For the past week or so I have been conducting an analysis of post-translational modifications of the huntingtin protein. After the protein is expressed in the cell, it is then often chemically modified by small moieties which can affect its fold and function.
In this instance, I have been looking at the phosphorylation of the protein through reanalysis of the peptides seen in the previous mass spectrometry experiments conducted as part of the domain mapping investigations. All of this work was done using the huntingtin protein sample derived from HEK293 cells, kindly provided by Stefan Kochanek and Bin Huang.
An example spectra showing phosphorylation on S2652.
Many phosphorylations can be found in the protein sample. A total of 89 different sites were detected, many of which have been previously reported but some we are reporting for the first time. All of the data can be downloaded and read on Zenodo.
It is important to note that the protein used in these experiments is derived from an environment most unlike that of native huntingtin in neuronal cells, so this work shows only the potential for these sites to be modulated by phosphorylation, not that they actually are under physiological conditions. Further experiments will definitely be needed to validate any of these sites for functional, structural or disease relevance. None-the-less, a good starting point.
This work was completed with a lot of help from Suzanne Ackloo, our in house mass spec guru, so a huge thanks to her for completely a large amount of the analysis of the mass spectrometry data output.
2 thoughts on “Huntingtin phosphorylation analysis by mass spectrometry”
Cool stuff, let us know if you want lysate from primary neurons to validate this, we can send you some.
Great – that would be awesome. I need to to talk to our mass spec guys about the technical specs for doing this kind of sample analysis and I will be in touch on email to talk specifics soon.
Thanks so much!