A Rapid One-Pot Workflow for Sensitive Microscale Phosphoproteomics.

Compared to advancements in single-cell proteomics, phosphoproteomics sensitivity has lagged behind due to low abundance, complex sample preparation, and substantial sample input requirements. We present a simple and rapid one-pot phosphoproteomics workflow (SOP-Phos) integrated with data-independent acquisition mass spectrometry (DIA-MS) for microscale phosphoproteomic analysis. SOP-Phos adapts sodium deoxycholate based one-step lysis, reduction/alkylation, direct trypsinization, and phosphopeptide enrichment by TiO2 beads in a single-tube format. By reducing surface adsorptive losses via utilizing n-dodecyl ß-d-maltoside precoated tubes and shortening the digestion time, SOP-Phos is completed within 3-4 h with a 1.4-fold higher identification coverage. SOP-Phos coupled with DIA demonstrated >90% specificity, enhanced sensitivity, lower missing values (<1%), and improved reproducibility (8%-10% CV). With a sample size-comparable spectral library, SOP-Phos-DIA identified 33,787 ± 670 to 22,070 ± 861 phosphopeptides from 5 to 0.5 µg cell lysate and 30,433 ± 284 to 6,548 ± 21 phosphopeptides from 50,000 to 2,500 cells. Such sensitivity enabled mapping key lung cancer signaling sites, such as EGFR autophosphorylation sites Y1197/Y1172 and drug targets. The feasibility of SOP-Phos-DIA was demonstrated on EGFR-TKI sensitive and resistant cells, revealing the interplay of multipathway Hippo-EGFR-ERBB signaling cascades underlying the mechanistic insight into EGFR-TKI resistance. Overall, SOP-Phos-DIA is an efficient and robust protocol that can be easily adapted in the community for microscale phosphoproteomic analysis.

Mapping Nanoscale-To-Single-Cell Phosphoproteomic Landscape by Chip-DIA.

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.