GAS-PHASE MUDPIT:?LC-FAIMS-MS/MS FOR PROTEOMICS ON AN ION TRAP MASS SPECTROMETER

气相 MUDPIT：离子阱质谱仪上的蛋白质组学 LC-FAIMS-MS/MS

• 批准号: 8171316
• 负责人: Michael MacCoss
• 金额: $ 0.67万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8171316
• 关键词: Cells; Charge; Complex Mixtures; Computer Retrieval of Information on Scientific Projects Database; Computer software; Coupled; Data; Data Set; Devices; Financial compensation; Funding; Gases; Grant; Housing; Institution; Ions; Peptides; Phase; Process; Proteomics; Research; Research Personnel; Resources; Scanning; Source; Spectrometry; Speed; System; Techniques; Trypsin; United States National Institutes of Health; Yeasts; base; cost; instrument; ion mobility; mass spectrometer; prototype; research study; ultra high resolution; voltage

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The analysis of complex mixtures in proteomics studies is limited by the overall peak capacity of the analysis technique. One way of increasing the peak capacity is by separating peptides prior to the mass spectrometer using long gradients or multidimensional separations. Another way to resolve more peptides is to use an ultra high-resolution mass spectrometer, at a cost of money and instrument complexity (e.g. FT-ICR MS). Here we describe the use of FAIMS (Field Asymmetric Ion Mobility Spectrometry) as a fast gas-phase separation technique interfaced with a bench-top ion trap mass spectrometer for increasing the peak capacity of proteomics analyses without losses in speed or revenue. In FAIMS, ions are separated based on their differential ion mobility in an asymmetric RF field. Ions are then selected by the application of a "compensation voltage" (CV). We have demonstrated the application of FAIMS to proteomics in experiments as follows. The soluble fraction of a whole-cell yeast lysate was digested to peptides using trypsin and analyzed using ?LC and a prototype FAIMS device coupled to an LTQ ion trap mass spectrometer, with preliminary experiments also performed on a homebuilt FAIMS system. We acquired several datasets using a 90 minute reversed phase gradient, with and without FAIMS. For FAIMS-MS experiments, an experimental cycle consisting of one mass-to-charge scan was performed at 8-12 values of CV, spanning the range of useful values of CV. For FAIMS-MS/MS experiments, the cycle consisted of one MS scan followed by two MS/MS scans, at 5 different values of CV. For non-FAIMS experiments, the cycle consisted of one MS scan followed by four MS/MS scans. All data were post-processed using SEQUEST and in-house analysis software.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 蛋白质组学研究中复杂混合物的分析受到分析技术的总体峰容量的限制。 增加峰容量的一种方式是在质谱仪之前使用长梯度或多维分离来分离肽。 解析更多肽的另一种方法是使用超高分辨率质谱仪，这需要花费大量资金和复杂的仪器（例如FT-ICR MS）。 在这里，我们描述了使用FAIMS（场不对称离子迁移谱）作为一种快速的气相分离技术与台式离子阱质谱仪接口，用于增加蛋白质组学分析的峰值容量，而不会损失速度或收入。 在FAIMS中，基于离子在非对称RF场中的微分离子迁移率来分离离子。 然后通过施加“补偿电压”（CV）来选择离子。 我们已经证明了FAIMS在蛋白质组学实验中的应用如下。 用胰蛋白酶消化全细胞酵母裂解物的可溶性部分的肽，并使用？LC和原型FAIMS设备耦合到LTQ离子阱质谱仪，初步实验也进行了自制FAIMS系统。 我们使用90分钟反相梯度采集了几个数据集，有和没有FAIMS。 对于FAIMS-MS实验，在8-12个CV值下进行由一次质荷扫描组成的实验循环，跨越CV的有用值的范围。 对于FAIMS-MS/MS实验，循环由一次MS扫描和随后的两次MS/MS扫描组成，在5个不同的CV值下。 对于非FAIMS实验，循环由一次MS扫描和随后的四次MS/MS扫描组成。 使用SEQUEST和内部分析软件对所有数据进行后处理。