Characterisation of Noncovalent AB5 toxin assemblies by means of Mass spectrometry and Tandem Mass Spectrometry
ASMS
Texas
2005
Susan E. Slade*1, Jonathan P. Williams1, Brian N. Green2, Daniel C. Smith1, James H. Scrivens1
1. Biological Mass Spectrometry and Proteomics Group, Department of Biological Sciences, University of Warwick, Coventry, UK, CV4 7AL.
2. Waters MS Technologies Centre, Micromass UK Ltd, Floats Rd, Wythenshawe, Manchester M23 9LZ, UK.
Experimental
The TOF instrument is not capable of tandem mass spectrometry experiments so further examination was
performed solely on the B chain pentamer.
The recombinant SLT AB5 holotoxin noncovalent complex and the noncovalent B5 homopentameric binding
subunit were expressed and purified according to the detailed procedure described by Williams et al. [1].
The CT AB5 holotoxin noncovalent complex and the noncovalent B5 homopentameric binding subunit were
purchased from Sigma.
(ii) Triple-Quadrupole MS for the B5 SLT homopentamer. The SLTx pentamer B5 consists of five identical
subunits that arrange into a pentameric ring structure (Figure 1A). Each subunit has a molecular mass of 7.7
kDa and is composed of 69 amino acids that form two three-stranded antiparallel β sheets and an α helix .
Analysis of the SLTx B-chain preparation (2 pmol/µL) in acetonitrile/H2O + 0.2 % formic acid confirmed the
homogeneity of the protein (Figure 3, inset). The Maximum Entropy program suggested the molecular weight
of 7688.5 Da for the B subunit which is in good agreement with the predicted mass. The mass spectrum
obtained for the noncovalent complex of SLTx-B5 buffered with ammonium acetate at pH~6.6 clearly shows
that the isolated B-pentamer remains intact during the ESI process (Figure 3 ).
Overview
Aim
To observe the Shiga-like (SLTx) and Cholera (CTx) AB5 holotoxin noncovalent complexes at a range of pH
and probe the gas phase dissociation pathways of the noncovalent B5 homopentameric binding subunit of
the toxins.
Method
A single ToF instrument provided the extended mass range capable for the observation of noncovalent
complexes in the gas phase. A triple quadrupole instrument facilitated tandem mass spectrometry (MS/MS)
experiments for the investigation of dissociation pathways.
Results
The intact SLTx and CTx AB5 holotoxin complexes were observed in the gas-phase of a time-of-flight mass
spectrometer at a range of pH, including one representing physiological conditions. Furthermore, we report
the dissociation of the noncovalent homopentameric binding subunit of the toxins by means of in-source
collision induced dissociation (CID) and CID in a collision cell of a triple quadrupole mass spectrometer
allowing us to probe the gas phase dissociation pathways.
Introduction
(SLTx) produced by enterohemorrhagic strains of Escherichia coli (EHEC) belongs to a family of structurally
and functionally related AB5 protein toxins that are associated with human disease. EHEC infection often
gives rise to hemorrhagic colitis, whilst toxin-induced kidney damage is one of the major causes of hemolytic
uremic syndrome (HUS) and acute renal failure in children. The A-subunit (~32 kDa) is positioned on one
face of the non-covalently-associated B-chain homopentamer (~7.8 kDa/subunit, Figure 1A), with the A-chain
C-terminus anchored non-covalently in its central pore (Figure 1B).
Mass Spectrometry.
To investigate the noncovalent AB5 and B5 toxin assemblies, a triple quadrupole mass spectrometer and a
single Time-of-Flight mass spectrometer (TOFMS) were utilised. Data acquisition and processing was
carried out using MassLynxTM (V3.5). Instrumental parameters used in the study were:
• Triple quadrupole MS – Quattro Ultima (Waters MS Technologies, Manchester, UK)
• ToFMS – LCT (Waters MS Technologies, Manchester, UK)
• Ion mode: ESI positive
• Capillary: 3.0kV
• Cone: 20-75V
• Source Temp: 110oc
• Desolvation Temp: 110oC
• Pressure: Optimised for observation of noncovalent complexes
• Collision gas: Argon (2.5 x 10-3 mbar) Quattro Ultima
• Concentration: 5-10µM in 10mM ammonium acetate
• Direct infusion: 4µL/min Harvard Apparatus (South Natick, MA, USA) Model 22 Syringe Pump
7600
7800
8000
1500
%
B4+
B6+
AB518+
2500
AB519+
AB517+
B5+
B6+
1
0
1
0
0
(B) CE= 25eV 5+
B
7+ B6+
%
B4+
B6+
0
0
0
1
%
0
0
1
0
AB518+
0
0
1
%
0
1000
pH~3.0
B4+
B6+
1500
2000
AB518+
2500
3000
3500
4000
4500
Figure 2: ESI-TOF mass spectra for the AB5 SLT holotoxin at various pH
5000
m /z
5
2
B5+
0
3000
B7+
%
B515+
m /z
3 5 00
B6+
B8+
1 0 0
0
0
14+
B3+ *B5
0
2
5
0
B38+
0
3
0
0
0
3
5
0
2
0
0
2
5
0
0
m
*B513+ B3
B513+ B37+
3
0
0
0
B49+
3
5
0
B37+
B37+ + (B27+)
B38+ + (B26+)
B513+
2 0 0 0
2 5 0 0
B2+
B25+ *B37+
3 0 0 0
3 5 0 0
m /z
B2+ + B25+
B3+ + B24+
Figure 5: ESI-Triple quadrupole pseudo-MS/MS/MS mass spectra and fragmentation pathways of the SLTB5 noncovalent complex. The precursor ion selected for MS/MS is marked with an asterix
Our data obtained by pseudo-MS/MS/MS experiments on the B49+ and B37+ ions show that these ions do
undergo further dissociation forming product ions which suggest the loss of a further B subunit as shown in
Figure 5. This sequential loss of individual subunits from the pentamer, tetramer and trimer ions indicate that
the noncovalent interactions maintaining the quaternary structure of the B-pentamer are extremely strong.
%
m
100
/ z
%
100
%
B7+
*B517+
CE= 15eV
B3+ + (B414+)
B4+ + (B413+)
B5+ + B412+
B6+ + (B411+)
B7+ + (B410+)
B8+ + (B49+)
%
B7+
B6+
B5+
B4+
B8+
B5+
B7+
B8+
%
0
1000
B518+
B8+
B6+
B517+B
B6+
AB519+
B5+
B518+
16+
5
B515+AB
%
20+AB 19+
5
5
1500
B6+
2000
B5+
2500
B517+ 16+ AB 20+
B5 15+ 5 AB 19+ pH~4.0
B518+
B5
5
3000
3500
4000
4500
5000
m/z
5500
B5+
B8+
0
1000
1500
2000
2500
B4+ B 7+
2
3000
3+
12+
*B517+ B /B4
3500
Figure 6: ESI-TOF mass spectra for the AB5 CT holotoxin at various pH
B27+ + (B310+)
m /z
Figure 8: ESI-Triple quadrupole MS/MS mass spectra and fragmentation pathway of the CT- B5 noncovalent
complex. The precursor ion selected for MS/MS is marked with an asterix
pH~5.0
B7+
B8+
B517+
B7+
B6+
pH~7.0
B517+
B516+ AB 20+
pH~6.0
5
B518+
B515+
AB519+
B7+
CE= 45eV
CE=45eV
16+
5
B515+ AB520+
Conclusions
It has been demonstrated that the SLT and CT AB5 holotoxins remain intact during the ESI process and can
be observed in the mass spectrum over a range of pH including including one representing physiological
conditions. As far as we know this is the first time the cholera holotoxin has been observed intact in the gas
by mass spectrometry. The increased sensitivity afforded by ToF mass analysers for observing noncovalent
assemblies has been shown. Pseudo-MS/MS/MS experiments for the SLT B5 homopentamer have shown
that the tetramer and trimer ions formed from the loss of a single B unit undergo further dissociation forming
product ions which suggest the loss of a further B subunit.
References
Figure 4: ESI-Triple quadrupole MS/MS mass spectra and fragmentation pathways of the SLT- B5
noncovalent complex. The precursor ion selected for MS/MS is marked with an asterix
m /z
3500
CE=15eV
100
B517+B
B6+
0
100
B37+ + (B26+)
B38+ + (B25+)
3000
0
B48+
B3+ + (B410+)
B4+ + B49+
B5+ + B48+
B6+ + (B47+)
2500
100
/ z
B48+
0
B5+
2000
(iii) Triple quadrupole MS/MS of the CT homopentamer B5. Tandem mass spectrometry (MS/MS) was
used to investigate the dissociation pathway of the homopentamer B517+ ion. The two dominant ions in the
mass spectrum of the homopentamer correspond to B516+ and B517+. The latter ion was selected as a
precursor ion to undergo CID in the collision cell of the instrument, the collision energy being varied to obtain
intense fragment ions. The multiply-protonated pentameric species B517+ selected for CID dissociated
almost entirely via the loss of one subunit to form monomer, dimer, trimer and tetramer ions but no peaks
which could be unequivocally assigned to trimer ions were observed, as shown in Figure 8. These ions
could still be formed, however as the ions B33+, B36+ and B39+, cannot be distinguished from the ions B+, B2+
and B3+, B22+, B24+ and B26+ and B44+, B48+ and B412+ respectively, to which some of the peaks have been
assigned. However, not all of the ions that are predicted to form were detected. This was either due to their
being present in very low abundance or due to the m/z ratio of the ion being beyond the upper m/z range of
the instrument, 4000, as discussed above. The multiply-protonated pentamer species B517+ selected for CID
dissociates almost entirely via the loss of one subunit as shown to form primarily abundant monomer ions. A
very low abundant dimer B27+ ion is also observed.
B8+
0
B3+ + (B411+)
B4+ + B410+
B5+ + B49+
B6+ + B48+
B7+ + (B47+)
1 5 0 0
1500
B518+
Figure 7: ESI-Triple quadrupole mass spectrum for the B5 cholera holotoxin
B48+
0
9+
7+ B4
B3+
0
B3+
%
0
B4+
B3+
0
1 0 0 0
100
B3+
*B37+
B2+
CE= 10eV
B. (i) TOFMS for the AB5 CT holotoxin. The individual A and B subunits but not the intact holotoxin have
been observed by ESI previously [4]. Here, as shown in Figure 6, we demonstrate that the intact AB5 cholera
holotoxin remains intact during the ESI process over a range of pH.
B48+
B49+
B410+ B37+
B5+
0
B36+
2500
B3+ + B36+
CE= 25eV
(E)
B514+
B5+
100
0
2000
1 0 0
B49+
B3+
B4+
B4+
(D) CE= 18eV
B6+
0
pH~3.5
B4+
B6+
*B49+
B3+
0
m/z
0
1
0
0
B6+
0
B5+
5
(C) CE= 15eV
0
pH~7.0
pH~6.6
AB518+
1500
%
3500
*B514+
B4+
B
0
%
B5+
100
3000
(A) CE= 15eV
%
0
B3+
0
1000
(B)
B512+
(iii) Triple quadrupole MS/MS of the SLT homopentamer B5. Tandem mass spectrometry (MS/MS) was
used to investigate the dissociation pathways of the SLTx B513+, B514+, B49+ and B37+ ions respectively. The
multiply-protonated pentamer species B514+ and B513+ selected for CID dissociate almost entirely via the loss
of one subunit (Figure 4) to form monomer, trimer and tetramer ions. However, not all of the ions that are
predicted to form were detected. This was either due to them being present in very low abundance or due to
the m/z ratio of the ion being beyond the upper m/z range of the instrument, 4000.
0
B4+
mass
The mass spectrum demonstrates that the homopentamer B5, the dominant form of the B subunit protein in
solution, remains intact during the ESI process. Mass spectra obtained previously, and during this
investigation for the noncovalent complex for the homopentamer B5 demonstrate that under the conditions
specified, the pentamer is stable and can be transferred from the liquid phase to the gas phase intact.
1
B5+
8200
2000
0
100
In this study ESI-MS and ESI-MS/MS has been used to examine the noncovalent interactions that exist
between the subunits of the complete SLTx and cholera holotoxins or the pentameric B-subunit. The ability to
rapidly identify and characterise such interactions will be very important for future research into this class of
pathogenic protein.
7400
B516+
B36+
B4+ + (B35+)
7200
Figure 3: ESI-Triple quadrupole mass spectrum for the B5 SLT holotoxin and inset showing the maximum
Entropy deconvoluted spectrum of the monomer subunit
%
Electrospray ionisation (ESI) is a powerful technique that aids the study of biological noncovalent assemblies.
ESI permits the assemblies to be transferred from the liquid phase to the gas phase intact allowing detection
by mass spectrometry (MS). In order to study noncovalent assemblies intact by means of ESI-MS, the
internal energy of the ions formed by the electrospray process must be kept to a minimum to avoid
dissociation. Collisional stabilisation is accomplished by increasing the backing pressure between the source
and ion guide interface region of the mass spectrometer
0
1000
B515+
Figure 1(B): Structure for theAB5 SLT holotoxin
100
CE= 15eV
%
B49+
0
1000
B513+
B514+
B515+
B4+
%
10 0
7688.50
7688.50
0
7000
%
CTx is also an AB5 protein toxin produced by the gram-negative bacillus Vibrio cholerae. The disease
resulting from infection and production of this toxin causes watery diarrhoea, and remains a major public
health problem in Africa, Asia and Latin America. Indeed there are over 200 000–500 000 reported new
cases of cholera each year, although the actual number of cases is likely to be much greater. Without
medical treatment, mortality associated with cholera infection is 20–50%. Furthermore, CTx has been
characterised as a potential ‘biowarfare’ agent by the US and UK defence agencies, and has been reported
to be of medical relevance as a novel immune adjuvant. The A-subunit (~27 kDa) is positioned on one face of
the non-covalently-associated B-chain homopentamer (~11.6 kDa/subunit), with the A-chain C-terminus
anchored non-covalently in its central pore, similar to the SLT holotoxin as shown above . CTx binds to, and
enters intestinal cells through interaction of the B subunit pentamer with the GM1 ganglioside receptor. The
interaction of the B subunit with its receptor is also thought to mediated the immunomodulatory functions of
the toxin. Within the cell cytosol the A subunit causes constitutive activation of adenylyl cyclase by activation
of the stimulatory G protein Gsα, resulting in elevated levels of intracellular cAMP. Elevation of cAMP
produces active secretion of salt and fluid through activation of Cl− channels in the apical plasma membrane
of intoxicated cells.
CE= 5eV
%
0
B517+
100
0
%
Results and Discussion
(ii) Triple-Quadrupole MS for the B5 CT homopentamer. The pentamer B5 consists of five identical
subunits that arrange into a pentameric ring structure. Each subunit has a molecular mass of 11.6 kDa and
is composed of 103 amino acids. The mass spectrum obtained for the noncovalent complex of B5 buffered
with ammonium acetate at pH~5.0 is shown in Figure 7. The mass spectrum obtained clearly shows that the
isolated B-pentamer remains intact during the ESI process.
%
10 0
B513+
B514+
100
A. (i) TOFMS for the AB5 SLT holotoxin. Previously, nanoelectrospray was performed on a 10 pmol/µL
aqueous solution of the holotoxin and at pH 3.5 AB5 ions were observed [2], however it was reported that at
neutral pH only a weak ion signal corresponding to the polyprotonated B5 pentamer was observed. It was
assumed that the AB5 ions were being generated by the nanoelectrospray process at neutral pH but at
mass-to-charge (m/z) ratios that exceeded the m/z 5000 capability of the FTICR mass spectrometer. In
order to test this assumption, a study of the toxin was carried out in the time-of-flight instrument at different
pH values (Figure 2). Our data confirms that of a previous investigation showing that the AB5 holotoxin
remains intact during the ESI process. However, in contrast to the results of the previous study, the mass
spectra obtained for the holotoxin shows a strong signal for both the B5 pentamer and the AB5 holotoxin at
pH~7. Our data clearly shows that within the pH range 3 – 7, the same three protonated holotoxin AB5 ions
are formed and no holotoxin ions were observed beyond m/z 5000. As far as we know this is the first time
the holotoxin has been observed intact at physiological pH.
*B49+
CE= 5eV (no gas)
10 0
(A)
0
100
1
Figure 1(A): Structure for the B5 SLT homopentamer
(iii) Triple quadrupole pseudo-MS/MS/MS of the SLT homopentamer B5. Previous results obtained via
BIRD in an FTICR mass spectrometer concluded that the B4 ions were resistant to further dissociation over
the temperature range studied [3]. The B49+ and the B37+ ions, formed from in-source CID, were therefore
subjected to CID in the collision cell in order to investigate their fragmentation under these conditions.
1. JP Williams et al. (2005) Biochemistry (in press).
2. EN Kitova, PI Kitov, DR Bundle, JS Klassen, (2001) Glycobiology, 11, 605-611
3. N Felitsyn, EN Kitova, JS Klassen, (2001) Anal.Chem., 73, 4647-4661.
4. BLM van Baar, AG Hulst, ERJ Wils, (1999) Toxicon 37, 85-108.