Evaluation of a Pooling Chemoproteomics Strategy with an FDA-Approved Drug Library.

Chemoproteomics is a key platform for characterizing the mode of action for compounds, especially for targeted protein degraders such as proteolysis targeting chimeras (PROTACs) 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.

Identifying Sex-Specific Serum Patterns of Alzheimer's Mice through Deep TMT Profiling and a Concentration-Dependent Concatenation Strategy.

Alzheimer's disease (AD) is the most prevalent form of dementia, disproportionately affecting women in disease prevalence and progression. Comprehensive analysis of the serum proteome in a common AD mouse model offers potential in identifying possible AD pathology- and gender-associated biomarkers. Here, we introduce a multiplexed, nondepleted mouse serum proteome profiling via tandem mass-tag (TMTpro) labeling. The labeled sample was separated into 475 fractions using basic reversed-phase liquid chromatography (RPLC), which were categorized into low-, medium-, and high-concentration fractions for concatenation. This concentration-dependent concatenation strategy resulted in 128 fractions for acidic RPLC-tandem mass spectrometry (MS/MS) analysis, collecting ∼5 million MS/MS scans and identifying 3972 unique proteins (3413 genes) that cover a dynamic range spanning at least 6 orders of magnitude. The differential expression analysis between wild type and the commonly used AD model (5xFAD) mice exhibited minimal significant protein alterations. However, we detected 60 statistically significant (FDR < 0.05), sex-specific proteins, including complement components, serpins, carboxylesterases, major urinary proteins, cysteine-rich secretory protein 1, pregnancy-associated murine protein 1, prolactin, amyloid P component, epidermal growth factor receptor, fibrinogen-like protein 1, and hepcidin. The results suggest that our platform possesses the sensitivity and reproducibility required to detect sex-specific differentially expressed proteins in mouse serum samples.

NIPSNAP protein family emerges as a sensor of mitochondrial health.

Since their discovery over two decades ago, the molecular and cellular functions of the NIPSNAP family of proteins (NIPSNAPs) have remained elusive until recently. NIPSNAPs 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.

Midkine Attenuates Aß Fibril Assembly and AmyloidPlaque Formation.

Proteomic profiling of Alzheimer's disease (AD) brains has identified numerous understudied proteins, including midkine (MDK), that are highly upregulated and correlated with Aß since the early disease stage, but their roles in disease progression are not fully understood. Here we present that MDK attenuates Aß assembly and influences amyloid formation in the 5xFAD amyloidosis mouse model. MDK protein mitigates fibril formation of both Aß40 and Aß42 peptides in Thioflavin T fluorescence assay, circular dichroism, negative stain electron microscopy, and NMR analysis. Knockout of Mdkgene in 5xFAD increases amyloid formation and microglial activation. Further comprehensive mass spectrometry-based profiling of whole proteome and aggregated proteome in these mouse models indicates significant accumulation of Aß and Aß-correlated proteins, along with microglial components. Thus, our structural and mouse model studies reveal a protective role of MDK in counteracting amyloid pathology in Alzheimer's disease.

Deep Multilayer Brain Proteomics Identifies Molecular Networks in Alzheimer's Disease Progression.

Alzheimer's disease (AD) displays a long asymptomatic stage before dementia. We characterize AD stage-associated molecular networks by profiling 14,513 proteins and 34,173 phosphosites in the human brain with mass spectrometry, highlighting 173 protein changes in 17 pathways. The altered proteins are validated in two independent cohorts, showing partial RNA dependency. Comparisons of brain tissue and cerebrospinal fluid proteomes reveal biomarker candidates. Combining with 5xFAD mouse analysis, we determine 15 Aß-correlated proteins (e.g., MDK, NTN1, SMOC1, SLIT2, and HTRA1). 5xFAD shows a proteomic signature similar to symptomatic AD but exhibits activation of autophagy and interferon response and lacks human-specific deleterious events, such as downregulation of neurotrophic factors and synaptic proteins. Multi-omics integration prioritizes AD-related molecules and pathways, including amyloid cascade, inflammation, complement, WNT signaling, TGF-ß and BMP signaling, lipid metabolism, iron homeostasis, and membrane transport. Some Aß-correlated proteins are colocalized with amyloid plaques. Thus, the multilayer omics approach identifies protein networks during AD progression.