MALDI SENSITIVITY ENHANCEMENT & OPTIMIZATION W/ UV & IR LASERS

MALDI 敏感性增强

• 批准号: 6120205
• 负责人: ALMA L BURLINGAME
• 金额: $ 0.66万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-03-01 至 2000-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6120205
• 关键词: biomedical resource; spectrometry; structural biology; technology /technique

Currently, the UCSF Mass Spectrometry Facility is equipped with two PerSeptive Biosystems Voyager and one AutoSpec MALDI instruments. Optimization of methods of sample preparation and laser configuration would enable the most efficient and productive use of the available instruments. Sensitivity enhancement would improve the data quality for researchers. Comparison of the two Voyager instruments has established equivalent sensitivity for both in linear and reflectron modes using standard dried droplet matrix preparation on a cze standard with chca matrix. The minimum requirement for detection with this method is from 10 to 100 fmol. To improve upon these limits, a protocol involving nitrocullulose/chca mixtures is being tested and has been demonstrated to allow detection at low fmol levels, with reproducibility in question at lower levels. Increased laser focus has been tested without improvement, but needs to be tested in conjunctuion with the nitrocullulose/chca sample preparation techniques. Usefulness for lab samples such as in gel digests will be evaluated once clear improvements on standards have been demonstrated. A second area of development is the use of alternative matrices and comparison of the UV and IR laser for MALDI. IR laser in conjunction with suitable matrices has been shown to reduce metastable decay of sialated and phosphorylated peptides. However, difficulties with laser stability and sample crystallization impede widespread use of the technique. Thus, development of appropriate sample preparation methods and optimal laser settings on the Voyager DE-STR IR system will be continued. Samples currently of interest to the group will take priority, such as sulfated peptides.

目前，UCSF质谱设备配备了 两台PerSeptive Biosystems Voyager和一台AutoSpec MALDI仪器。 样品制备方法和激光器配置的优化 能够最有效和最富有成效地利用现有的 仪器. 提高敏感度将提高数据质量 为研究人员。 两个旅行者仪器的比较 建立了线性和反射器的等效灵敏度 在CZE上使用标准干燥液滴基质制备的模式 标准CHCA矩阵。 检测的最低要求 该方法为10 - 100 μ mol。 为了改善这些限制， 正在测试涉及硝化纤维素/氯氰尿酸混合物的方案， 已经证明允许在低fmol水平下检测， 在较低水平上的再现性问题。 增强激光聚焦 已经过测试，没有改进，但需要在 与硝化纤维素/CHCA样品制备液结合 技术. 实验室样品（如凝胶电泳）的灵敏度将 一旦在标准方面有明显的改进，就进行评价。 第二个发展领域是使用替代矩阵， 用于MALDI的紫外和红外激光的比较。 红外激光器 用合适的基质已经显示出减少亚稳态衰减 唾液酸化和磷酸化肽。 然而， 激光稳定性和样品结晶阻碍了 技术。 因此，开发适当的样品制备 旅行者DE-STR红外系统的方法和最佳激光设置 将继续进行。 小组目前感兴趣的样品将 优先考虑，如硫酸化肽。