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

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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.