The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Copenhagen 2200, Denmark.
Department of Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, Martinsried 82152, Germany.
J Proteome Res. 2022 Nov 4;21(11):2815-2826. doi: 10.1021/acs.jproteome.2c00376. Epub 2022 Oct 26.
In recent years, the concept of cell heterogeneity in biology has gained increasing attention, concomitant with a push toward technologies capable of resolving such biological complexity at the molecular level. For single-cell proteomics using Mass Spectrometry (scMS) and low-input proteomics experiments, the sensitivity of an orbitrap mass analyzer can sometimes be limiting. Therefore, low-input proteomics and scMS could benefit from linear ion traps, which provide faster scanning speeds and higher sensitivity than an orbitrap mass analyzer, however at the cost of resolution. We optimized an acquisition method that combines the orbitrap and linear ion trap, as implemented on a tribrid instrument, while taking advantage of the high-field asymmetric waveform ion mobility spectrometry (FAIMS) pro interface, with a prime focus on low-input applications. First, we compared the performance of orbitrap- versus linear ion trap mass analyzers. Subsequently, we optimized critical method parameters for low-input measurement by data-independent acquisition on the linear ion trap mass analyzer. We conclude that linear ion traps mass analyzers combined with FAIMS and Whisper flow chromatography are well-tailored for low-input proteomics experiments, and can simultaneously increase the throughput and sensitivity of large-scale proteomics experiments where limited material is available, such as clinical samples and cellular subpopulations.
近年来，生物学中细胞异质性的概念受到越来越多的关注，同时也推动了能够在分子水平上解析这种生物学复杂性的技术的发展。对于使用质谱（scMS）进行单细胞蛋白质组学和低投入蛋白质组学实验，轨道阱质谱分析仪的灵敏度有时可能会受到限制。因此，低投入蛋白质组学和 scMS 可以从线性离子阱中受益，线性离子阱比轨道阱质谱分析仪具有更快的扫描速度和更高的灵敏度，但代价是分辨率降低。我们优化了一种采集方法，该方法结合了轨道阱和线性离子阱，在三联仪器上实现，同时利用高场非对称波形离子淌度谱（FAIMS） pro 接口，主要关注低投入应用。首先，我们比较了轨道阱与线性离子阱质谱分析仪的性能。随后，我们通过在线性离子阱质谱分析仪上进行数据非依赖性采集来优化低投入测量的关键方法参数。我们得出结论，线性离子阱质谱分析仪与 FAIMS 和 Whisper 流色谱相结合，非常适合低投入蛋白质组学实验，可以在有限的材料（如临床样本和细胞亚群）的情况下同时提高大规模蛋白质组学实验的通量和灵敏度。
