Department of Chemistry, Institutes of Biomedical Sciences, Fudan University, Shanghai 200438, China.
Pharmacy Department, Shanghai Pudong Hospital, Fudan University Pudong Medical Center, Shanghai 201399, China.
J Proteome Res. 2024 Jul 5;23(7):2441-2451. doi: 10.1021/acs.jproteome.4c00117. Epub 2024 Jun 4.
Global profiling of single-cell proteomes can reveal cellular heterogeneity, thus benefiting precision medicine. However, current mass spectrometry (MS)-based single-cell proteomic sample processing still faces technical challenges associated with processing efficiency and protein recovery. Herein, we present an innovative sample processing platform based on a picoliter single-cell reactor (picoSCR) for single-cell proteome profiling, which involves in situ protein immobilization and sample transfer. PicoSCR helped minimize surface adsorptive losses by downscaling the processing volume to 400 pL with a contact area of less than 0.4 mm. Besides, picoSCR reached highly efficient cell lysis and digestion within 30 min, benefiting from optimal reagent and high reactant concentrations. Using the picoSCR-nanoLC-MS system, over 1400 proteins were identified from an individual HeLa cell using data-dependent acquisition mode. Proteins with copy number below 1000 were identified, demonstrating this system with a detection limit of 1.7 zmol. Furthermore, we profiled the proteome of circulating tumor cells (CTCs). Data are available via ProteomeXchange with the identifier PXD051468. Proteins associated with epithelial-mesenchymal transition and neutrophil extracellular traps formation (which are both related to tumor metastasis) were observed in all CTCs. The cellular heterogeneity was revealed by differences in signaling pathways within individual cells. These results highlighted the potential of the picoSCR platform to help discover new biomarkers and explore differences in biological processes between cells.
单细胞蛋白质组的全局分析可以揭示细胞异质性，从而有益于精准医学。然而，目前基于质谱（MS）的单细胞蛋白质组学样品处理仍然面临与处理效率和蛋白质回收相关的技术挑战。在此，我们提出了一种基于皮升级单细胞反应器（picoSCR）的单细胞蛋白质组分析的创新样品处理平台，该平台涉及原位蛋白质固定和样品转移。通过将处理体积缩小到 400 pL，接触面积小于 0.4 mm，picoSCR 有助于最大限度地减少表面吸附损失。此外，picoSCR 在 30 分钟内实现了高效的细胞裂解和消化，这得益于最佳试剂和高反应物浓度。使用 picoSCR-nanoLC-MS 系统，通过数据依赖型采集模式，从单个 HeLa 细胞中鉴定出超过 1400 种蛋白质。鉴定到拷贝数低于 1000 的蛋白质，表明该系统的检测限为 1.7 zmol。此外，我们还对循环肿瘤细胞（CTC）的蛋白质组进行了分析。通过 ProteomeXchange 可获取标识符 PXD051468 来获取数据。在所有 CTC 中均观察到与上皮-间充质转化和中性粒细胞细胞外陷阱形成（均与肿瘤转移相关）相关的蛋白质。通过单个细胞内信号通路的差异揭示了细胞的异质性。这些结果突出了 picoSCR 平台在发现新的生物标志物和探索细胞间生物学过程差异方面的潜力。
