以凝集素磁性奈米探針結合質譜法解析抗藥性非小細胞肺癌細胞中之變異醣蛋白體

Abstract

Technological advancements in the field of mass spectrometry (MS) have enabled scientists to understand the biological system at the molecular level. Simultaneous identification and quantitation of thousands of proteins by mass spectrometry-based proteomics allows qualitative and quantitative description of a cellular proteome. Glycosylaton on protein plays important role in disease progression. Understanding the functional role of glycosylation-mediated pathogenesis requires deep characterization of glycoproteome, which remains tecnically challenging due to the inherently complex nature of glycoproteins. Thus, the thesis aims to utilize a new strategy for glycopeptide enrichment; glycotope-specific enrichment is achieved using lectins and rapid separation of analytes is facilitated using magnetic nanoparticles. Importantly, the method focuses on large-scale analysis of intact glycopeptides. The established method is then used to characterize the glycosylation in non-small cell lung cancer cells (NSCLCs). To facilitate the analysis of the seriously under-explored glycoproteome, we designed an enrichment strategy that employs lectin-conjugated magnetic nanoprobe coupled with Orbitrap HCD-CID-MS/MS, in the first part of this study. Three nanoprobes, MNP@ConA (high-mannose glycotopes), MNP@AAL (fucosylated glycotopes) and MNP@SNA (sialylated glycotopes), were used for complementary glycotope-specific enrichment and site-specific glycosylation analysis. Our results revealed the first large-scale identification of glycosylation in NSCLCs; 2290 and 2767 nonredundant glycopeptides were confidently identified (Byonic score ≥100) in tyrosine kinase inhibitor (TKI)-sensitive PC9 and -resistant PC9-IR cells, respectively. The multi-lectin approach allowed complementary profiling of the glycoproteome of the NSCLCs with only 5 common glycopeptides identified among > 20000 non-redundant intact glycopeptides from the three nanoprobes. By the advantages of high lectin density and methoxy ethylene glycol (MEG)-protection to minimize non-specific binding, glycotope enrichment specificity of 79% and 62% were achieved using MNP@AAL and MNP@SNA, respectively. Our results also show elevated site-specific terminal fucosylation and core fucosylation of various glycoproteins in TKI-resistant PC9-IR cells. Without immuno-precipitation, the sensitivity of our approach allows identification of 50 glycopeptides from 10 out of 12 potential N-glycosylation sites from the therapeutic target EGFR as well as increased terminal fucosylation and α1,6-fucosylation and sialylated structures at N-175, 413, 444 sites of EGFR in the resistant PC9-IR cells. In the second part, we pursued site-specific quantitation to compare the glycoproteome profiles of PC9-IR and PC9 cells using MNP@AAL. Label-free quantitation revealed predominant fucosylation in PC9-IR cells, suggesting the potential role of fucosylation associated with NSCLC resistance. A total of 914 glycopeptides (10-fold: 373 glycopeptides, 5-fold: 190 glycopeptides, 2-fold: 351 glycopeptides) were upregulated in PC9-IR cells, whereas 272 glycopeptides (10-fold: 72 glycopeptides, 5-fold: 71 glycopeptides, 2-fold:129 glycopeptides) have higher abundance in PC9 cells. Proteins including MHCs, lysosomal membrane proteins, and the oncogene receptor EGFR have been identified with significantly upregulated fucosylation in PC9-IR cell. We have also identified site-specific alterations, particularly in the case of ALCAM 1, where glycans with potential terminal bisecting HexNAc were increased in PC9-IR cells. Our results not only demonstrated a sensitive approach to analyze the vastly under-represented N-glycoproteome but also may reveal a glycoproteomic atlas to further explore the site-specific function of glycoproteins associated with drug resistance in NSCLC. This may lead us to select targeted glycoprotein for further structural validation, functional analysis, and biological examinations. For the technical advancement, the developed methodology can be generally applied to other types of samples.