METHODS OF IONIZATION IN MASS SPECTROSCOPY

蛋白质结构的分析方法正在得到改进，并在蛋白质组学的独立和合作研究项目中得到应用和测试。正在测试在线液相色谱和基于凝胶的层析系统。识别翻译后修饰或独特基因产物的合作研究正在进行中。正在设计的方法用于选择性地分离和表征通常不能用基于凝胶的方法检测到的低分子蛋白质。靶向功能蛋白质组学的新方法已经设计出来，并正在进行测试。定量检测丝氨酸或苏氨酸残基磷酸化变化的策略已在模型多肽中得到验证。磷酸丝氨酸的β-消除反应之后是硫代乙酰化反应。在水解后，半胱氨酸硫与生物素化的可切割标签反应，这有助于分离先前含有磷丝氨酸的多肽家族。通过将来自一个试验组的蛋白质混合物与来自非氚试剂组的氚生物素化标签和来自对照组的蛋白质反应，可以对来自处理细胞和对照细胞的多肽进行质量标记和区分。等量的多肽混合后，可以用质谱仪测量比率，并根据细胞事件区分来自管家蛋白质的多肽和新形成或修饰的多肽。亲和标记的多肽可以有效地从总多肽中分离出来，然后用MALDI/TOF/MS或LC/MS进行分析。采用液-色-电喷雾-质谱法对13C-色氨酸代谢产物13C标记的NAD进行了定量分析。改进了毛细管区带电泳法的进样和浓缩方法。经过表面涂层的磁珠被洗入柱中，磁性被捕获，然后施加样品。多肽附着在珠涂层上，允许稀释样品的浓缩，从而增强了毛细管电泳法对稀溶液的适用性。

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 on the identification of post-translational modifications or unique gene products are in progress. Methods are being devised for the selective isolation and characterization of low molecular weight proteins not usually detected by gel based methods. New methods for targeted functional proteomics have been devised and are being tested. Strategies for the quantitative detection of changes in phosphorylation of serine or threonine residues have been verified in model peptides. Beta-elimination of phosphoric acid from phosphoserine is followed by thioacetylation. After hydrolysis, the cysteine sulfur reacts with a biotinylated cleavable tag which facilitates separation of the family of peptides that previously contained phosphoserine. By reacting a protein mixture from one experimental group with deuterated biotinylated tag and proteins from a control group with non-deuterated reagent, it is possible to mass label and distinguish peptides from treated vs. control cells. Admixture of equal quantities of peptides allows a ratios to be measured mass spectrometrically and distinctions to be made between peptides from housekeeping proteins vs. those newly formed or modified with respect to cellular events. The affinity tagged peptides can be effectively separated from the total peptide mass and then analyzed by MALDI/TOF/MS or LC/MS. Liquid-chromatography-electrospray mass spectrometry methods have been applied for the quantitative analyses of 13C-labeled NAD as product of 13C-tryptophan metabolism. Improved methods of sample introduction and concentration have been devised 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.