Investigating the effect of source temperature on protein structure

With native-like MS experiments careful optimisation of the instrument parameters is important for retention of protein structure. The intent of this study was to revisit data previously published by Scarff et al (Scarff, Patel et al. 2009) in light of recent improvements in to native MS experiments. Previous publications on native haemoglobin used elevated source temperatures which could lead to partial denaturation. The effect of temperature of the source on the structure of HbA and HbS were investigated using a combination of native MS and IMS-MS. The results are shown in Figure 4-5.

100 Native MS

Figure 4-5 – Native MS spectra of HbA illustrating the effect of varying source temperature on the MS spectrum. Increasing the source temperature appears to increase the relative intensity of the monomer and dimer compared to the tetramer, suggesting dissociation of the tetramer oligomers Figure 4-5 shows an increased peak width at half maximum (PWHM) for all samples compared with spectra shown in Figure 4-3. This peak width increase arises as a result of increased pressures throughout the system induced by operating with IMS separation on and a source pressure of 6.8 mBar (consistent with Scarff et al (Scarff, Patel et al. 2009)). It can be observed, however, that as the temperature increases the PWHM narrows. This improved resolution comes from improved desolvation induced at elevated temperatures. Figure 4-5 also suggests that at increased temperatures the relative intensity of the αholo

monomer 7+ charge state increases, most prominently at 80oC and 100oC. This observation could be explained as by dissociation of the dimer. At 80oC there is, however, also an increase in the relative intensity of the dimer suggesting that at elevated source temperatures there is either better transmission of the dimer ions, or disruption of the tetramer to generate more monomer/ dimer ions.

L10 Normal, 37oC m/z 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900 3000 3100 3200 3300 3400 3500 3600 3700 3800 3900 4000 4100 4200 4300 4400 4500 4600 4700 4800 4900 % 0 100

MJE100213_04_dt_01 37 (4.617) TOF MS ES+

692 T14+ T15+ T16+ T17+ T18+ D10+ D11+ D12+ αh₇+ αh₈+ 37o_C 50o_C 80o_C 110o_C HbA

101 There also appears to be a minor increase in relative intensity of the T18+ charge state with an increase in temperature, coinciding with a minor decrease in the intensity of the T14+ charge state. This is also observable in the dimer; as temperature increases so does the relative intensity of D12+ compared to D10+. As a protein unfolds more basic sites become exposed, leading to an increase in the number of sites available for protonation on the molecule. A previous study by Justin Benesch (Benesch, Sobott et al. 2003) explored the thermal unfolding of a protein by using a heated capillary to unfold a protein in solution prior to electrospray analysis. It was observed that with increasing capillary temperature TaHSP16.9, a dodecamer, thermally dissociated into monomeric and dimeric subunits. It was also observed that for monomeric lysozyme, increasing the capillary temperature led to a wider charge state distribution. The data presented here shows a similar trend, supporting the theory that increasing the source temperature adversely affects the folded state of the protein. It should be noted, however, that heating a sample using a thermally- controlled capillary results in a higher degree of dissociation of the complex compared to varying the temperature of the source region, suggesting that source temperature has a limited effect on structure.

102

Figure 4-6 - Native MS spectra of HbS illustrating the effect of varying source temperature on the MS spectrum. Increasing the source temperature appears to increase the relative intensity of the monomer and dimer compared to the tetramer, suggesting dissociation of the tetramer oligomers Figure 4-6 shows a similar trend to that shown in Figure 4-5. With increasing temperature the intensity of monomer and dimer peaks increases, suggesting a temperature-dependant dissociation of the tetramer. There is also an observed shift in charge state distribution, for both the tetramer and the dimer, towards more extended conformations, consistent with unfolding of both species. The increase in intensity of the T18+ ion is more pronounced in the HbS sample than for HbA, suggesting a possible difference in thermal stability.

It is likely that the observed low mass peak shouldering observed in both Figure 4-6 and Figure 4-7 is as a result of the peak broadening of the proposed HSA peaks via solvent adduct formation.

Ion mobility mass spectrometry

Figure 4-7 shows the arrival time distributions of HbA charge states 14+ to 17+. The ATD for the 18+ charge state is not included since it had a poor signal to noise ratio under these experimental conditions.

E34 110oC m/z 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800 % 0 100

MJE101513_07 728 (12.393) TOF MS ES+

1.35e3 T14+ T15+ T16+ T17+ T18+ D10+ D11+ D12+ αh₇+ αh₈+ 37o_C 50o_C 80o_C 110o_C HbS

103 The 14+ charge state, which is proposed to be representative of the native structureshows that with increasing temperature there is no major change in the drift time, suggesting that the protein undergoes no large conformational changes as a result of temperature. There is evidence, however, of an increase in the ATD for the 14+ charge state with increasing temperature, with broadening occurring beyond 50oC. Increased peak broadening is consistent with an increase in the number of conformations a protein is able to adopt, suggesting an increased flexibility with the molecule, consistent with a higher degree conformational freedom.

For the 37oC data, increasing charge leads to a decrease in arrival time, as a more highly charged molecule traverses the mobility cell faster, as well as increased tailing of the peak until a shoulder appears at 17+. Peak shouldering is consistent with the emergence of an additional conformation as a result of partial unfolding of the protein.

Looking at both sets of the data together, increasing temperature leads to a minor increase in drift time, followed by a decrease in drift time across all charge states. These observations suggest an expansion followed by a relaxation of the protein in the gas phase. Interestingly, the decrease in drift time coincides with an increase in the proteins ATD suggesting that the relaxation that occurs may facilitate the protein adopting a wider range of conformations. Increasing both source temperature and charge leads to peak broadening and shouldering to an increased amount when considered together. The effects of both temperature and charge have an additive effect of destabilising the protein to the point that, at the 17+ charge state at 50oC, 80oC and 110oC HbA has two distinct peaks in its ATD consistent with two distinct conformations.

104

Figure 4-7 – The arrival time distributions for the 14+  17+ charge state of HbA at four different temperatures: 37oC, 50oC, 80oC and 110oC.

Increasing the temperature appears to have a limited effect on the arrival time distributions of the 14+ and 15+ charge states. 16+ and 17+ charge states show evidence of increased peak shouldering, consistent with the appearance of an additional conformation, suggesting that elevated temperatures may have a deleterious effect on the conformation of the higher charge states.

37o_C 50o_C 80o_C 110o_C 14+ ₁₅+ ₁₆+ ₁₇+

HbA

105 Figure 4-8 shows the observed ATD’s for HbS. There is evidence for increased peak broadening of each charge state with increased temperature consistent with an increase in the dynamics of the protein at higher temperatures. There is also evidence for a more extended conformation of HbS observable at higher charge states, suggested by the shouldering present in 16+ and 17+ charge states at all temperatures.

Whilst the trend shown by the HbS data is similar to that observed for HbA, there is evidence to suggest that HbS is affected to a greater degree by charge and temperature effects. Both HbS and HbA show changes in the drift time profile of the 14+ charge state; HbA shows a minor increase, followed by a decrease. HbS, however, shows a significant decrease in drift time, consistent with a gas phase collapse of the protein. Both HbA and HbS also show increased peak broadening as a factor of temperature; HbS shows a much more severe peak broadening at FWHM compared to HBA suggesting that it is more susceptible to temperature-dependant effects. Both proteins show evidence of peak shouldering as a factor of charge, again with HbS showing a greater extended profile than HbA.

106

Figure 4-8 - arrival time distributions for the 14+  17+ charge state of HbS at four different temperatures: 37oC, 50oC, 80oC and 110oC.

Increasing the temperature appears to have a limited effect on the arrival time distributions of the 14+ and 15+ charge states. 16+ and 17+ charge states show evidence of increased pear shouldering, consistent with the appearance of an additional conformation, suggesting that elevated temperatures may have a deleterious effect on the conformation of the higher charge states.

37o_C 50o_C 80o_C 110o_C 14+

HbS

107