Travelling Wave Ion Mobility Mass Spectrometry-based Conformational Studies of Prion Protein - Comparison of Truncated Recombinant Mouse with Recombinant Syrian Hamster

摘要

Travelling wave-based ion mobility mass spectrometry is a powerful method for the study of conformational changes in recombinant prion proteins. Estimated cross-sections may be easily obtained by calibration against previously published drift cell-based experimental cross-sections. The secondary structure of all the proteins was determined using far-UVCDprior to mass spectrometry analysis. It has been previously published that small, but significant, differences were observed in the estimated cross-sections between the alpha-helical and beta sheet-rich SHa PrP (90-231) isoforms at pH 5.5 but not at pH 7.0. (Data not shown) The experimentally obtained estimated cross-sections for SHa PrP (90-231) and SHa PrP (23-231) are very similar. The biologically relevant charge states, [M+6H]~(6+) to [M+9H]~(9+), of the experimental data is in good agreement with the theoretical cross-sections. This suggests that the flexible N-terminal sequence may not be as unstructured as previously predicted. Significant differences in the estimated cross-sections were observed between the helical and beta sheet-richMoPrP (91-230) isoforms at physiological pH, pH 5.5 and pH 7.0. The differences in estimated cross-section were greater than those previously in Syrian hamster samples. Acomparison of the rodent sequences revealed that there is a high sequence identity between SHa PrP (90-231) andMoPrP (91-230). There are eight different amino acid residues in total between the species resulting in a 229 Da mass difference. Acomparison of the estimated cross-sections for alpha-helical SHa PrP (90-231) and alpha-helical MoPrP (91-230) at pH 5.5 and pH 7.0 was made. It was observed that alpha-helical SHa PrP (90-231) had a 5percent difference in cross-section than alpha-helical Mo PrP (90-230). This is despite the relatively small difference in mass, 229 Da, between the proteins (1.5percent difference). This observation was true at both pH 5.5 and pH 7.0. Both hamster and mouse prion proteins had a similar number of conformations. At least two stable conformations were measured at charge states [M+7H]~(7+), [M+9H]~(8+) and [M+10H] ~(10+). The unfolding patterns of alpha-helical SHa PrP (90-231) is parallel to that of alpha-helical Mo PrP (91-230). This suggests the proteins could be structurally similar.