METHODS OF IONIZATION IN MASS SPECTROSCOPY

质谱中的电离方法

• 批准号: 6432768
• 负责人: SANFORD P MARKEY
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF MENTAL HEALTH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6432768
• 关键词: aminoacid metabolism; anandamide; biomarker; biomedical equipment development; brain metabolism; eicosanoid metabolism; electrospray ionization mass spectrometry; ion cyclotron resonance spectrometry; kynurenine; liquid chromatography; mass spectrometry; method development; nitric oxide; trace elements; tryptophan

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.

在蛋白质组学的独立和协作研究项目中，正在改善，应用和测试蛋白质结构的分析方法。 色谱系统，即在线液态色谱和基于凝胶的液体色谱系统。关于鉴定翻译后修饰或独特基因产品的协作研究正在进行中。正在设计用于选择性隔离和表征低分子量蛋白的方法，通常无法通过基于凝胶的方法检测到。针对目标功能蛋白质组学的新方法已设计并正在测试中。在模型肽中已经验证了丝氨酸或苏氨酸残基磷酸化变化的定量检测策略。磷酸磷酸磷酸的β-脱肽之后是硫乙酰化。水解后，半胱氨酸硫与可裂解的可裂解标签反应，该标签促进了先前含有磷酸碱的肽家族的分离。通过将一个实验组的蛋白混合物与具有非脱氧试剂的对照组的氘代生物素化的TAG和蛋白质反应，可以将肽和区分肽与处理过的对照细胞区分开。 相等数量的肽的混合可以通过光谱法测量比率，并从家政蛋白与肽之间的肽之间进行区分，而与细胞事件相对于新形成或修饰的蛋白质。亲和力标记的肽可以有效地与总肽质量分离，然后通过Maldi/TOF/MS或LC/MS进行分析。液态染色体学 - 电喷雾质谱法已应用于13C标记的NAD作为13C- tryptophan代谢的产物的定量分析。为毛细管电泳设计了改进的样品引入和浓度方法。 将表面涂层的磁珠洗净成柱，磁捕获，并施加样品。 肽粘在珠涂层上，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。