在分子水平上直接表征人类细胞系的电喷雾萃取电离质谱研究

Isolated human cell lines are important in vitro models in biomedical studies. There is currently a lack of analytical methods that allow rapid and simple characterization of growing cell cultures with high information depth. Here the development of analytical platforms is proposed that will offer direct metabolic phenotyping of growing cell lines at the molecular level. Metabolites from incubated cultures will be sampled by extractive electrospray ionization (EESI) interface and analyzed with mass spectrometry (MS). The approach is featured by the high speed of analysis and simplicity of operation, absence of sample preparation, noninvasive sampling, and the possibility to profile metabolic changes in real time. The method is versatile, enabling sampling of both volatile and nonvolatile metabolites from cells grown in liquid as well as in gel media. MS detection can be performed on any type of mass spectrometer with atmospheric ionization. Proposed research activities are presented in time sequence of their implementation. Major steps include the design and construction of experimental platforms for cell analysis with EESI-MS, characterization and optimization of analytical performance, and model studies with human cell lines. Construction of experimental setups will largely rely on resources available in the Key Laboratory for Mass Spectrometry and Instrumentation at East China Institute of Technology. The major instrument building blocks will include a mass spectrometer with high sensitivity and molecular range of detection (Orbitrap-MS and LTQ-MS), EESI ionization source built in house, cell incubator and sampling interfaces. With model human cell and metabolite systems, analytical performance will be tested with regard to sensitivity, molecular range and reproducibility of detection. The major experimental variables will include sampling geometry, ionization parameters, neutral gas flow, chemical composition, temperature, liquid/headspace volume, and medium chemical composition. Model experiments will emphasize potential of the method for the reliable classification of cell lines and its sensitivity to metabolic changes in real time. Specifically, an array of closely related cancer cell lines (e.g., melanoma cancers) will be screened by EESI-MS. Cell lines will be differentiated based on characteristic patterns in produced spectra together with statistical analysis if needed. Change in the metabolic profile of a cancer cell line will be probed in response to therapy treatment (nutrient deprivation) or deliberate chemical contamination (with HeLa cells). The proposed project will be accomplished within a timeframe of three years. In perspective, EESI-MS analysis of cell lines in vitro can benefit research in very different areas, including but not limited to the analysis of metabolic pathways and pathology mechanisms, drug/therapy development, clinical diagnostics, and cell line authentication.

人类细胞系分析在生命科学及医药研究中具有重要作用。因此，开发在分子水平上对细胞进行直接分析的质谱学新方法具有积极意义。在前期研究的基础上，本项目拟着重研究适宜于细胞分析的中性解吸采样（ND）及其与电喷雾萃取电离质谱（EESI-MS）直接分析的联用技术，研制与细胞培养、直接采样及质谱分析相适应的仪器装置，构建适合于直接获取细胞挥发性、非挥发性物质深度分子信息的中性解吸电喷雾萃取电离质谱（ND-EESI-MS）研究平台，采用人类细胞系模型对ND-EESI-MS平台进行表征，并优化ND-EESI-MS直接分析细胞系的实验条件及参数，从而获得最佳分析性能，创建一种直接快速获得细胞代谢产物分子信息的新型质谱分析方法。本项研究将拓展ND-EESI-MS在细胞直接分析中的应用，为深入研究细胞生命活动规律的化学基础、探讨其癌变的分子机制提供实验基础，在生物医药方面具有重要的应用前景。

人类细胞系分析在生命科学及医药研究中具有重要作用。因此，开发在分子水平上对细胞进行直接分析的质谱学新方法具有重要意义。本项目采用适宜于微生物细胞代谢产物分析的顶空采样及其与常压电离质谱（包括常压化学电离APCI、常压萃取化学电离EAPCI、电喷雾萃取电离EESI）直接分析的联用技术，研制与细胞培养、直接采样及质谱分析相适应的仪器装置，构建适合于直接获取细胞挥发性、非挥发性物质深度分子信息的直接电离质谱研究平台，采用人类细胞系模型对平台进行表征，并优化直接分析细胞系的实验条件及参数，从而获得最佳分析性能，创建一种直接快速获得细胞代谢产物分子信息的新型质谱分析方法。在本项目的支持下，发表SCI收录论文6篇；获国家授权专利1件，申请发明1件；达到项目考核指标。具体内容如下：.1.在前期DAPCI-MS相关研究的基础上，以创新思维自行设计、制作了一种简便的电晕放电电离装置，能够满足微生物细胞挥发性代谢产物的直接质谱分析需求，并采用不同的进样方式考察了该装置平台的稳定性与电离机理的研究。.2.以电晕放电电离技术实现了临床上常见致病菌挥发性代谢产物的在线监测分析，对其质谱指纹谱图进行了分析，通过其生物标志物进行快速分析鉴别。该方法灵敏性高特异性强，有望应用于临床微生物的快速识别鉴定。.3.采用电晕放电电离装置直接分析了临床上实际样品（血液、尿液、唾液、呼出气体等），对实际样品微生物细胞的感染进行快速分析诊断，与临床传统方法比较具有较高的可靠性，大大缩短了对疾病的确诊时间，有助于指导医生用药和减轻患者病痛。该方法有望在实际临床分析诊断得到更大推广与应用。.4. 以不同铀矿区的耐强辐射微生物嗜酸性氧化亚铁硫杆菌为研究对象，考察了三种离子化方式：常压化学电离（APCI）、常压萃取化学电离（EAPCI）、电喷雾萃取电离（EESI）对微生物挥发性化合物的检测，以及顶空采样（Headspace Sampling）、界面采样（Interface Sampling）和中性解吸采样（ND Sampling）三种进样方式对电离效果的影响。有望实现在生物冶金上的不同矿区菌种快速分析鉴定。