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 achieving hundred-fold sensitivity gains that extend phosphoproteomics to nanogram scale, making it compatible with cell-type-resolved spatial analysis. nanoPhos employs loss-less solid phase extraction capture (SPEC) for sample preparation, followed by automated phosphopeptide enrichment using Fe(III)-NTA cartridges. The method identifies over 57,000 unique phosphorylation sites from 1 µg cell lysate and over 4,000 from only 10 ng—a hundred-fold improvement over recent protocols. Combined with Deep Visual Proteomics (DVP), nanoPhos enables region- and cell-type resolved phosphoproteomics of mouse brain tissue with spatial fidelity and a depth of 13,000 phosphosites from only 1,000 cell shapes.
We demonstrate nanoPhos performance across multiple sample types and biological contexts. From FACS-sorted cells, we identified more than 52,000 phosphosites from 3,000 cells and nearly 10,000 from just 100 cells. In mouse embryonic stem cells cultured under different conditions, nanoPhos captured biologically meaningful phosphorylation differences distinguishing pluripotency states and lineage specification from as few as 500 sorted cells. The method extends to archived formalin-fixed paraffin-embedded (FFPE) tissues, recovering over 25,000 phosphosites from 1 µg of FFPE mouse brain with high correlation to fresh-frozen samples. Applied to clinical lung adenocarcinoma specimens via laser capture microdissection, nanoPhos identified over 20,000 phosphosites from pathologically defined tumor and healthy tissue regions, revealing kinase signatures and pathway alterations characteristic of cancer metabolism and signaling.
Most notably, integration of nanoPhos with DVP (phosphoDVP) enables global phosphoproteomic measurements in spatially and cell-type-resolved tissue contexts. From laser-microdissected mouse brain neurons, we achieved comprehensive phosphoproteome coverage distinguishing cortical versus subcortical regions and excitatory versus inhibitory cell types, with phosphorylation patterns reflecting distinct synaptic architectures and regional specialization.
Finally, we validate the biological significance and quantitative accuracy of nanoPhos-derived phosphorylation data, we perform targeted verification using parallel reaction monitoring (PRM) using the recently released Sciex 8600 mass spectrometer. This orthogonal validation will confirm key cell-type-specific phosphorylation changes identified in our discovery experiments.