The study of a non-covalent protein complex by means of electrospray
ionisation mass spectrometry.
Susan E. Slade, James H. Scrivens, Keith R. Jennings, Daniel Smith and Wendy A. Foxall.
Biological Mass Spectrometry and Proteomics Group, University of Warwick, Coventry, CV4 7AL, U.K
OVERVIEW
A study was undertaken to optimise the experimental conditions for the analysis of a biologicallyimportant non-covalent protein complex by means of nanoflow (NF) electrospray ionisation mass
spectrometry (ESI-MS) on a Q-Tof Ultima Global (Waters MS Technologies, Manchester, U.K.)
Instrument.
Methods
At higher pH, peaks were observed in the 2700 - 4500 m/z range due to the formation of multiply
charged species. The measured peak width at half height was >100 Da when the voltage on the first
RF lens was 45 V. The peak widths were reduced considerably by increasing the voltage to 200 V but
were still >10 Da at half height. The widths of the poorly resolved peaks were ascribed to the
presence of sodiated species. After a 25-fold reduction in the NaCl concentration, the peak widths
were reduced to < 2 Da. The reconstructed mass spectrum revealed three major peaks including the
monomer, the pentamer complex (38,446 Da) and a third species of molecular mass 12,814 Da,
which has yet to be identified (see Figure 4). It was not possible to increase the abundance of
pentamer complex further by adjusting the source temperature, pressure and RF voltage.
The molecular mass of the monomeric species was determined for the protein used in the study.
A number of experimental parameters were varied including:
Solution pH
Source pressure
Source temperature
Voltage applied to the 1st RF transport lens
to facilitate the detection of the intact oligomeric complex and increase the information content of the
spectra.
Results
By careful control of experimental conditions, protein-protein complexes may be observed under
favourable conditions in a commercial mass spectrometer.
INTRODUCTION
Electrospray ionisation mass spectrometry has enabled one to demonstrate the presence of a number
of weak protein interactions (see Loo for review). This study aims to investigate a biologicallyimportant non-covalent protein complex by means of ESI-MS and observe the effects of varying the
experimental conditions on the resulting spectra obtained. Previous observations suggest that it forms
a non-covalent multimeric protein complex in solution.
Shiga-like toxins (SLTs) are produced by enterohemorrhagic strains of Escherichia coli and are
associated with disease in both humans and animals. SLT-1 is an AB toxin consisting of a 32 kDa
enzymatic A subunit that inhibits protein synthesis and a doughnut shaped B-component that appears
to be composed of five identical 7.7 kDa subunits (see Figure 1) based on crystallographic evidence
(Ling et al.).
One face of the pentameric B-component is involved in toxin attachment to the host cell while the Asubunit is situated on the opposite face with its hydrophobic C-terminus anchored in the central pore.
The B-component was chosen for study, as it is non-toxic and can be purified in the absence of the Acomponent by means of affinity chromatography. Elution from the column with a denaturant followed
by dialysis results in the formation of the complex. A Western blot of the refolded B-component
analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), probed with
anti-SLT-1 antibody, clearly shows that even under denaturing conditions, the pentamer is still present
(see Figure 2).
Figure 4. Reconstructed spectrum of the SLT-1 B component in 10 mM ammonium acetate identifying
the monomer and pentamer species.
The inset shows the ESI spectrum for the 850-4000 m/z range with the multiply charged species of the
pentamer identified.
Further preparations of protein were purified and extensively dialysed against ammonium acetate
buffer prior to analysis. A considerable improvement in the quality of the raw data was evident in these
samples with the measured peak width at half height of m/z 3200 reduced to 1.15 Da when the voltage
on the first RF lens was optimised at 150 V. Variation of the voltage on the first RF lens resulted in an
increase in the monomeric species observed and concomitant decrease in the pentamer complex
(data not shown).
Under experimental conditions of elevated pressure in the source region, the ESI spectrum obtained
indicates a reduction in the abundance of the protein monomer charge state envelope (see Figure 5).
Differential scanning calorimetry and circular dichroism (CD) studies by Pina et al. on the formation of
the protein complex suggest that the protein exists in solution predominantly in two forms, the
unfolded monomer and the pentamer complex. It was shown that the folding/unfolding process is a
reversible highly cooperative transition between unfolded monomers and folded pentamer complex.
Under all the conditions studied, the concentrations of intermediates could not be detected by
calorimetry and CD data were consistent with the presence of only two populated states.
Evidence for the formation of the pentamer complex by the use of mass spectrometry would enable an
investigation of the intact AB toxin, also an example of a non-covalent protein-protein interaction.
Figure 5. ESI spectrum of the SLT-1 B component in 10 mM ammonium acetate for the 850-4000 m/z
range obtained under elevated source pressure conditions.
This observation is reflected in the reconstructed spectrum showing the presence of only the pentamer
complex in the absence of monomer and other oligomeric species (see Figure 6).
Figure 1. Crystal structure of the pentameric
B-component of Shiga-like toxin-1 from E. Coli.
Figure 2. Western blot of a SDS-PAGE analysis
of denatured SLT-1 B-component showing both
the monomer and pentamer complex.
MATERIALS AND METHODS
The protein samples were diluted to a final concentration within the range 0.3-10 µ
M in a volatile aqueous buffer (10 mM ammonium acetate) and analysed by the use of ESI mass
spectrometry on a Q-Tof Ultima Global instrument (Waters MS Technologies, Manchester, U.K.). The
pH of each of the sample solution was adjusted by the addition of 0.1% or 1% formic acid as required.
Samples were infused using NF capillaries (Waters MS Technologies, Manchester, U.K.) maintained at
1.5 kV. In order to obtain a stable flow of sample from the capillary, a low backpressure of nitrogen gas
was applied as necessary.
The molecular mass of the monomeric species was determined with the first RF lens maintained at
45 V. During the optimisation experiments the RF lens voltage, source temperature and pressure were
varied.
Data were obtained over the mass/charge (m/z) range 500-8000 using a 2.4-second scan. Data
collected over a 20 minute period were combined, smoothed and the background subtracted prior to
processing by the deconvolution algorithm MaxEnt.
Figure 6. Reconstructed spectrum of the SLT-1 B component in 10 mM ammonium acetate identifying
the pentamer species obtained under elevated source pressure conditions.
It was not possible to increase the abundance of pentamer complex further by decreasing the source
temperature.
The SLT-1 B-component was desalted by a 25-fold dilution into 10 mM ammonium acetate and
concentrated using a Micron centrifugal filter device (Millipore, Billerica, U.S.A.) with a 3 kDa molecular
weight exclusion limit or by multiple dialysis steps against 10 mM ammonium acetate.
CONCLUSIONS
RESULTS
Molecular mass measurements were performed for the SLT-1 B-component dissolved in ammonium
acetate buffer containing 1% formic acid. Two major peaks were observed in the reconstructed mass
spectrum (see Figure 3), the monomer (7,688 Da) and the less abundant dimer (15,374 Da). It was
apparent from the data that the B component was multiply sodiated resulting from insufficient
desalting during the purification step.
The above results demonstrate that by careful control of experimental conditions, protein-protein
complexes may be observed under favourable conditions in a commercial mass spectrometer.
Careful desalting of samples is essential in working with samples from biological systems in order to
minimize peak broadening of high mass complexes.
The observation of the pentamer in the absence of the dimer, trimer and tetramer is taken as evidence
of complex formation as opposed to simple aggregation. These observations are in agreement with
previous studies that demonstrated the presence of only two species in solution, the pentamer
complex and unfolded protein monomer.
REFERENCES
Ling, H., Boodhoo. A., Hazes, B., Cummings, M. D., Armstrong, G. D., Brunton, J. L. and Read, R.J.
Structure of the Shiga-like toxin I B-pentamer complexed with an analogue of its receptor Gb3.
Biochemistry 1998 (37) 1777-1788.
Loo, J. A. Studying non-covalent protein complexes by electrospray ionisation mass spectrometry.
Mass
Spec. Reviews 1997 (16) 1-23.
Figure 3. Reconstructed mass spectrum of the SLT-1 B-component in 10 mM ammonium acetate
with 1% formic acid identifying the monomer and dimer species.
The inset shows the ESI spectrum for the 850-4000 m/z range with the multiply charged species of
the monomer identified
Pina, D. G., Gomez, J., Villar, E., Johannes, L. and Shnyrov, V. L. Thermodynamic analysis of the
structural stability of the Shiga toxin B-subunit. Biochemistry 2003 (42) 9498-9506.