Huntington’s disease (HD) is a debilitating progressive neurodegenerative disorder that has profound effects on an individual’s cognitive, motor, and behavioral functions. HD is caused by mutation in the huntingtin (HTT) gene that results in CAG repeat expansion and production of an aggregation-prone polyglutamine-containing protein (mHTT). The expression of mHTT causes selective degeneration, mainly in the striatum and cortex brain regions. Yet, the molecular signatures that underlie their selective sensitization remain incompletely characterized.

In HD mouse models, we systematically employed immunopurification-mass spectrometry (IP-MS) using HTT antibodies targeting non-overlapping epitopes to capture distinct sub-cellular pools of HTT complexes and postulate domain-specific interactions. Targeted MS was used for validation of selected mHTT- and tissue-dependent interactions at different disease stages.

IP-MS samples from huntingtin-containing complexes isolated with two sets of antibodies (NT and E10) from the striatum, cortex, and cerebellum of mice harboring an Htt gene with knock-in of human exon1 containing either a normal polyglutamine (Q20) or expanded polyglutamine region (Q140). Isolated proteins were reduced with TCEP, alkylated with chloroacetamide, then desalted and digested with trypsin using S-Trap devices. Peptides (~1 ug) were analyzed using PRM on an Ultimate 3000 nanoLC coupled to a Q Exactive HF instrument.