Evaluating Linear Ion Trap for MS3-Based Multiplexed Single-Cell Proteomics.
Evaluating Linear Ion Trap for MS3-Based Multiplexed Single-Cell Proteomics.
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DOI：
10.1021/acs.analchem.2c03739
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发表时间：
2023-01
影响因子：
7.4
通讯作者：
Junho Park;Fengchao Yu;J. Fulcher;Sarah M. Williams;Kristin M. Engbrecht;Ronald J. Moore;G. Clair;V. Petyuk;A. Nesvizhskii;Ying Zhu
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文献类型：
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作者：
Junho Park;Fengchao Yu;J. Fulcher;Sarah M. Williams;Kristin M. Engbrecht;Ronald J. Moore;G. Clair;V. Petyuk;A. Nesvizhskii;Ying Zhu
人们越来越需要开发高通量和高灵敏度的单细胞蛋白质组学质谱方法。常用的基于同量异位素标记的多路复用单细胞蛋白质组学方法由于在MS/MS裂解期间共分离的干扰离子而遭受扭曲的蛋白质定量，也称为比率压缩。我们推断使用基于MS 3的量化可以减轻比率压缩并提供更好的量化。然而，以前的研究表明，减少蛋白质组覆盖率的MS 3方法，可能是由于长的占空比时间和离子损失在多级离子选择和碎片。在本文中，我们描述了一种改进的MS采集方法，用于基于MS 3的单细胞蛋白质组学，通过采用线性离子阱来测量报告离子。我们证明了线性离子阱可以增加单细胞水平肽的蛋白质组覆盖率，通过MS 3方法获得更高的增益。将优化的实时搜索MS 3方法进一步应用于单个巨噬细胞的免疫激活研究。在总共研究的126个单细胞中，当需要至少50%和75%的非缺失数据时，分别有超过1200和1000种蛋白质可定量。我们的评估还揭示了多路复用单细胞蛋白质组学的低分辨率离子阱检测器的几个局限性，并提出了实验解决方案，以尽量减少其对单细胞分析的影响。
There is a growing demand to develop high-throughput and high-sensitivity mass spectrometry methods for single-cell proteomics. The commonly used isobaric labeling-based multiplexed single-cell proteomics approach suffers from distorted protein quantification due to co-isolated interfering ions during MS/MS fragmentation, also known as ratio compression. We reasoned that the use of MS3-based quantification could mitigate ratio compression and provide better quantification. However, previous studies indicated reduced proteome coverages in the MS3 method, likely due to long duty cycle time and ion losses during multilevel ion selection and fragmentation. Herein, we described an improved MS acquisition method for MS3-based single-cell proteomics by employing a linear ion trap to measure reporter ions. We demonstrated that linear ion trap can increase the proteome coverages for single-cell-level peptides with even higher gain obtained via the MS3 method. The optimized real-time search MS3 method was further applied to study the immune activation of single macrophages. Among a total of 126 single cells studied, over 1200 and 1000 proteins were quantifiable when at least 50 and 75% nonmissing data were required, respectively. Our evaluation also revealed several limitations of the low-resolution ion trap detector for multiplexed single-cell proteomics and suggested experimental solutions to minimize their impacts on single-cell analysis.