High-resolution H/D exchange studies on the HET-s218-295 prion protein.
In a search for improved resolution of hydrogen/deuterium (H/D) exchange experiments analyzed by mass spectrometry (HXMS), we evaluated two methodologies for a detailed structural study of solvent accessibility in the case of the HET-s(218-295) prion protein. For the first approach, after incubation in the deuterated solvent, aggregated HET-s(218-295) was digested with pepsin and the generated peptides were analyzed by nanospray mass spectrometry in an ion trap, with and without collision-induced dissociation (CID). We compared deuterium incorporation in peptides as determined on peptide pseudomolecular ions and on b and y fragments produced by longer peptides under CID conditions. For both b and y fragment ions, an extensive H/D scrambling phenomenon was observed, in contrast with previous studies comparing CID-MS experiments and (1)H NMR data. Thus, the spatial resolution of HXMS experiments could not be improved by means of MS/MS data generated by an ion trap mass spectrometer. In a second approach, the incorporation of deuterium was analyzed by MS for 76 peptides of the HET-s(218-289) peptide mass fingerprint, and the use of shared boundaries among peptic peptides allowed us to determine deuteration levels of small regions ranging from one to four amino acids. This methodology led to evidence of highly protected regions along the HET-s(218-295) sequence.
Nazabal
Alexis
A
Institut Européen de Chimie et de Biologie, UMR CNRS 5144, Pessac, France. nazabal@org.chem.ethz.ch
Bonneu
Marc
M
Saupe
Sven J
SJ
Schmitter
Jean-Marie
JM
D000595:Amino Acid Sequence; D041961:Deuterium Exchange Measurement; D005656:Fungal Proteins / Q000032:analysis* / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011328:Prions / Q000032:analysis* / Q000737:chemistry*; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D012680:Sensitivity and Specificity
D005656:Fungal Proteins; C083891:HET-S protein, Podospora anserina; D011328:Prions