Mass Spectrometry as a means to identify structural proteins in a newly characterised virus.
Susan E. Slade; Martha R. J. Clokie; Nicholas H. Mann; Keith R. Jennings; James H. Scrivens
Overview
Biological Mass Spectrometry and Proteomics, Department of Biological Sciences, University of Warwick, Coventry, CV4 7AL, U.K.
Purpose
To use mass spectrometric analysis to investigate cyanobacteria - cyanophage
interactions in greater detail including:
•identification of the genes encoding the proteins involved in host
recognition.
•Identification of locations within the genome encoding other viral structural
proteins.
Introduction
What are bacteriophage?
Bacteriophage are viruses that only infect bacteria.
Methods
They are one of the main factors influencing its population dynamics.
Large quantities of cyanophage were grown and the structural proteins purified
using polyacrylamide gel electrophoresis.
A number of phages infecting marine Synechococcus have been isolated and
characterized (Waterbury and Valois, 1993; Suttle and Chan 1993; Wilson et al.
1993).
Enzymatic digestion of a 68kDa protein band was performed and the tryptic
peptides extracted.
A tryptic mass fingerprint and amino acid sequence tags were obtained by
electrospray ionisation mass spectrometry and collision induced decomposition
experiments.
They are composed of:
protein head - containing genome
body sheaths
tail fibres - important in host recognition (see Figure 3).
Figure 5. A phage particle attached to a bacterium
Why are we particularly interested in S-PM2?
S-PM2 is a double stranded DNA myovirus (see Figure 6).
It was isolated from Plymouth Sound (UK) from Synechococcus.
It is the first virus of its type to be sequenced in March 2002.
Results
Seven tryptic peptides were observed and three amino acid sequence tags
obtained for comparison against the genome sequence.
capsid
When grown on Synechococcus (WH7803) it forms clear plaques (see
Figure 7).
The gene encoding this 68kDa protein has been unequivocally identified within
the genome.
Similarity searches have tentatively identified the protein as being involved in
the organisation of tail fibres.
body sheath
Introduction
tail fibre
What are Synechococcus?
Discovered in 1979, they are Gram negative bacteria that are extremely
important in terms of carbon fixation in the environment (Paerl,2000).
The cells are predominantly coccoid in shape and measure 0.6 - 0.8 m in
diameter (see Figure 1).
They are abundant in the tropical, sub-tropical and temperate regions
of the oceans (see Figure 2).
Figure 3. Diagrammatic representation of a phage particle.
Host - virus interactions
Figure 6. Electron micrograph of cyanophage S-PM2.
High concentrations of bacteria and virus particles can be isolated from
marine waters (see Figure 4) (Bergh et al., 1989; Proctor and Fuhrman,
1990; Suttle et al., 1990).
Figure 1. Light microscope image of Synechococcus
(By courtesy of John Waterbury).
Figure 7. Cyanophage S-PM2 grown on a lawn of Synechococcus
(red). Clear areas (plaques) show host cell lysis following infection.
Figure 4. Sea water sample stained with cyber green showing
bacterial cells surrounded by smaller phage particles.
0.01
1
2
0.05
0.1
0.3
0.5
3 5
10
30
50
Chlorophyll a [mg/m3]
The infection cycle
Traditional techniques for gene identification involve comparing the
DNA sequence of S-PM2 with highly characterised similar viruses
and locating regions of homology.
1. The phage recognises a suitable host (see Figure 5) through interactions
between the virus tail fibres and a suitable receptor on the host cell surface.
Genome sequences are available for over 100 phages (typically
infecting Escherichia coli or other bacteria of medical importance).
2. The phage injects its genome into the host.
S-PM2 shows no homology to any previously sequenced phage
(see Figure 8).
3. The phage uses the cell’s machinery to replicate.
Figure 2. Distribution of chlorophyll a in the oceans, Synechococcus
are dominant in the dark blue areas.
Why mass spectrometry?
4. Either the host cell is lysed or the phage inserts its genome into the host’s.
Analysis using mass spectrometry will provide amino acid sequence
tags which can be used to probe the genome to identify the genes
of interest.
K
T4 24.3 24 segD 23 22 21 68 67 20 19 18 17 16 15 14 13 wac 12 11
46 47 55 uvsW uvsY 3 23 22 21 20 19 18 17 16 15 14 13
wedge
21 kb
Analysis by mass spectrometry (continued)
W
V
Y
G
S
A
G
S
A
A
V
I
A
T
A
yMax
1552.84
100
Suitable peptides for collision induced decomposition (CID) experiments were
isolated using the first quadrupole and allowed into the collision cell.
Instrumental conditions for CID analysis:
•Mass range 50-2400 m/z
•Argon collision gas
•Collision energy 18-45eV (sample dependent)
776.42
1549.72
%
775.44
72 kb
Product ion spectra were processed using the MaxEnt3 deconvolution algorithm
prior to de novo sequencing to derive an amino acid sequence tag.
Figure 8. Comparison of S-PM2 and E. coli T4 genetic organisation in the
head and tail assembly region.
Methods
The DNA genome sequence was translated into potential amino acid sequences
in all six reading frames. The sequence tags produced by CID experiments
were compared against the hypothetical phage protein complement to identify
the gene encoding each sample protein.
867.45
954.49
y8
y9
371.10
333.19
y2
244.14
147.11 b3 291.04
y1
445.13 527.32
b6
598.38
b7
739.39 777.26
y6
1554.17
1096.57
y11
944.49 1025.53;y10
900.49
b11
959.51
1195.62
1549.21
y12
1219.67 1308.71
b14
y13 1440.73
1554.55
1561.23
0
M/z
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
Figure 11. MaxEnt3 processed product ion spectrum of [M+2H] 2+
precursor ion 776.36 including amino acid assignments.
Results
Cyanophage growth, purification and protein separation
RQN
Cultures containing 1L of Synechococcus (7803) were grown under ambient
conditions using artificial sea water.
During the exponential growth phase, S-PM2 was added in a ratio of 1 phage
particle : 1 bacterial cell.
DLP
VP
P
I
D
E
T
AF
100
Phage protein isolation and purification
yMax
1711.84(M+H) +
1035.56
y9
The cyanophage structural proteins were successfully resolved using SDSPAGE analysis (see Figure 9).
%
Following bacterial cell lysis, sodium chloride was added to precipitate cell
debris, which was then removed during centrifugation.
The phage particles were precipitated using cold acetone at
-20oC
677.31
b6
68 kDa
overnight.
659.30
291.04
122.08
144.06
68kDa
Following centrifugation to harvest the phage particles were resuspended in
protein extraction buffer.
172.88
417.19
y3
200
400
518.26 546.22
1709.89
742.38
y6
856.65
839.41 861.42
970.49
b9
1713.47
1036.53 1148.63
y10 1317.56
1392.70
y12 1478.76 1566.74
1693.84
0
1715.07
M/z
600
800
1000
1200
1400
1600
Figure 12. MaxEnt3 processed product ion spectrum of [M+2H] 2+
precursor ion 856.40 including amino acid assignments.
Samples were boiled in sodium dodecyl sulphate (SDS) loading buffer for 10
minutes and loaded onto a 10% polyacrylamide gel.
Conclusions
The gel was electrophoresed at 150V for 2 hours.
The gel was visualised using either Coomassie Brilliant Blue or silver stain
dependent on sample loading.
Figure 9. Silver and Coomassie blue stained polyacrylamide gels showing
purified cyanophage structural proteins
It is extremely difficult to produce sufficient quantities of purified cyanophage
proteins to perform CID experiments (1011 phage per sample).
Seven tryptic peptide masses were obtained and three amino acid sequence tags.
Analysis of tryptic peptides
Sample preparation for analysis by mass spectrometry
Coomassie stained protein bands were excised from the gel and cut into
small pieces (~2mm3).
Seven tryptic peptides were detected following digestion of the 68kDa protein
band. Three were selected for CID experiments, yielding the amino acid sequence
tags TTGASA(I/L), VAASGAS and (I/L)P(PV or VP), see Table 1 and Figures 1012.
Gel pieces were washed sequentially with 50mM ammonium bicarbonate pH
8.0 and acetonitrile at least three times.
A comparison of the expressed sequence tags with the genome identified the
region encoding this protein as ORF 27.
Gel pieces were dried at ambient temperature in a vacuum centrifuge.
Tryptic peptide
theoretical Mr
Mr detected
Amino Acid
Sequence Tag
Amino acid
residue #
Amino acid sequence of ORF 27
The protein was reduced with 10mM dithiothreitol and alkylated with 100mM
iodoacetamide.
1164.56
1550.81
1164.54
1550.72
TTGASA(I/L)
VAASGAS
210-222
275-290
SSTTGASALGDAK
ATAIVAASGASGYVWK
1602.85
1602.76
-
621-634
AADQIEDIKLVIEF
1686.88
1686.84
-
368-383
IVVSGGQIESTEVVDR
1710.86
1710.80
(I/L)P(PV or VP)
140-154
FATEDIPPVPLDNQR
2083.94
2083.90
-
255-274
TNPSGGEGTYNPSTGIFTER
2473.24
2473.20
-
502-526
IQNATAGFVADEEITQNLAGGGVAK
Gel pieces were sequentially washed with acetonitrile and 50mM ammonium
bicarbonate pH 8.0 at least twice.
Gel pieces were dried at ambient temperature in a vacuum centrifuge and
rehydrated in a solution containing 10ngml-1 trypsin in 50mM ammonium
bicarbonate pH 8.0 on ice for 40 minutes.
The identification has enabled more specific searches to be carried out with
known phage genomes. Thus, due to similarity at the C terminal end with the
protein from the T4 E. coli phage, we have tentatively identified our protein as
being involved in the organisation of tail fibres.
Most importantly we hope to identify the tail fibre proteins involved in host
specificity. We can then carry out a range of genetic and molecular biological
experiments to determine the exact nature of this phage – cyanobacteria
interaction.
Mass spectrometry has given us the starting point from which to try to understand
this fascinating problem.
Table 1. Comparison of theoretical and detected tryptic masses from 68kDa
protein. Sequence tags with corresponding genome sequence from ORF 27 are
also shown.
The peptides were freeze-dried and resuspended in 2% formic acid.
K
A
D
G
L
A
100
Each sample was desalted prior to analysis using a C18 ZipTip (Millipore
Corporation, Bedford, U.S.A.) according to the manufacturer’s instructions
except the peptides were eluted with 60% acetonitrile containing 1% formic
acid.
583.29
S
A
G
T
T
SS
References:
Bergh, O., Borsheim K.Y., Bratbak, G. et al (1989). High abundance of viruses found in aquatic
environments. Nature 340 (6233): 467-468
yMax
Paerl, H. W. (2000). Chapter 5 Marine Plankton. In The ecology of cyanobacteria: their diversity
in Time and Space. Ed. Whitton, B. A., and Potts, M. Kluwer Academic Publishers, Amsterdam.
Proctor, I. M. and Fuhrman, J. A. (1990). Viral mortality of marine-bacteria and cyanobacteria.
Nature 343 (6253): 60-62
582.34
Suttle, C. A., Chan, A.M. and Cottrell, M. T. (1990). Infection of phytoplankton by viruses and
reduction of primary productivity. Nature 347 (6292): 467-469
%
Analysis by mass spectrometry
789.42
y9
133.08
All samples were analysed using a Q-Tof mass spectrometer (Micromass,
Manchester,U.K.) fitted with a nanoflow (NF) capillary infusion inlet.
Calibration was achieved using a solution of sodium iodide.
89.06
218.15
y2
175.11;b2
258.12
661.35
y7
359.15
390.20
y4
503.28 565.26
y5
425.18
584.43
613.05
890.47
y10
732.40
y8
973.49
1073.54
1055.54
801.89
100
200
300
400
500
600
700
800
900
Suttle, C. A. and Chan, A. M. (1993). Marine cyanophages infecting oceanic and coastal strains of
Synechococcus - abundance, morphology, cross-infectivity and growth-characteristics. Mar. EcolProg. Ser. 92 (1-2): 99-109
1165.16
955.48
680.34
1167.59
1111.53
0
Instrumental conditions for MS analysis:
NF capillary (Protana Engineering, Sweden) voltage 1000V
•Cone voltage 40V
•Scan time 2.4sec
•Mass range 400-2400 m/z
From the data obtained, we have unequivocally identified the gene in the S-PM2
genome (ORF 27), which codes for the 68 kDa protein.
The procedure will be repeated on the remaining viral structural proteins and we
hope to determine where proteins involved in the capsid and tail fibres are coded
for in the genome of S-PM2.
Each tryptic digestion was incubated at 37oC for 20 hours.
Peptides were extracted three times with a solution containing
H2O/acetonitrile/formic acid (50/50/5 v/v/v).
The sequence tags obtained searched against the genome and the matching
sequences identified .
1000
1100
1186.60
1195.61
M/z
1200
Figure 10. MaxEnt3 processed product ion spectrum of [M+2H] 2+ precursor
ion 583.27 including amino acid assignments.
Waterbry, J. B. and Valois F. W. (1993). Resistance to co-occurring phages enables marine
Synechococcus communities to coexist with cyanophages abundant in seawater. Appl. Environ.
Micro. 59 (10): 3393-3399
Wilson, W. H., Joint I. R. Carr, N. G. et al (1993). Isolation and molecular characterization of five
marine cyanophages propagated on Synechococcus sp. strain WH7803. Appl. Environ. Micro. 59
(11): 3736-3743