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

在数据独立采集 (DIA) 质谱分析中使用捕获离子淌度同步离子

• 批准号: RGPIN-2019-06833
• 负责人: Rost, Hannes
• 金额: $ 2.48万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=750829
• 关键词: Using; trapped; ion; mobility; synchronize

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.

质谱法（MS）已广泛用于蛋白质组学中，用于分析复杂样品，例如临床或环境样品以及全细胞裂解物。具体而言，在自下而上的蛋白质组学中，蛋白质是从生物学样品中提取的，并使用液体色谱与串联质谱法（LC-MS/MS）的在线液体色谱系统进行分析之前进行分析。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，但仅利用一小部分离子束进行质量分析。在传统的数据依赖性采集（DDA）中，仅选择单个前体离子进行碎片化，因此有效地过滤了超过2000 da的质量范围的99.9％。为了比DDA方法更全面和可重复地对片段离子进行采样，我们最近开发了使用与数据无关的采集（DIA）的Swath-MS。但是，还使用相对较小的质量隔离窗口来提高特异性（例如25 m/z），从而在典型的占空比中使用了所有可用离子的1.25％。尽管DIA通过DIA提高了可重复性和灵敏度，但典型的DIA方法（例如Swath-MS）仍然具有相对较低的离子束效率，而超过98％的离子无法达到质量分析仪。但是，原则上，使用在分析精确时间释放离子的诱捕设备可以实现100％的占空比（直到TOF分析仪）。该原理已通过我们的合作者在“平行积累序列片段化”（DDA-PASEF）方法中实施了DDA分析。在这里，我们建议使用独立于数据的采集来实施一种称为DIA-PASEF的新方法，以增加DIA方法的离子使用情况，从而进一步提高其敏感性和吞吐量。这将在以下三个目标中实现：AIM 1：计算模拟并实施一种新颖的采集方案，该方案利用了DIA-PASEF的离子迁移率和M/Z之间的耦合。目标2：为DIA-PASEF数据制定目标分析策略，以允许分析四个维数据空间（信号强度，保留时间，离子迁移率和M/Z）。 AIM 3：：开发一种无靶方法的方法，用于对DIA-PASEF数据的无偏分析。这项研究的潜在影响是MS（一种用于研究中使用的核心分析技术）的至关重要的改进，具有通过改善占空比的改善来提高灵敏度，分析速度和准确性。这项研究的预期结果在于一种改进的方法，可以全面采样离子化的分子及其碎片。影响范围从实验室条件下生物系统的分子行为的基础研究到临床和药物研究中的应用领域，在该研究中，MS可用于直接分析临床样本，例如组织和血浆。