Mapping Nanoscale‐To‐Single‐Cell Phosphoproteomic Landscape by Chip‐DIA

Abstract Protein phosphorylation plays a crucial role in regulating disease phenotypes and serves as a key target for drug development. Mapping nanoscale‐to‐single‐cell samples can unravel the heterogeneity of cellular signaling events. However, it remains a formidable analytical challenge due to the low detectability, abundance, and stoichiometry of phosphorylation sites. Here, we present a Chip‐DIA strategy, integrating a microfluidic‐based phosphoproteomic chip (iPhosChip) with data‐independent acquisition mass spectrometry (DIA‐MS) for ultrasensitive nanoscale‐to‐single‐cell phosphoproteomic profiling. The iPhosChip operates as an all‐in‐one station that accommodates both quantifiable cell capture/imaging and the entire phosphoproteomic workflow in a highly streamlined and multiplexed manner. Coupled with a sample size‐comparable library‐based DIA‐MS strategy, Chip‐DIA achieved ultra‐high sensitivity, detecting 1076±158 to 15869±1898 phosphopeptides from 10±0 to 1013±4 cells, and revealed the first single‐cell phosphoproteomic landscape comprising druggable sites and basal phosphorylation‐mediated networks in lung cancer. Notably, the sensitivity and coverage enabled the illumination of heterogeneous cytoskeleton remodeling and cytokeratin signatures in patient‐derived cells resistant to third‐generation EGFR therapy, stratifying mixed‐lineage adenocarcinoma‐squamous cell carcinoma subtypes, and identifying alternative targeted therapy for late‐stage patients. With flexibility in module design and functionalization, Chip‐DIA can be adapted to other PTM‐omics to explore dysregulated PTM landscapes, thereby guiding therapeutic strategies toward precision oncology.