Glycoproteomic Alterations in Drug-Resistant Nonsmall Cell Lung Cancer Cells Revealed by Lectin Magnetic Nanoprobe-Based Mass Spectrometry Juanilita T. Waniwan Yi-Ju Chen Rey Capangpangan Shao-Hsing Weng Yu-Ju Chen 10.1021/acs.jproteome.8b00433.s002 https://acs.figshare.com/articles/dataset/Glycoproteomic_Alterations_in_Drug-Resistant_Nonsmall_Cell_Lung_Cancer_Cells_Revealed_by_Lectin_Magnetic_Nanoprobe-Based_Mass_Spectrometry/7218794 Understanding the functional role of glycosylation-mediated pathogenesis requires deep characterization of glycoproteome, which remains extremely challenging due to the inherently complex nature of glycoproteins. We demonstrate the utility of lectin–magnetic nanoprobe (MNP@lectin) coupled to Orbitrap HCD-CID-MS/MS for complementary glycotope-specific enrichment and site-specific glycosylation analysis of the glycoproteome. By three nanoprobes, MNP@ConA, MNP@AAL, and MNP@SNA, our results revealed the first large-scale glycoproteome of nonsmall cell lung cancer (NSCLC) with 2290 and 2767 nonredundant glycopeptides confidently identified (Byonic score ≥100) in EGFR-TKI-sensitive PC9 and -resistant PC9-IR cells, respectively, especially with more fucosylated and sialylated glycopeptides in PC9-IR cells. The complementary enrichment was demonstrated with only five glycopeptides commonly enriched in three MNP@lectins. Glycotope specificity of 79 and 62% for enrichment was achieved using MNP@AAL and MNP@SNA, respectively. Label-free quantitation revealed predominant fucosylation in PC9-IR cells, suggesting its potential role associated with NSCLC resistance. Moreover, without immunoprecipitation, this multilectin nanoprobe allows the sensitive identification of 51 glycopeptides from 10 of 12 reported sites from onco-protein EGFR. Our results not only demonstrated a sensitive approach to study the vastly under-represented <i>N-</i>glycoprotome but also may pave the way for a glycoproteomic atlas to further explore the site-specific function of glycoproteins associated with drug resistance in NSCLC. 2018-09-28 00:00:00 site-specific function sialylated glycopeptides EGFR-TKI-sensitive PC 9 glycosylation-mediated pathogenesis Glycoproteomic Alterations drug resistance site-specific glycosylation analysis NSCLC resistance PC 9-IR cells MNP glycoproteome HCD-CID-MS Nanoprobe-Based Mass Spectrometry nonsmall cell lung cancer onco-protein EGFR Label-free quantitation glycoproteomic atlas 51 glycopeptides Drug-Resistant Nonsmall Cell Lung Cancer Cells Revealed 2767 nonredundant glycopeptides multilectin nanoprobe glycotope-specific enrichment