Huntingtin methylation analysis by mass spectrometry

For the past week or so I have been continuing to conduct an analysis of post-translational modifications or PTMs of the huntingtin protein. After the protein is expressed in the cell, it is often chemically modified by small moieties which can affect its fold and function called PTMs. In my last post I looked at one type of PTM called phosphorylation which is when a PO43group is added to the amino acids serine, threonine or tyrosine. This time I have been looking at methylation which is when arginine or lysine amino acids are modified with a CH3 group.

In this instance, I have been looking at the methylation 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.

Site Modification Previously Reported Sequence Methylated in our samples? How many of peptide MS experiments?
K234‑ac TLAAAVPkIMASFGN Yes 1 – monomethylated, 1 – dimethylated
K343‑ac GSFGVTRkEMEVSPS Yes 2 – monomethylated
K941‑ub LVPKLFYkCDQGQAD Yes 2 – monomethylated
K1262‑ub DLQNSTEkFGGFLRS Yes 2 – monomethylated
K1337 – ub (in mouse) GLSSNPSKSQGRAQR Yes 1 – monomethylated
K1431‑ub LFEPLVIkALKQYTT Yes 1 – monomethylated
K1730 – ub (in mouse) LEEHSEGKQIKNLPE Yes 1 – monomethylated
R1801 – me2 (in mouse) RITAAATRLFRsDGC Yes 1 – dimethylated

Methylation sites which overlaps with other modification sites of huntingtin – NB all data of previous reported modifications retrieved from Phosphosite

Many methylations are found in the protein sample which is interesting as these are not widely reported previously. A total of 118 different sites were detected across the 3 datasets although only 9 were found in triplicate across the datasets. Some of the sites detected overlap with ubiquitination sites, which could implicate huntingtin methylation as being connected to ubiquitination although this would require a HUGE amount more work to ascertain and unpick. All of the data can be downloaded and read on Zenodo.

As with the phosphorylation analysis, 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 methylation, 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.

As with previous mass spectrometry data analysis, 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 completing a large amount of the analysis of the mass spectrometry data output.

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