Metal ion-enhanced ZIC-cHILIC StageTip for N-Glycoproteomic and Phosphoproteomic Profiling in EGFR-mutated Lung Cancer Cells.

Surface glycosylation and intracellular phosphorylation regulates the cell-cell communication and signaling cascades. Due to complex glycosylation and dynamic phosphorylation, exploring their interplay remains technically challenging. In this study, we reported a tandem ZIC-cHILIC StageTip strategy for streamlined and simultaneous (sialo)glycoproteomic and phosphoproteomic profiling. We first demonstrated that Fe ions expand the utility of ZIC-cHILIC strategy to phosphoproteomic analysis with greatly enhanced >4-fold coverage and high specificity for mono-phosphopeptides (95%). The Fe-ZIC-cHILIC tandem tips, leveraging stepwise fractionation, enable large-scale coverage of 10,536 glycopeptides, including highly confident 4,285 sialoglycopeptpides, and 11,329 phosphopeptides in a single cell type. To study the mechanism underlying the tyrosine kinase inhibitor (TKI) resistance in non-small cell lung cancer (NSCLC), application of the strategy to 4 NSCLC cells harboring different EGFR mutations reveals significantly differential 1,559 glycopeptides and 1,949 phosphopeptides either in EGFR mutation or TKI resistant cells. Without protein immunoprecipitation, the approach identified FDA-approved drug targets, such as EGFR, ERBB2, MET, and integrin family members. Most prominent alterations were observed in EGFR (auto-phosphorylation Y1197 and 10 bi- and triantennary fucosyl-sialo glycans at N603), downstream PI3K-Akt pathway (ERBB2-T1240, MET-S990/T992, AKT-S124/S126) and integrin family (sialo-fucosyl glycans), suggesting site-specific alteration between N-glycosylation and phosphorylation interplay in the TKI resistant L858R-T790M mutant NSCLC cells. The glycoproteomic and phosphoproteomic landscape may help to unravel the complex modification alterations underlying the resistant mechanism, offering insights for improving therapeutic strategies and patient outcomes.