Investigation into the feasibility of subcellular proteomics

这项研究的主要目的是利用纳米操纵器控制的提取质谱学方法来研究亚细胞蛋白质组学(蛋白质分析)的可能性。现有的蛋白质组质谱技术通常涉及细胞/细胞器的混合。分析单个细胞或单个细胞器的优点是，特别是它允许观察相同功能的不同单个细胞的异质性，或相同功能的不同单个细胞器的异质性。这在疾病诊断和监测中具有潜在的应用。我们希望通过纳米操纵器控制的提取技术实现亚细胞分析，例如直接分析探针纳米提取(DAPNe)。DAPNe利用安装在显微镜载玻片上的纳米操纵器单元。毛细管尖端固定器连接到纳米操纵器单元，压力注射器连接到毛细管尖端固定器。显微镜被用来寻找一个离散的感兴趣区域，纳米操纵器被用来在这个离散的感兴趣区域中定位毛细血管尖端。毛细管尖端的内径为1微米，填充有大约10微升的溶剂。压力注射器允许将溶剂注入到样品表面或内部，并且所得到的溶剂化样品被吸入回毛细管末端。然后，毛细管尖可以被转移到纳米电喷雾电离源(NanoES，NSI)以直接引入质谱仪，或者溶剂化样品可以被分配到井或小瓶中，并在被引入质谱仪之前进行液相色谱(LC)分离。此外，研究的目的是：优化蛋白质的提取条件(已经使用该技术成功地提取了用于类脂组学(脂质分析)的蛋白质，和代谢组学(代谢物分析)。-在不牺牲灵敏度的情况下进一步减少提取的规模。-评估NSI和LC的好处以及这两种样品引入方法如何适用。-比较DAPNe等技术与成熟的蛋白质组学技术的有效性。

The main aim of this research is to investigate the potential for subcellular proteomics (protein analysis) by using nanomanipulator-controlled extraction mass spectrometry methods.Existing proteomic mass spectrometry techniques generally involve the pooling of cells/organelles. The advantage of analysis of single cells or single organelles is that, in particular, it allows observation of heterogeneity of different single cells of the same function, or of different single organelles of the same function. This has potential uses in disease diagnostics and monitoring.We hope to achieve subcellular analysis through nanomanipulator-controlled extraction techniques such as Direct Analyte Probed Nanoextraction (DAPNe). DAPNe utilises a nanomanipulator unit mounted on a microscope slide. A capillary tip holder is attached to the nanomanipulator unit, and a pressure injector is attached to the capillary tip holder. The microscope is used to find a discrete region of interest, and the nanomanipulator is used to position the capillary tip in this discrete region of interest. The capillary tip has a 1 um internal diameter and is filled with approximately 10 uL of solvent. The pressure injector allows for the solvent to be injected either onto the surface, or inside a sample, and the resulting solvated sample is aspirated back into the capillary tip.The capillary tip can then be transferred to a nano-electrospray ionisation source (NanoES, NSI) for direct introduction to the mass spectrometer, or the solvated sample can be dispensed into a well or a vial and subjected to liquid chromatograpy (LC) separation before being introduced to the mass spectrometer.Further objectives of the research are to:- Optimise the extraction conditions for proteins (successful extractions have been carried out using this technique for lipidomics (lipid analysis), and metabolomics (metabolite analysis)).- Further reduce the dimensions of extraction without trading off with sensitivity.- Evaluate the benefits of both NSI and LC and how both sample introduction methods are applicable.- Compare the efficacy of techniques such as DAPNe against well-established proteomic techniques.