I have a question about HDX-MS to assay peptide-protein interactions that you may kindly help me.
I have tested a peptide interaction with its targeted protein using HDX-MS. All the data showed that peptide alone didn’t show retained H-D exchange, but peptide showed very strong H-D exchange when mixing with protein. I did a negative control peptide, which didn’t show slower H-D exchange when mixing with same protein. I concluded that this peptide interacted with its targeted protein. While when validation using biotinylated peptide to interact with this protein such as SPR techniques, the interaction was not found.
Could your guys please give me some advises whether HDX-MS give me false positive data (From my understanding, HDX-MS is enough to conclude its interaction if H-D exchange is significantly retained)? From my understanding, the labelled peptide in SPR system has the possibility to affect the accessibility of its targeted protein.
You are correct that SPR can suffer from artifacts caused by limited accessibility due to binding your peptide to the surface. Because of this, you can't use a technique such as SPR to disprove the presence of a protein-ligand interaction. HDX is a solution technique, and is therefore not prone to surface affects like SPR. Due to this fact, I'd tend to believe that there is an interaction between your protein and peptide.
Regarding your questions of false positives in HDX, it's really hard for me to imagine many ways for this to occur. Here are some of the possibilities that I can come up with:
1) If you're at VERY high concentrations of protein/peptide, you could force non-physiological interactions
2) If the pH of your protein and peptide solutions are different, or if one or more of them contain a different amount of a viscosogen like glycerol, or even different concentrations of salt, then you have the possibility of perturbing the exchange rates and stability of the hydrogen bonds in your peptide in the absence of any interaction with your protein.
Thanks a lot for your comments, James. Both peptides and proteins are buffer exchanged very well. It is not possible from the different buffer. I have a negative control peptide with same concentration, and also a positive control peptide complex in the same concentration. The positive control H-D exchange is comparable to the test one, while the negative control peptide didn't show any H-D exchange. I will try longer linker for this peptide to test SPR, and wish to provide additional proof to convince my colleagues who are not in this field.
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