Methods Of Ionization In Mass Spectroscopy

蛋白质组学的独立和合作研究项目正在改进、应用和测试蛋白质结构分析方法。设计并实现了一种自动化的二维液相色谱系统，用于电喷雾质谱分析复杂的多肽混合物。该系统使用强阳离子交换分馏成六个陷阱，然后在单个反相柱上梯度洗脱。该系统已经用酵母的可溶性蛋白质进行了测试，并能够在所得的复合提取物中识别数百种蛋白质。从人类细胞分离的DNA多蛋白复制复合体中鉴定核蛋白的合作研究一直在继续。合作者从细胞提取物中分离出完整和功能的DNA复制复合体。我们已经确认了11种已知的复制蛋白的存在，但需要进一步的分离步骤来减少蛋白质背景。二维液相色谱与各种蛋白水解酶已被使用。许多蛋白质与哺乳动物合成体复合物共纯化，bbb200已被确定。合成体复合体的一个成分是PCNA。恶性细胞的PCNA似乎有特定的翻译后结构变化。分离PCNA的方法正在研究中，包括微尺度溶液等电聚焦。

Methods for the analysis of protein structures are being improved, applied and tested in independent and collaborative research projects in proteomics. An automated two dimensional liquid chromatographic system has been designed and implemented for the analyses of complex mixtures of peptides using electrospray ionization mass spectrometry. The system fractionates using strong cation exchange into six traps, followed by gradient elution on a single reverse phase column. The system has been tested with soluble proteins from yeast and is capable of identifying hundreds of proteins in the resulting complex extract. Collaborative studies have been continued on the identification of nuclear proteins in a DNA multi-protein replication complex isolated from human cells. Collaborators isolate an intact and functional DNA replication complex from cell extracts. We have confirmed the presence of 11 known replication proteins but further separation steps are required to reduce protein background. Two dimensional liquid chromatography with various proteolytic enzymes have been used. Many proteins co-purify with the mammalian synthesome complex, and >200 have been identified. One component of the synthesome complex is PCNA. There appear to be specific post-translational structural changes in PCNA characteristic of malignant cells. Methods for the isolation of PCNA are being pursued, including micro scale solution isoelectric focusing.