Simultaneous profiling of sialoglycoproteome and phosphoproteome in TKI-resistant NCSLC cells using metal ion-decorated ZIC-cHILIC StageTip

The cellular plasma membrane proteins are densely decorated with surface glycosylation and intracellular phosphorylation whose interplay determines the cell-cell communication and signaling cascades. However, their concomitant characterization remains extreme challenges due to complexity of glycan structures, low stoichiometry (particularly sialylation), labile, dynamic nature and low detectability of both phosphopeptides and glycopeptides in mass spectrometry. In this study, we introduce a streamlined metal ion-decorated ZIC-cHILIC strategy for simultaneous, sensitive and large-scale (sialo-)glycoproteomic and phosphoproteomic profiling. We previously reported a stepwise ZIC-cHILIC StageTip with high enrichment efficiency and specificity for large-scale glycoproteomic characterization (Chen, et al., Anal. Chem. 2021). Based on different degree of negative charge on phosphate and glycans, we further implemented metal ions into a tandem ZIC-cHILIC StageTip and simple pH control as a streamlined strategy for simultaneous enrichment and stepwise fractionation of both (sialo)glycopeptides and phosphopeptides profiles. Despite tyrosine kinase inhibitor (TKI) demonstrated precision oncology for EGFR-mutant patient in non-small cell lung cancer (NSCLC), nearly every patient develop resistance. The strategy was demonstrated on patient-derived TKI-resistant NSCLC cells, identifying 13,714 high-confident glycopeptides (including 3,142 sialo-glycopeptides) and 15,164 phosphopeptides in 4 different EGFR mutated NSCLC cells. Compared to naked ZIC-cHILIC, the metal ion under pH control increases 5.6-fold specificity (from 15% to 84%) and 10-fold phosphopeptides (17,036). Without protein immunoprecipitation, the approach identified important glycosylation and phosphorylation on druggable protein, such as 10 of 12 N-glycosylation sites and driver autophosphorylation sites, such as Y1172 and Y1110 of EGFR protein, the target therapy in lung cancer. Quantitative comparison also showcase the sensitivity with high coverage of many oncoproteins and their downstream signaling cascades, such as dysregulated glycosylation and phosphorylation under different EGFR downstream cascades in PI3K and MAPK pathway in these patients. Our approach may offers a high performance approach to reveal the dynamic interplay of multi-PTMomic landscape in cell membrane.