School of Basic Medical Science, Anhui Medical University, Hefei 230032, China.
State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing 102206, China.
Anal Chem. 2022 Jun 28;94(25):8827-8832. doi: 10.1021/acs.analchem.1c05365. Epub 2022 Jun 14.
Peripheral blood mononuclear cells (PBMCs) play vital roles in physiological and pathological processes and represent a rich source for disease monitoring. Typical molecular profiling on PBMCs involves the sorting of cell subsets and thus requires a large volume of peripheral blood (PB), which impedes the clinical practicability of omics tools in PBMC measurements. It would be clinically invaluable to develop a convenient approach for preparing PBMCs from small volumes of PB and for deep proteome profiling of PBMCs. To this end, here, we designed an apparatus (PBMC-mCap) for microscale enrichment and proteome analysis of PBMCs, which pushed the needed PB volume from the normal 2 mL or higher to 100 μL or lower, comparable to the volume of a drop of finger blood. A PBMC-specific mass spectra library containing 8869 proteins and 121,956 peptides was further built, which, in combination with the optimized data-independent acquisition strategy, helped to identify 6000 and 6500 proteins from PBMCs with 100 μL and 1 mL of PB as initial materials, respectively. Further application of the strategy for PBMC proteomes revealed a steady difference between gender (male vs female) and upon stimulus (COVID-19 vaccination). For the latter, we observed differentially expressed genes and pathways involving the activation of immune cells, including the NF-κB pathway, inflammation response, and antiviral response. Our strategy for the proteome analysis of microscale PBMCs may provide a convenient clinical toolkit for disease diagnosis and healthy state monitoring.
外周血单个核细胞（PBMCs）在外周血单个核细胞（PBMCs）在生理和病理过程中起着至关重要的作用，是疾病监测的丰富来源。典型的 PBMC 分子谱分析涉及细胞亚群的分选，因此需要大量的外周血（PB），这阻碍了 PBMC 测量中组学工具的临床实用性。开发一种从少量 PB 中制备 PBMCs 的便捷方法，以及对 PBMCs 进行深度蛋白质组学分析，将具有重要的临床价值。为此，我们设计了一种用于 PBMC 微尺度富集和蛋白质组分析的装置（PBMC-mCap），将所需的 PB 体积从正常的 2 毫升或更高降低到 100 微升或更低，与一滴指尖血的体积相当。进一步构建了一个包含 8869 种蛋白质和 121956 种肽段的 PBMC 特异性质谱文库，该文库与优化的数据非依赖性采集策略相结合，有助于从 100μL 和 1mL 的 PBMCs 中分别鉴定出 6000 种和 6500 种蛋白质。该策略在外周血单个核细胞蛋白质组学中的进一步应用揭示了性别（男性与女性）和刺激（COVID-19 疫苗接种）之间的稳定差异。对于后者，我们观察到涉及免疫细胞激活的差异表达基因和途径，包括 NF-κB 途径、炎症反应和抗病毒反应。我们用于微尺度 PBMC 蛋白质组分析的策略可能为疾病诊断和健康状态监测提供一种便捷的临床工具包。
