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: 7957858
• 负责人: Michael MacCoss
• 金额: $ 2.77万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7957858
• 关键词: Biology; Cells; Charge; Complex Mixtures; Computer Retrieval of Information on Scientific Projects Database; Computer software; Coupled; Data; Data Set; Devices; Financial compensation; Funding; Fungal Genome; 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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 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法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，但代价是金钱和仪器复杂性（例如 FT-ICR MS）。 在这里，我们描述了使用 FAIMS（场不对称离子淌度光谱法）作为一种快速气相分离技术，与台式离子阱质谱仪连接，以提高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，离子根据其在不对称 RF 场中的微分离子迁移率进行分离。 然后通过施加“补偿电压”(CV) 选择离子。 我们在实验中展示了 FAIMS 在蛋白质组学中的应用，如下所示。 使用胰蛋白酶将全细胞酵母裂解物的可溶性部分消化成肽，并使用 LC 和与 LTQ 离子阱质谱仪耦合的原型 FAIMS 装置进行分析，还在自制的 FAIMS 系统上进行了初步实验。 我们使用 90 分钟反相梯度（使用或不使用 FAIMS）获取了多个数据集。 对于 FAIMS-MS 实验，由一次质荷扫描组成的实验周期以 8-12 个 CV 值进行，跨越了 CV 有用值的范围。 对于 FAIMS-MS/MS 实验，循环包括一次 MS 扫描，然后是两次 MS/MS 扫描，采用 5 个不同的 CV 值。 对于非 FAIMS 实验，周期包括一次 MS 扫描，然后是四次 MS/MS 扫描。 所有数据均使用 SEQUEST 和内部分析软件进行后处理。