Highlights d Deep profiling of proteome and phosphoproteome in AD progression d Validation of protein alterations in two independent AD cohorts d Identification of Ab-induced protein changes in AD and the 5xFAD mouse model d Prioritization of proteins and pathways in AD by multi-omics

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Since their discovery over two decades ago, the molecular and cellular functions of the NIPSNAP family of proteins (NIPSNAPs) have remained elusive until recently. NIP-SNAPs interact with a variety of mitochondrial and cytoplasmic proteins. They have been implicated in multiple cellular processes and associated with different physiologic and pathologic conditions, including pain transmission, Parkinson's disease, and cancer. Recent evidence demonstrated a direct role for NIPSNAP1 and NIPSNAP2 proteins in regulation of mitophagy, a process that is critical for cellular health and maintenance. Importantly, NIPSNAPs contain a 110 amino acid domain that is evolutionary conserved from mammals to bacteria. However, the molecular function of the conserved NIPSNAP domain and its potential role in mitophagy have not been explored.It stands to reason that the highly conserved NIPSNAP domain interacts with a substrate that is ubiquitously present across all species and can perhaps act as a sensor for mitochondrial health.

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Chemoproteomics is a key platform for characterizing the mode of action for compounds, especially for targeted protein degraders such as proteolysis targeting chimeras (PRO-TACs) and molecular glues. With deep proteome coverage, multiplexed tandem mass tag-mass spectrometry (TMT-MS) can tackle up to 18 samples in a single experiment. Here, we present a pooling strategy for further enhancing the throughput and apply the strategy to an FDA-approved drug library (95 best-in-class compounds). The TMT-MS-based pooling strategy was evaluated in the following steps. First, we demonstrated the capability of TMT-MS by analyzing more than 15 000 unique proteins (> 12 000 gene products) in HEK293 cells treated with five PROTACs (two BRD/BET degraders and three degraders for FAK, ALK, and BTK kinases). We then introduced a rationalized pooling strategy to separate structurally similar compounds in different pools and identified the proteomic response to 14 pools from the drug library. Finally, we validated the proteomic response from one pool by reprofiling the cells via treatment with individual drugs with sufficient replicates. Interestingly, numerous proteins were found to change upon drug treatment, including AMD1, ODC1, PRKX, PRKY, EXO1, AEN, and LRRC58 with 7-hydroxystaurosporine; C6orf64, HMGCR, and RRM2 with Sorafenib; SYS1 and ALAS1 with Venetoclax; and ATF3, CLK1, and CLK4 with Palbocilib. Thus, pooling chemoproteomics screening provides an efficient method for dissecting the molecular targets of compound libraries.

Background Alzheimer’s disease (AD) displays a long asymptomatic stage prior to development of dementia. Identification of molecular alterations during AD progression would provide insight into temporal alterations associated with AD. Method AD stage‐associated molecular networks were characterized by mass spectrometry in 90 frontal cortical tissue samples in five groups, including controls with low/high amyloid plaque and tau tangle pathology (LPC and HPC, respectively), cases with high Aβ pathology and slight mild cognitive impairment (MCI), late‐stage AD with high plaque/tangle pathology, and samples with progressive supranuclear palsy (PSP). We additionally performed whole proteome comparisons between human AD and the 5XFAD and Tau P301S mouse models. Protein targets implicated as altered by AD progression in the proteomic analyses were validated by affinity assays and immunostaining in human tissue and mouse models. Results AD stage‐associated molecular networks were characterized by profiling 14,513 proteins and 34,173 phosphosites in human brain with mass spectrometry, highlighting 173 protein alterations in 17 pathways. In total 16,128 proteins in 5xFAD and wild‐type mouse cortex were profiled, and share 97, 89, and 169 protein alterations with HPC, MCI, and AD, respectively, whereas Tau P301S mice share less than 15 protein alterations with each group. Neither mouse model shares more than 15 non‐tau phosphopeptide alterations with each AD sub‐group, although Tau P301S shares up to 78 phospho‐Tau peptides with human AD. During validation of proteins implicated in the proteome characterization, Netrin‐1 levels were found to increase in AD stage progression by immunoblotting but was not augmented in any other neurodegenerative disease cases tested. Additionally, affinity assays demonstrated a direct Aβ‐Netrin‐1 interaction, and immunostaining demonstrated Netrin‐1 and amyloid plaque colocalization in human AD and 5X‐FAD brain tissues, but not in Tau P301S. Conclusion Our proteomic analyses reveal altered proteins in AD and related mouse models. Human‐mouse comparisons suggest 5xFAD mice resemble symptomatic AD and that Tau P301S mice reproduce only AD‐related Tau phosphorylation. Netrin‐1 accumulates in AD, shows direct Aβ binding, and colocalizes with amyloid plaques in human AD and mouse 5XFAD brain tissue, and may be vital to AD pathogenesis. These results provide novel insights into AD‐associated proteins and pathways.