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bioRxiv · 10.64898/2026.09.22.753649

Tracking cellular biomolecular condensate dynamics under proteostatic stress with middle-down phosphoproteomics

Pandi, B.·Ng, D. C. M.·Schaal, P.·Alamillo, L.·Binti, S.·Lau, E.·Lam, M. P. Y.
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Abstract

Coordination of biological function requires the partition of cellular components including into biomolecular condensates, but an overall landscape of how protein compartmentalize into higher-order assemblies under stress is still emerging. We apply proteome-wide solubility profiling to compare the compositions of NP-40-insoluble proteins and their phosphorylation status, using a new mass spectrometry-based hybrid bottom-up and chemical middle-down proteomics approach to analyze the solubility behavior of 8,740 proteins and 31,647 phosphopeptides under normal and ER stress conditions. Cell stress induces a pervasive differential partition of proteins in and out of detergent-insoluble cellular compartments. This differential partition is partially orthogonal to stress-induced abundance changes and comprises both phosphorylation-dependent and phosphorylation-independent mechanisms. Whereas phosphorylation-independent partition changes involve largely secretory pathway proteins and implicate higher-order assemblies of chaperones and clients, phosphorylation-based partitions suggest a dynamic rearrangement of biomolecular condensate compositions across cytoplasmic and nuclear ribonucleoprotein assemblies. The accumulation of serine/arginine rich (SR) proteins and other annotated nuclear speckle members in the condensate-rich proteome fractions emerges as a central feature of stress-induced remodeling. Our results establish global solubility dynamics as an integral component of proteome stress response and implicates broad involvements of splice factor spatial reorganization as a prominent facet of ER stress response.

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Pandi, B., Ng, D. C. M., Schaal, P., Alamillo, L., Binti, S., Lau, E., Lam, M. P. Y.. 2026-09-23. Tracking cellular biomolecular condensate dynamics under proteostatic stress with middle-down phosphoproteomics. https://doi.org/10.64898/2026.09.22.753649

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