Investigation into the feasibility of subcellular proteomics

本研究的主要目的是探讨利用纳米操纵器控制的提取质谱方法进行亚细胞蛋白质组学（蛋白质分析）的潜力。现有的蛋白质组学质谱技术通常涉及细胞/细胞器的合并。分析单细胞或单个细胞器的优点在于，特别地，其允许观察具有相同功能的不同单细胞或具有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方面具有潜在的用途。我们希望通过纳米操纵器控制的提取技术，如直接分析物探针纳米提取（DAPNe），实现亚细胞分析。DAPNe利用安装在显微镜载玻片上的纳米操纵器单元。毛细管尖端保持器附接到纳米操纵器单元，并且压力注射器附接到毛细管尖端保持器。显微镜用于找到感兴趣的离散区域，并且纳米操纵器用于将毛细管尖端定位在该感兴趣的离散区域中。毛细管尖端具有1 μ m内径，并且填充有约10 μ L溶剂。压力进样器可将溶剂注入样品表面或样品内部，溶剂化后的样品被吸回毛细管尖端，然后可将毛细管尖端转移至纳米电喷雾电离源（NanoES，NSI）直接导入质谱仪，或将溶剂化的样品分配到孔或小瓶中，并在引入质谱仪之前进行液相色谱（LC）分离。研究的进一步目标是：- 优化蛋白质的提取条件（使用该技术已成功进行了脂质组学（脂质分析）和代谢组学（代谢物分析）的提取）。进一步减少提取的维度，而不会牺牲敏感性。评估NSI和LC的优点，以及两种进样方法的适用性。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.