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。色谱系统，在线液相色谱仪和凝胶色谱仪正在测试中。合作研究已经开始鉴定从人类细胞分离的DNA复制复合体中的蛋白质。这种被称为合成体的核多蛋白复合物就像一台合成或复制DNA的机器。合作者从粗提取物中纯化了5200倍的完整功能DNA复制复合体，并使用Western blotting鉴定了许多复制蛋白。DNA聚合酶α和γ， DNA引物酶，RF-C，以及易于提取的DNA连接酶I和拓扑异构酶II，被发现专门与人类细胞来源的DNA合成组共同纯化。凝胶、免疫亲和和柱层析分离被用于结合电喷雾和基质辅助激光解吸电离离子阱和四极杆/飞行时间质谱来表征合成体中的复杂蛋白质混合物。必须设计和采用额外的分离技术，因为许多蛋白质与哺乳动物合成体复合物共同纯化。恶性细胞中PCNA特有的翻译后结构变化需要完整的初级结构表征，而合成体复合体中的蛋白质鉴定可能需要通过质谱法识别少量的色氨酸片段。另外三个分离项目正在进行中。首先，电泳方法正在被测试用于选择性分离和表征低分子量蛋白质，这些蛋白质通常是通过凝胶方法检测不到的。在第二项研究中，通过二维凝胶蛋白分离和凝胶内胰蛋白酶消化，然后进行液相色谱/质谱分析，进行全细胞匀浆蛋白质组分析。这些结果将与蛋白质鉴定和序列覆盖范围进行比较，这些蛋白质鉴定和序列覆盖范围使用新开发的串联液相色谱分离色氨酸片段与自动质谱检测。第三项研究改进了毛细管区带电泳的进样和浓缩方法。表面涂覆的磁珠被洗涤到一个柱中，被磁捕获，然后应用样品。多肽粘附在珠状涂层上，允许稀释样品富集，从而增强了毛细管电泳对稀释溶液的适用性。

Methods for the analysis of protein structures are being improved, applied and tested in independent and collaborative research projects in proteomics. Chromatographic systems, both on-line liquid chromatographic and gel-based are being tested. Collaborative studies have been initiated on the identification of proteins in a DNA replication complex isolated from human cells. This nuclear multi-protein complex known as the synthesome acts as a machine that synthesizes or replicates DNA. Collaborators have purified 5200-fold an intact and functional DNA replication complex from crude extracts and identified a number of the replication proteins using Western blotting. DNA polymerases alpha and gamma, DNA primase, RF-C, and the readily extractable forms of DNA ligase I and topoisomerase II were found to exclusively co-purify with the human cell-derived DNA synthesome. Gel, immunoaffinity and column chromatographic separations are being used in combination with electrospray and matrix assisted laser desorption ionization with ion trap and quadrupole/time-of-flight mass spectrometers to characterize the complex protein mixture in the synthesome. Additional separation techniques must be devised and employed because many proteins co-purify with the mammalian synthesome complex. Specific post-translational structural changes characteristic of PCNA in malignant cells requires complete primary structure characterization, whereas identification of proteins in the synthesome complex may proceed from mass spectrometric recognition of small numbers of tryptic fragments. Three additional separation projects are in progress. In the first, electrophoretic methods are being tested for the selective isolation and characterization of low molecular weight proteins not usually detected by gel based methods. In a second study, total cell homogenate proteome analyses are being performed by two-dimensional gel protein separation and in-gel tryptic digestion followed by liquid chromatography/mass spectrometry. These results are being compared with protein identifications and sequence coverage attained using a newly developed tandem liquid chromatography separation of tryptic fragments with automated mass spectrometric detection. A third study has improved methods of sample introduction and concentration for capillary zone electrophoresis. Magnetic beads that have been surface coated are washed into a column, magnetically trapped, and the sample applied. Peptides stick to the bead coating, permitting enrichment of dilute samples, consequently enhancing the applicability of capillary electrophoresis to dilute solutions.