOF (32) and ESI/TOF (33) mass
spectrometers. Some of the initial measurements of the proteolytic
fragments produced by digestion with trypsin or Lys-C proteinases were
carried out on the same instruments. However, mass determinations of
proteolytic fragments and MS/MS measurements on the daughter ions in
the more difficult cases were made on the Manitoba/Sciex prototype
tandem quadrupole/TOF mass spectrometer (QqTOF) (34) coupled to a MALDI source (35, 36), shown in Fig. 1. The
MALDI matrix consisted of 2,5-dihydroxybenzoic acid, and argon or
nitrogen were used as collision gases; energies of 50-180 eV were
applied. For comparison, monoisotopic mass values of the peptide
fragments from either enzymatic digestions or collision-induced
dissociation (CID) were calculated using the computation program ProMac
(Sciex, Concord, ON).

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Fig. 1.
Schematic diagram of the QqTOF mass
spectrometer with a MALDI ion source (36). In this instrument the
mass-selecting quadrupole Q was modified to give an
m/z range of 6000.
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In addition, some ESI measurements, including parent ion scans (37,
38), were made on a Micromass Quattro-LC ESI triple quadrupole
instrument. Here the parent ion scans were carried out for arginine,
i.e. the third quadrupole was set to detect m/z 175 (diagnostic of Arg), while the first
quadrupole was scanned, thus yielding an m/z
spectrum of all parent ions containing arginine. In addition, such
scans often give better signal/background ratios than ordinary MS spectra.
 |
RESULTS |
Preliminary Characterization of the BMV Isolates
Typical MALDI and ESI spectra of the
intact protein subunits are shown in Figs. 2 and
3. The measured average masses for the coat proteins of the isolates (Table I) all differed from the value of
20,253 Da calculated from the published coat protein sequence of the
type isolate (24-26) (derived from the cloned nucleic acid sequence,
assuming deletion of the N-terminal methionine), and the masses of most
isolates differed from each other. Clearly a number of modifications in
the coat proteins had occurred. To define these modifications, we
carried out a series of MS and MS/MS measurements after proteolytic
digestion of the proteins, as described above. These digests provided
progressively more detailed information that finally made it possible
to characterize the differences among the BMV isolates.

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Fig. 2.
MALDI/TOF m/z
spectrum of the BMV-P coat protein. The measurement was
performed on the MALDI/TOF mass spectrometer (32) using myoglobin
(16,952 Da) as an internal standard and sinapinic acid as matrix.
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Fig. 3.
ESI/TOF m/z
spectrum of the BMV-P coat protein. The protein solution was
prepared in methanol/water solution (v/v, 1:1) containing 5% acetic
acid at pH 2.5. The m/z spectrum shown was
acquired on the ESI/TOF mass spectrometer (33), and a deconvoluted mass
spectrum is shown in the inset.
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In the initial protein sequencing experiments, ESI/TOF MS was used to
analyze tryptic digests of BMV-P. Peptide mapping and MS/MS
measurements on the tryptic fragments led to confirmation of the type
sequence (derived from the published cDNA sequence of BMV (24-26))
for 180 of 189 amino acids (17). The undefined residues were contained
within two short regions near the N terminus (residues 2-8 and
20-26), and we deduced from the measurements that these stretches
included at least two differences from the type sequence.
To resolve this uncertainty, further digestions of the BMV-P samples
(with Lys-C proteinase as well as trypsin) were carried out, and the
masses of the peptide fragments from each digest set were measured by
MALDI as well as ESI mass spectrometry.
MS spectra from BMV-P (Lys-C digest) are shown in Figs. 4 and
5, and the observed fragments are listed
in Table II. Consistent with the initial
analyses, no ions corresponding to the predicted masses of the (2-8)
and (20-26) fragments were found. However, a prominent singly charged
ion could be observed at m/z = 679.4 in the
ESI spectrum, although not in its MALDI counterpart, and we realized
that its mass is 42 Da greater than the predicted mass of the fragment
encompassing residues 2-8. This is consistent with acetylation at the
N terminus, a common post-translational modification. The acetylation
was confirmed by the MS/MS spectrum of this parent ion shown in Fig.
6, which contains a complete set of
acetylated b ions together with a complete set of y ions, of which only
y7 (i.e. the parent ion [M+H]+) is
acetylated. Moreover, the diagnostic ion at
m/z = 679.4 is present in the ESI spectra
from the digests of all samples tested, consistent with N-terminal
acetylation of the BMV coat protein (and deletion of the N-terminal
methionine) for all the BMV isolates. This observation proved to be the
key that allowed us to determine the complete coat protein sequences of
all the isolates examined.

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Fig. 4.
MALDI/TOF m/z
spectrum of a Lys-C digest of the BMV-P coat protein. Data
were recorded by the MALDI/TOF mass spectrometer (32), and the
principal ions are labeled by the range of residues included. The
inset shows an amplified profile of the low
m/z region.
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Fig. 5.
Partial ESI m/z
spectrum of a Lys-C digest of the BMV-P coat protein.
Peptide mapping was carried out on a Micromass triple quadrupole mass
spectrometer with an ESI source. The ion at
m/z = 679.4, observed here with high
intensity, was not seen in the MALDI spectrum of BMV-P (Fig. 4).
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Fig. 6.
CID mass spectrum of the singly charged ion
at m/z 679.4 measured by ESI MS/MS on
the triple quadrupole instrument. An apostrophe denotes
an acetylated ion (e.g. b1' in place of b1), i.e.
an ion 42 Da heavier than the value predicted by the nucleic acid
sequence.
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Determination of the Coat Protein Sequences of the Isolates
BMV-V--
The Valverde isolate (BMV-V) (30) is the simplest case.
The N-terminal of its coat protein is acetylated, but its mass and the
masses of its peptide digest fragments are otherwise identical with
those deduced from the published type sequence (24-26), consistent with the absence of any other modification. MS/MS measurements confirmed this result, as well as the absence of zero-sum mutations.
BMV-P--
Prominent ions at m/z = 445 are found in both ESI and MALDI spectra (Figs. 4 and 5) and in the ESI
parent ion spectrum (Fig. 6), consistent with the predicted mass of
residues 20-22, and thus reducing the unknown region to residues
23-26. The only alteration in that region consistent with the overall
mass is a substitution of Arg for Trp at residue 23, and this
interpretation is supported by the observation of ions at
m/z = 347.0 (sequence 24-26,
TAR), 503.2 (sequence 23-26, RTAR) and 601.4 (sequence 20-23, RNRR) (see also BMV-T measurements below).
The modification is consistent with a single base change of the nucleic
acid sequence (UGG to CGG). Together with acetylation at the N
terminus, it then accounts fully for the observed mass differences
(Tables I and II) between BMV-P and the published type sequence
(24-26).
It is interesting to note that Wang et al. (39, 40) have
reported a BMV isolate from Nebraska whose sequence is identical with
that of BMV-P, indicating that the same mutation has arisen at
different geographic locations.
BMV-T--
The ions mentioned above are also observed in the MS
spectra of the BMV-T isolate. In addition, they appear in the BMV-T
parent ion scan for m/z = 175, an Arg marker
(Fig. 7). However, the most convincing
evidence for the Trp
Arg modification was obtained when fragments
from a Lys-C digestion of BMV-T (pv47) were analyzed in the MALDI-QqTOF
instrument (Table III), enabling high
mass accuracy to be obtained for both parents and daughters. The ion
observed at m/z = 3685.106 has a mass 29.973 Da smaller than the value deduced from the published nucleotide
sequence for fragment 9-41, consistent with the modification Trp
Arg (calculated mass difference
m = 29.978 Da). MS/MS
measurements confirmed that Trp had indeed mutated to Arg at residue 23 in BMV-T, as well as in BMV-P.

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Fig. 7.
Parent ion scans of
m/z 175 on the triple quadrupole
instrument for isolates of BMV-T (type pv47), BMV-W (Western
wheatgrass), BMV-P (Portage), and BMV-V (Valverde), digested by Lys-C
protease.
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Another anomalous fragment ion was observed at
m/z = 2886.503 (Table III). This is 14.024 Da larger than the mass of the deduced fragment encompassing residues
166-189, suggesting the substitution of Leu or Ile for Val (calculated
m = 14.016 Da). Comparison of BMV-T and BMV-P spectra in the
ESI parent ion scans gave
(m/z) = 4.6 (962.8-958.2) for the triply charged ion and
(m/z) = 3.7 (722.5-718.8) for the quadruply
charged ion (Fig. 7), consistent with this interpretation. It was
confirmed and localized by MALDI-MS/MS measurements on the ion at
m/z = 2886, and ESI-MS/MS measurements of
the corresponding triply charged ion at m/z = 963 (Table IV). All of the observed b
ions from b2 to b24 have masses of 14 Da greater than predicted, localizing the modification at residue 167. Consistent with this observation, the C-terminal ions up to
y22 have m/z values that agree with
calculations made for the unmodified protein, whereas y24
(i.e. the parent ion [M+H]+) is modified.
Substitution of Ile or Leu for Val at residue 167 is therefore the only
solution consistent with the MS measurements. It corresponds to a
single base change of the nucleic acid sequence (GUC to AUC or CUC; see
below). As expected, similar results were obtained from the BMV-1
(pv47) isolate.
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Table IV
CID fragmentation of the singly charged ion of m/z 2886.50 (MALDI) and
the triply charged ion of m/z 963.6 (ESI) from the BMV-T (pv47) peptide
(residues 166-189, AVVVHLEVEHVRPTFDDFFTPVYR)
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BMV-W--
The overall mass of BMV-W is smaller than that of BMV-P
by about 100 Da (Table I), and a prominent doubly charged ion appears in the parent ion spectrum (Fig. 7) of BMV-W at
m/z = 868.2 Da, corresponding to a mass of
99 Da less than the calculated mass of fragment 24-41. The mass of
this ion was determined accurately by a MALDI QqTOF measurement of the
tryptic digest, which yielded the spectrum shown in Fig.
8. In this spectrum, the ion
corresponding to fragment 24-41 (expected mass 1834.055 Da) is
replaced by another peak at m/z = 1734.981, with a monoisotopic mass 99.074 Da smaller than predicted. Because
valine has a mass of 99.068 Da, this suggests that one of the three
valines in the segment (TARVQPVIVEPLAAGQGK) has been deleted. MS/MS
measurements on the m/z = 1735 ion, shown in
Fig. 9a, indicated that both
valine residues 30 or 32 were present, but at first inspection a
deletion of Val-27 appeared to give good agreement between the MS/MS
data and calculations, as shown in Table
V. Indeed there would be almost perfect
agreement if the mass were measured to only one or two decimal places
(typical accuracies for some other methods of MS measurement). However, closer examination showed that all the b-ion mass differences (observed
calculated) are negative, with an average
=
11 mDa,
whereas the average
for the y ions is only
0.1 mDa (note that the
latter figure is much less sensitive to residue assignment errors near
the N terminus).

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Fig. 8.
MALDI QqTOF mass spectrum of a tryptic digest
of the BMV-W coat protein. The inset shows an expanded
view of the abnormal peptide fragment ion with monoisotopic mass
1734.981 Da.
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Fig. 9.
CID mass spectra of the parent ion with
m/z = 1734.98 measured by MALDI MS/MS
on the QqTOF instrument. a, overall spectrum.
b, expanded views of the above spectrum near 230 and 1506 Da.
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Table V
Comparison of CID fragmentation of the singly charged ion (m/z 1734.98)
from the BMV-W peptide (residues 24-41) with mass values calculated
assuming Val27 deletion, i.e. from TARQPVIVEPLAAGQGK
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We then realized that another possibility lay in replacing Arg with Gly
at residue 26, which gives an almost identical calculated mass change
(
m = 99.080 Da). This stimulated a more careful search of the
MS/MS spectrum of Fig. 9a, resulting in the discovery of the
small peaks shown in Fig. 9b at
m/z = 230.118 and 1505.853 (initially
unassigned), corresponding to b3 and y15 in the
breakup of the peptide with the R
G substitution. Furthermore, this substitution yields the daughter ion masses shown in Table
VI, where the b-ion mass differences now
alternate between positive and negative, with an average
= 0.4 mDa, a considerable improvement. Thus we conclude that residue 26 in
BMV-W has undergone an R
G mutation, corresponding to a single
genetic code change of AGG to GGG in the nucleic acid sequence.
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Table VI
Comparison of CID fragmentation of the singly charged ion (m/z 1734.98)
from the BMV-W peptide (residues 24-41) with mass values calculated
assuming a mutation of Arg to Gly at position 26, i.e. from
TAGVQPVIVEPLAAGQGK
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Effect of Change of Propagation Host on BMV Coat Protein
Sequences
A change in virulence of a virus by passaging through a different
host is a well established phenomenon (41). It is interesting to see if
such passaging is accompanied by a change in the coat protein sequence,
a possible explanation of the change in virulence.
In this case, the BMV-P isolate had been propagated in Little Club
wheat in 1989 (28), after which the purified lyophilized virus was used
for the ESI mass measurement listed in Table I. To provide material for
additional analyses, BMV-P was propagated in 1999 from the same source
used in 1989 (preserved in the Cereal Research Center collection),
except that BW155 wheat was used, as Little Club was no longer readily
available. This repropagated virus is denoted as BMV-P*.
Two distinct masses appeared in the deconvoluted ESI spectrum of BMV-P*
(Fig. 10). The minor peak (at 20,265 Da) corresponded to the one previously observed for BMV-P, but the
predominant peak had a mass indistinguishable from that previously
determined for BMV-T (20,279 Da), suggesting a mutation of Val to Ile
or Leu at position 167, as deduced above for BMV-T. This was confirmed by mass mapping and by MS/MS measurements (not shown).

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Fig. 10.
Change in the mass spectrum of BMV-P
produced by a change in propagation host. Proteins were dissolved
in methanol/water solution (v/v, 1:1) containing 5% acetic acid, and
m/z measurements were carried out on the ESI/TOF
mass spectrometer (33). The deconvoluted mass spectra are shown.
A, BMV-P*, i.e., after the host change to BW155
wheat; B, BMV-P before the host change, i.e.
propagated in Little Club wheat.
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A further change in the coat protein sequence of BMV-P was observed
when the propagation host was changed from BW155 wheat (BMV-P*) to AC
Assiniboia oat (yielding BMV-P*2). In addition to peaks
corresponding to sequences identical to BMV-P (20,265 Da) and BMV-T
(20,279 Da), BMV-P*2 had a new peak at 20,315 Da (Table
VII). Analysis by tandem QqTOF mass
spectrometry showed that this new sequence had a mutation of Leu to Phe
at position 35, again accounted for by a single nucleic acid base
change (CUC to UUC).
When the BW155 wheat propagation host (BMV-P*) was changed instead to
Seneca maize (generating BMV-P*3), the same three peaks
were observed, but there was a higher proportion of the 20,315 Da peak.
After BMV-P*3 was in turn inoculated on TR241 barley and
propagated (generating BMV-P*4), the purified virus no
longer contained the protein of 20,315 Da, but had a main peak at
20,279 Da and a new small peak at 20,295 Da. The sequence of the 20,295 Da peak was identical to that of BMV-V.
A similar phenomenon was observed when BMV-N, like BMV-P, was
propagated in 1999 in BW155 (rather than Little Club) wheat, yielding
BMV-N*. Two protein peaks were observed in the deconvoluted ESI
spectrum of BMV-N* (Table VII). The larger peak (20,194 Da) corresponded to the mass previously measured for BMV-N, but there was a
new peak (at 20,225 Da) whose mass did not correspond to that of any of
the previously analyzed BMV isolates. The MS spectrum of a BMV-N* Lys-C
digest was similar to that for BMV-W with two exceptions. First, the
monoisotopic ion corresponding to residues 9-15 in BMV-W (and BMV-P,
see Table II) appeared at m/z = 946.542 instead of the calculated value of 918.506 Da, and MS/MS measurements (not shown) indicated that this was caused by a substitution of Ala
Val at residue 12 (corresponding to a single base change GCG to GUG).
Second, the ion at m/z = 1904.984 corresponding to residues 112-130 in BMV-W (and BMV-T, see Table
III) was present, but there was an additional peak at
m/z = 1934.993. MS/MS measurements on this
ion (not shown) indicated that it also corresponded to residues
112-130, but with an Ala
Thr substitution at position 122 (corresponding to the single base change GCA to ACA). Thus the 30.009 Da mass difference observed in the 112-130 doublet (calculated
separation 30.011 Da) is accounted for, as well as the difference in
the overall masses between BMV-N and BMV-N*.
BMV-N appeared less responsive than BMV-P to further changes in
propagation host. After the initial set of changes induced by the use
of BW155 rather than Little Club wheat (Table VII), which generated
BMV-N*, no further changes were observed when the propagation host was
subsequently shifted from BW155 wheat to AC Assiniboia oat, then from
BW155 wheat to Seneca maize, and finally re-propagated in Seneca maize.
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DISCUSSION |
Mass Spectrometric Methods--
The measurements reported here
were carried out over a period of several years (15-20), during which
techniques and available instrumentation improved greatly. In
particular, the mass measurement accuracy for both proteolytically
generated fragments and daughter ions improved by about two orders of
magnitude from the original observations in 1996 to the recent
measurements with the QqTOF instrument. This led to a marked increase
in the level of confidence in the sequence assignments, notably in
distinguishing between Val deletion and Arg
Gly substitution in
BMV-W, as described above. In that case, the ability to obtain a mass
accuracy better than ~10 mDa for the daughter ions was clearly important.
In addition, it is useful to note that the peak that defined the
N-terminal acetylation for all the BMV samples was observed only
in the ESI spectrum, not in the MALDI spectrum, emphasizing the
advantage of having both modes of ionization available (42, 43).
Significance of Polymorphism in BMV Coat Protein (CP)
Sequences--
Coat protein molecular weights inferred from the
sequence analysis of cDNA clones, while accurate, do not show the
complete profile of the viral coat protein population actually present in infected plant tissue. Comparisons of the CP sequences of a set of
distinct BMV isolates not only show consistent discrete polymorphisms,
but also identify a specific host-attenuation effect on the profile of
the CP population of the BMV-N and BMV-P isolates. If CP sequences of
defined BMV isolates remained invariant, we would expect the type
isolate BMV-T (pv47) CP sequence to be identical with that reported in
the literature (27, 29). Instead, the BMV-T (pv47) sequence differs
from the reported one by a Trp
Arg substitution at position 23 and
a Val
Ile/Leu substitution at position 167, as indicated above. To
ensure that this was not because of a mix-up of the isolates, the virus
purifications and TOFMS analyses were repeated using double-blind
coding (BMV-1), and the same results were obtained. The CP sequence of
the BMV-V isolate (30), however, was identical with that previously
reported for BMV-T (pv47) (29). The fact that BMV-P and the Nebraska isolate reported by Wang et al. (39, 40) have identical CP sequences, emphasizes that similar mutations may occur independently at
different geographic sites.
Fig. 11 shows the modifications
observed in the isolates of Table I. These probably arise because
replication of viral RNA occurs at a high error rate (10
4
per cycle), much higher than DNA. There is thus a numerically large
pool of viruses with minor mutations produced with each round of viral
replication; minor mutations are those that do not affect critical
functions of viral replication, packaging, or transport. A particular
minor mutant may easily gain predominance after hundreds of
replication cycles, even though it is only favored slightly in a given
host.

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Fig. 11.
Measured amino acid sequences of the BMV
coat proteins. The top line shows the type amino acid
sequence deduced from a cDNA clone (24-26). The mutations observed
are shown; dashes indicate agreement with the type sequence.
N-terminal acetylation is indicated by the symbol @. I at
residue 167 in BMV-T indicates Ile or Leu, but most likely Ile
(see "Discussion"). The modifications induced by passaging through
various hosts are not shown but are described in the text.
|
|
The inability to distinguish whether Val is substituted by Ile or Leu
at residue 167 in BMV-T is a chronic difficulty in MS measurements with
low energy CID. Because Ile and Leu have the same mass, MS can only
distinguish between them by methods that are sensitive to the detailed
structure of the side chains, such as high energy CID. Nevertheless, in
this case we do have some additional information available, the initial
nucleic acid sequence (GUC). Substitution of Ile requires a codon
change from GUC to AUC (i.e. G
A, a purine
purine
transition), whereas substitution of Leu requires a change from GUC to
CUC (i.e. G
C, a purine
pyrimidine transversion). In
general, transitions are significantly more probable than transversions
(44), consistent with examinations of viral coat proteins; 20 of 28 nucleic acid substitutions that we have observed in these compounds
have been transitions. We conclude that the substituent at residue 167 in BMV-T is probably Ile.
Role of Mass Spectrometry in Plant Virus Research--
Analysis by
MALDI or ESI mass spectrometry of the viral coat protein is an approach
that can distinguish virus isolates or closely related virus strains
rapidly and definitively. Moreover, this method reveals directly any
post-translational modifications, which of course could not be deduced
from nucleic acid sequencing. Here, for example, the N-terminal residue
in all the BMV isolates studied was modified by acetylation, a common
phenomenon in ssRNA plant virus coat proteins, reported elsewhere for
members of the tobamo-, potex-, and poty-virus groups. Structural
information of this kind may contribute to understanding how the virus
functions, and what factors account for variations in virulence among
different isolates.
Moreover, reverse transcription of viral RNA and amplification of the
reverse-transcribed cDNA will only yield the protein that is coded
by the particular nucleic acid sequence selected. Mass spectrometry, by
contrast, gives a reasonably accurate picture of the distribution of
sequences in a mixed population of virus coat proteins, because the
relatively small changes in sequence observed here are unlikely to
produce much change in MS sensitivity. The data shown in Table VII and
in Figs. 10 and 11 show that it is now possible to measure by TOFMS the
change in profile of the population of coat proteins of a given virus
isolate when the propagation host is changed. The changes we observe in
the coat protein sequence provide one possible explanation for the
change in virulence produced by passaging through a different host.
 |
ACKNOWLEDGEMENTS |
We thank A. N. Krutchinsky, R. J. McNabb, V. Spicer, and M. Bromirski for skillful assistance in the TOF
laboratory, and J. Ackerman and B. Gillis for excellent work in the
propagation and purification of virus preparations.
 |
FOOTNOTES |
*
The work at the University of Manitoba was supported by
grants from the Natural Sciences and Engineering Research Council of
Canada and Grant GM59240 from the National Institutes of Health.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Physics & Astronomy, University of Manitoba, Winnipeg, MB R3T 2N2, Canada. Tel.: 204-474-9358; Fax: 204-474-7622; E-mail:
standin@cc.umanitoba.ca.
Published, JBC Papers in Press, March 26, 2001, DOI 10.1074/jbc.M100189200
 |
ABBREVIATIONS |
The abbreviations used are:
ESI, electrospray
ionization;
BMV, brome mosaic virus;
CID, collision-induced
dissociation;
CP, coat protein;
MALDI, matrix-assisted laser
desorption/ionization;
TOFMS, time-of-flight mass spectrometry.
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