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 monophosphopeptides (95%). The Fe-ZIC-cHILIC tandem tips, leveraging stepwise fractionation, enable large-scale coverage of 10,536 glycopeptides, including highly confident 4285 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 four NSCLC cells harboring different epidermal growth factor receptor (EGFR) mutations reveals significantly differential 1559 glycopeptides and 1949 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 biantennary 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.