I have been continuing to characterise the huntingtin protein samples I am generating in the lab. You can read about my first attempts to map post-translational modifications by mass spec here. Previously I found phosphorylation modifications on the huntingtin protein which are located on the same sites as huntingtin protein derived from human cells which was good news! However, the experiment was only able to map a small percentage of the entire protein so I have been trying to improve the number of peptides seen in the mass spec by altering the way I fragment the sample. Previously I would digest the protein into short peptides with trypsin but I was keen to try lysarginase which cleaves the mirror image of motif of trypsin.

Trypsin/Lysarginase cleavage sites

Using this enzyme drastically increased the coverage of huntingtin and we can see more phosphorylations most of which correspond to those found on mammalian derived expression systems. This means that the huntingtin protein is likely being phosphorylated in a physiologically relevant manner. All of the data can be found on Zenodo.