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

Mass spectrometry (MS)-based phosphoproteomics has transformed our understanding of cell signaling, yet current workflows face limitations in sensitivity and spatial resolution when applied to sub-microgram scale protein inputs. Here, we present nanoPhos, a robust method for ultra-sensitive phosphoproteomics, which allows deep coverage at high throughput and is compatible with Deep Visual Proteomics (DVP). It employs loss-less solid phase extraction capture (SPEC) for sample preparation and protein processing, followed by automated zero-volume phosphopeptide enrichment using Fe(III)-NTA cartridges. nanoPhos identifies over 55,000 unique phosphorylation sites from 1 ug cell lysate and over 8,000 from as little as 10 ng, a hundred-fold more identifications than recent protocols. In combination with laser microdissection, it enables cell-type and anatomically resolved phosphoproteomics of mouse brain tissue with spatial fidelity and depth of more than 17,000 phosphosites from only 1000 cell shapes. This establishes nanoPhos as a versatile and ultra-sensitive platform for probing cell types dispersed in heterogenous tissue and extends DVP to post-translational modifications.