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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 蛋白质组学研究中对复杂混合物的分析受到分析技术总体峰容量的限制。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多肽。另一种解析更多多肽的方法是使用超高分辨率质谱仪，但成本较高，仪器复杂(例如FT-ICR MS)。在这里，我们描述了FAIMS(场不对称离子迁移率光谱)作为一种快速气相分离技术的使用，该技术与台式离子陷阱质谱仪相连接，可在不损失速度或收入的情况下提高蛋白质组学分析的峰容量。在FAIMS中，根据离子在非对称射频场中的不同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。 我们在以下实验中展示了FAIMS在蛋白质组学中的应用。全细胞酵母裂解物的可溶性部分用胰酶消化成多肽，用LC和连接LTQ离子陷阱质谱仪的FAIMS原型装置进行分析，并在自制的FAIMS系统上进行了初步实验。我们使用90分钟的反向相位梯度获得了几个数据集，其中有FAIMS和没有FAIMS。对于FAIMS-MS实验，在8-12个CV值下执行一个实验周期，包括一个质量-电荷扫描，跨越CV的有用值范围。对于FAIMS-MS/MS实验，周期由一次MS扫描和两次MS/MS扫描组成，CV值为5个不同值。对于非FAIMS实验，周期由一次MS扫描和四次MS/MS扫描组成。所有数据均使用SEQUEST和内部分析软件进行后处理。

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.