Using trapped ion mobility to synchronize ions in data-independent acquisition (DIA) mass spectrometry

质谱（MS）已广泛用于蛋白质组学分析复杂的样品，如临床或环境样品以及全细胞裂解物。具体来说，在自下而上的蛋白质组学中，从生物样品中提取蛋白质，并在使用液相色谱-串联质谱（LC-MS/MS）在线系统进行分析之前进行酶裂解。******在基于LC-MS/MS的自下而上的蛋白质组学中，最先进的质谱仪有效地将离子转移到真空中，但仅利用离子束的一小部分进行质量分析。在传统的数据依赖采集（data-dependent acquisition， DDA）中，只选择一个前体离子进行碎片化，因此在2000 Da以下的质量范围内，可以有效滤除99.9%以上的质量。为了比DDA方法更全面和可重复性地采样碎片离子，我们最近开发了使用数据独立采集（DIA）的SWATH-MS。然而，SWATH-MS方法也采用相对较小的质量隔离窗口来增加特异性（例如25 m/z），因此在典型的占空比内利用不到1.25%的所有可用离子。因此，虽然DIA提高了再现性和灵敏度，但典型的DIA方法（如SWATH-MS）仍然具有相对较低的离子束效率，98%以上的离子永远无法到达质谱分析仪。然而，原则上，100%的占空比（直到TOF分析仪）可以通过在分析的精确时间释放离子的捕获装置来实现。我们的合作者已经在“并行积累串行碎片”（DDA- pasef）方法中实现了这一原理。******在这里，我们提出实现一种称为DIA- pasef的新方法，使用数据无关的采集，以增加DIA方法的离子使用率，从而进一步提高其灵敏度和吞吐量。这将在以下三个目标中实现：******目标1：计算模拟和实现一种新的获取方案，该方案利用DIA-PASEF的离子迁移率和m/z之间的耦合。******目标2：为DIA-PASEF数据制定有针对性的分析策略，以便对四维数据空间（信号强度、保留时间、离子迁移率和m/z）进行分析。******目标3：开发对DIA-PASEF数据进行无偏分析的无目标方法。******这项研究的潜在影响是对MS的重要改进，MS是研究中使用的核心分析技术，通过我们对占空比的改进，有可能提高灵敏度、分析速度和准确性。本研究的预期结果在于改进了对电离分子及其片段进行全面采样的方法。其影响范围将从实验室条件下生物系统分子行为的基础研究到临床和药理学研究的应用领域，在这些领域中，质谱可以用于直接分析临床样本，如组织和血浆。

Mass spectrometry (MS) has been used extensively in proteomics for the analysis of complex samples, such as clinical or environmental samples as well as whole cell lysates. Specifically, in bottom-up proteomics, proteins are extracted from a biological sample and enzymatically cleaved before analysis using an on-line system of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS).******In LC-MS/MS based bottom up proteomics, state-of-the-art mass spectrometers efficiently transfer ions into the vacuum, but make use of only a small fraction of the ion beam for mass analysis. In traditional data-dependent acquisition (DDA), only a single precursor ion is selected for fragmentation, thus effectively filtering out over 99.9% of the mass range up to 2000 Da. To sample fragment ions more comprehensively and reproducibly than DDA methods, we have recently developed SWATH-MS which uses data-independent acquisition (DIA). However, also SWATH-MS methods employ relatively small mass isolation windows to increase specificity (e.g. 25 m/z) and thus utilize less than 1.25% of all available ions within a typical duty cycle. Thus, while reproducibility and sensitivity is increased with DIA, typical DIA methods such as SWATH-MS still have relatively low ion beam efficiency and over 98% of the ions can never reach the mass analyzer. However, in principle, a 100% duty cycle (until the TOF analyzer) could be achieved using trapping devices that release the ions at the precise time of analysis. This principle has been implemented for DDA analysis in the “Parallel Accumulation SErial Fragmentation” (DDA-PASEF) method by our collaborators. ******Here we propose to implement a novel method termed DIA-PASEF using data-independent acquisition in order to increase the ion usage of DIA methods and thus further increase their sensitivity and throughput. This will be achieved in the following three aims:******Aim 1: Computationally simulate and implement a novel acquisition scheme that exploits the coupling between ion mobility and m/z for DIA-PASEF.******Aim 2: Develop a targeted analysis strategy for DIA-PASEF data to allow analysis of the four dimensional data space (signal intensity, retention time, ion mobility and m/z).******Aim 3: : Development of an untargeted approach for unbiased analysis of DIA-PASEF data.******The potential impact of this research is a crucial improvement of MS, a core analytical technology used in research, with the potential to increase sensitivity, speed of analysis and accuracy through our improvements in duty cycle. The anticipated result of this research lies in an improved method to comprehensively sample ionized molecules and their fragments. The impact will range from basic research into the molecular behavior of biological systems under laboratory conditions to applied areas in clinical and pharmacological research, where MS can be used to directly analyze clinical samples such as tissue and blood plasma.