Development and Applications of Negative Ion Mode nanoLC−MS/MS-Based Sulfoglycomics
| Autor: | Chu-Wen Cheng, 鄭筑文 |
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| Rok vydání: | 2015 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 103 Mass spectrometry (MS)-based glycomics aims to comprehensively map all the glycan constituents of a cell or tissue under a particular patho-physiological state. Sulfated glycans are known to participate in many important biological processes but often under-detected or represented in MS-based glycomics due to their lower abundance and ionization problems. Thus, in my study, we aim to extend and complement our MALDI-MS and MS/MS-based sulfoglycomic workflow with advanced nanoLC-MS/MS-based strategy to improve the detection limit. By the anion-exchange solid-phase extraction method established previously in our laboratory, permethylated sulfated glycans could be efficiently extracted from the total pool of glycans. After applying the strategy to the cultured human bronchial epithelial cells, we now showed that these can be analyzed by nanoLC-ESI-MS/MS in negative ion mode, which is more conducive than MALDI-MS for sensitive detection of doubly charged di-sulfated glycans. The additional coupling of nanoLC separation prior to MS improves the detection sensitivity and handling capacity for comprehensive analysis, therefore, more MS/MS spectra could be obtained. We have now systematically investigated the most useful fragmentation characteristics afforded by higher energy collision dissociation (HCD) versus ion trap CID, based on a panel of synthetic standards and a complex pool of sulfated glycans derived from cultured human bronchial epithelial cells. We show that an efficient mapping of various isomeric fucosylated, sialylated, sulfated glycotopes by negative ion mode nanoLC-MS/MS analysis of permethylated glycans can benefit from data dependent parallel acquisition of both HCD and ion trap CID MS2, supplemented further by a product ion dependent MS3 scan function, and how the generated data can be productively utilized. The manually verified dataset of over hundred glycan entries, each represented by a distinct m/z value and hence glycosyl composition, additionally serves to guide current development of much needed computational tool for sulfoglycomic data analysis and presentation. When applied to both mouse and human eosinophils, we could observe novel sulfated glycans otherwise not detected by MALDI-based strategy, and additionally identified the occurrence of sulfate on terminal sialic acid. Therefore, we further utilized glycolipid derived from sea urchin, which is already known to contain sulfate at C8 position of NeuNAc and NeuGc to establish the characteristic ion of sulfated sialic acid on mass spectrometry. Moreover, in order to map the occurrence of sulfated sialic acid on the surface of cells in different tissues, series of cell line were applied to do the glycomic analysis. In addition, to evaluate the ratio of non-sulfated versus sulfated glycans, we have successfully adapted the well-established GC-MS linkage analysis method of partially methylated alditol acetate (PMAA) to targeted LC-MS/MS analysis in positive ion mode and quantified the ratio among non-sulfated, mono-sulfated, and di-sulfated glycans in bovine thyroglobulin as 82:13:8. In conclusion, by the newly developed nanoLC-MS/MS strategies, we can now obtain more qualitative and quantitative information of the sulfated glycome at higher sensitivity, which would be important in understanding their functional expression and physiological relevance in biological systems. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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