Frere, G. A. | bioRxiv Science

bioRxiv2025

Next-Generation Multiplexed Targeted Proteomics Quantifies Post-Translational Modifications, Compound-Protein Interactions, and Disease Biomarkers with High Throughput

The GoDig platform enables sensitive, multiplexed targeted pathway proteomics without manual scheduling or synthetic standards. Here we present GoDig 2.0, which increases sample multiplexing to 35-fold, improves time efficiency and reduces scan delays for higher success rates, and allows flexible spectral and elution library generation from different mass spectrometry data types. GoDig 2.0 measures 2.4x more targets than GoDig 1.0, quantifying >99% of 800 peptides in a single run. We compiled a library of 23,989 human phosphorylation sites from a phosphoproteomic dataset and used it to profile kinase signaling differences across cell lines. In human brain tissue, we established a hyperphosphorylated tau assay including pTau127, revealing potential biomarkers for Alzheimers disease. We also quantified diglycyl-lysine peptides to assess polyubiquitin branching. Finally, we built a library of 20,946 reactive cysteines and profiled covalent compound-protein interactions spanning diverse pathways. GoDig 2.0 enables high-throughput analyses of site-specific protein modifications across many biological contexts.

biochemistry↗

bioRxiv2025

Thyroid Hormone Receptor Beta Signaling is a Targetable Driver of Prostate Cancer Growth

Thyroid hormone (TH) signaling plays a major role in the development, energy homeostasis, and metabolism of most tissues. Recent observations have identified THs as drivers of prostate cancer (PCa) tumor development and progression. We reported that the T3-scavenger protein {micro}-crystallin (CRYM) regulates the development and progression of PCa and that this involved crosstalk with the androgen receptor (AR) signaling. However, the mechanisms remain incompletely understood. Here, we explored the role of thyroid hormone receptor {beta} (TR{beta}), which is the main effector of TH signaling, in the context of PCa. The use of the TR{beta}-selective antagonist NH-3 inhibited PCa cell proliferation in vitro and reduced tumor size in PCa xenograft models. Notably, NH-3 was highly effective in the engrafted 22Rv1 cell line, a model for castration-resistant PCa (CRPC). Mechanistic studies revealed that NH-3 downregulates AR and the AR target genes Nkx3.1 and KLK3 (PSA). NH-3 was a more effective anticancer agent than enzalutamide and showed synergistic properties in combined use. Evidence from human datasets corroborates our findings whereby elevated TR{beta} expression and mutations in TH signaling pathways are associated with the onset of PCa. Collectively, these results establish TR{beta} as a mediator of tumorigenesis in PCa and identify NH-3 as a promising therapeutic agent for targeting AR signaling, particularly in CRPC.

cancer biology↗