nanoPhos breaks the sensitivity barrier in phosphoproteomics — profiling 4,000+ phosphosites from just 10 ng input — and, integrated with Deep Visual Proteomics, resolves cell-type-specific signaling networks directly within intact tissue sections.
nanoPhos achieves nanogram-scale phosphoproteomics through loss-less solid phase extraction and zero-dead-volume enrichment, identifying over 57,000 phosphosites from 1 μg input with >90% quantitative precision, and when integrated with Deep Visual Proteomics enables cell-type-resolved spatial phosphoproteomics that captures over 13,000 phosphosites from 1,000 laser-microdissected neuronal shapes, revealing distinct signaling signatures across neuronal populations and brain regions.
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 µg 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.