Stress granules (SGs) are membrane-less cellular condensates that form in response to physiological stressors such as viral infection, hypoxia, and oxidative stress. Their dysregulation has been linked to neurodegeneration, cancer, and infectious disease. Defining the protein composition of SGs has remained technically challenging; they’re transient, lack a membrane boundary, and their resident proteins represent only a small fraction of total cellular protein. Bulk purification methods sacrifice spatial resolution and introduce cytosolic contamination, while existing single-cell approaches have lacked the sensitivity required for subcellular proteomic profiling.

To address these gaps, Davison et al. developed a capillary-based subcellular sampling workflow coupled to LC-MS for targeted, spatially resolved proteomics of individual SGs in live cells. Samples were separated using a Vanquish Neo in nano-flow mode, with peptides separated on an Aurora® Rapid™ 8×75 XT C18 UHPLC column before nano-electrospray ionisation (nESI) and DIA on an Orbitrap Astral. This global, exploratory DIA proteomics approach identified 5,115 proteins across the full dataset, with a high-confidence SG-associated proteome of 405 proteins defined after stringent filtering.

Leveraging this workflow, the researchers identified and validated four previously unreported SG-resident proteins: AHNAK2, DDX39B, NUDT1, and FKBP2, confirming their localisation by immunofluorescence across two independent stress conditions. This work establishes capillary-based subcellular sampling as a viable strategy for organelle-resolved proteomics from single cells, with implications for understanding condensate biology in disease contexts.

Publication
bioRxiv

Authors

Claire Davison, Nicolas Locker, Mariana Marques, Sam Kelly, Emily Relton, Tanavi Sharma, Emily Fraser, Pedro Aragon Fernandez, Erwin Schoof, Mia Petersen, Jordan Pascoe, Kathryn S. Lilley, Sneha Pinto, Matt Spick, Melanie Bailey

Title

Capillary-based subcellular sampling uncovers the stress granule proteome in single cells