, 2008), and hence both forms should share the majority of in vivo interacting proteins. Human and/or murine fl-Htt complexes were immunoaffinity purified from the cortex, striatum, and cerebellum of BACHD and WT mice at 2 or 12 months of age using HDB4E10 (Figure 1B). As a negative control, a mock IP for each sample condition (defined by specific brain region, age, and genotype) was performed in the absence of the Htt antibody. Therefore, a total of 30 independent IP experiments were performed, and approximately 765 trypsin-digested gel slices

were subjected to LC-MS/MS analysis (Figure 1C). Using the MASCOT (Matrix Science) sequence database-searching tool, we identified a total of 747 high-scoring, putative Htt-interacting proteins Compound Library from BACHD and WT NVP-AUY922 ic50 mouse brains (Table S1 available online). Consistent with the claim that we are surveying fl-Htt-interacting proteins, we identified Htt peptides spanning the entire sequence of Htt in both BACHD and WT samples (Figure 1D

and Table S2). As confirmation that HDB4E10 more efficiently immunoprecipitates human Htt than murine Htt, we observed more Htt peptides and more extensive sequence coverage of fl-Htt in BACHD mice compared to WT mice. We next performed several standard bioinformatic analyses to determine the validity of our approach in isolating Htt interactors in vivo and to define potential biological pathways that Htt may be engaged in at various ages within different brain regions. We compared our list of 747 candidates

with previously reported Htt interactors. Among a curated list of 876 proteins previously identified as putative ex vivo Htt interactors, most of which were obtained from Y2H experiments (Goehler et al., 2004 and Kaltenbach et al., Thymidine kinase 2007; http://HDbase.org), we found 139 proteins also present in our in vivo Htt interactome (Figure 1E and Table S3). This represents a highly significant enrichment (p = 7.00 × 10−50; hypergeometric test). The disease specificity of our interactome is supported by the comparison of our data set with that of another polyQ disease protein, Ataxin-1 (Lim et al., 2006). Despite the fact that one-third of our samples originated from the cerebellum, the same tissue source as the Ataxin-1 interactome study, only 38 proteins were present in both data sets with relatively modest enrichment (p = 0.0005; Figure 1E and Table S3). Thus, our in vivo fl-Htt interactome appears to be specific to Htt and provides a valuable list of Htt-interacting proteins, both in vivo and ex vivo, for further investigation to determine their roles in Htt biology and HD pathogenesis. Gene Ontology (GO) analysis (Huang et al., 2009a and Huang et al.