nanoPhos enables ultra-sensitive and cell-type resolved spatialphosphoproteomics

Oliinyk, D.; Heymann, T.; Henneberg, L.; Bardziukova, A.; Thielert, M.; Kjaergaard, J.; Vasconez, S.; Oeller, M.; Rodriguez, E.; Rosenberger, F. A.; Mann, M.

doi:10.1101/2025.05.29.656770

bioRxiv · 10.1101/2025.05.29.656770

nanoPhos enables ultra-sensitive and cell-type resolved spatialphosphoproteomics

Oliinyk, D.·Heymann, T.·Henneberg, L.·Bardziukova, A.·Thielert, M.·Kjaergaard, J.·Vasconez, S.·Oeller, M.·Rodriguez, E.·Rosenberger, F. A.·Mann, M.

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Abstract

Mass spectrometry (MS)-based phosphoproteomics has transformed our understanding of cell signaling, yet current workflows face limitations in sensitivity and spatial resolution at sub-microgram inputs. Here, we present nanoPhos, a robust method that extends phosphoproteomics to nanogram scale, making it compatible with cell-type-resolved spatial analysis. It employs loss-less solid phase extraction capture (SPEC) for sample preparation, followed by automated phosphopeptide enrichment using Fe(III)-NTA cartridges. nanoPhos identifies over 57,000 unique phosphorylation sites from 1 {micro}g cell lysate and over 4,000 from only 10 ng, a hundred-fold improvement from recent protocols. Combined with Deep Visual Proteomics (DVP), it enables region- and cell-type resolved phosphoproteomics of mouse brain tissue with spatial fidelity and a depth of 13,000 phosphosites from only 1000 cell shapes. This establishes nanoPhos as a versatile and ultra-sensitive platform that extends DVP to post-translational modifications and opens up for cell-type-specific signaling analysis in intact tissue.

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Oliinyk, D., Heymann, T., Henneberg, L., Bardziukova, A., Thielert, M., Kjaergaard, J., Vasconez, S., Oeller, M., Rodriguez, E., Rosenberger, F. A., Mann, M.. 2025-06-01. nanoPhos enables ultra-sensitive and cell-type resolved spatialphosphoproteomics. https://doi.org/10.1101/2025.05.29.656770

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