The cutting of huntingtin protein by enzymes in the cell into shorter fragments is linked to cellular toxicity and therefore is thought to contribute to the phenotype and symptoms of Huntington’s disease. By searching through Pubmed, I have found a number of published papers which detail the sites at which this happens in the huntingtin amino acid sequence. I can then compare these sites with the fragment start and stop sites from my previous limited proteolysis experiment and mass spec analysis. Some of these sites overlap which corroborates the idea that these could be domain boundaries or at least that this region of the protein is unfolded and not a discretely folded domain. All the details of these analyses are uploaded and free for anyone to read at Zenodo.
All of the mass spec data (in .raw format) is now available to download so that anyone can analyse or critique it. MaxQuant is freely available software which can allow you to do this should you feel so inclined. I have also uploaded a more detailed description of the mass spec protocols with the raw data files – thanks to Paul Taylor at SPARC, Sick Kids for providing this information. Suzanne Ackloo, chemical probes coordinator at SGC Toronto, gave me a super mass spec 101 tutorial this week. I have quite limited previous experience using these methods whereas Suzanne has previously experience in the field of proteomics, frequently using such mass spec methodologies. This has really helped me understand a lot more about the data processing procedure, the limitations of such analyses as well as understanding some of the more technical details of the mass spec methods.
I am looking forward to receiving the next round of mass spec data from Paul Taylor shortly to see if we can get further corroboration of putative fragment boundaries or not for the huntingtin protein.