Pick-up single-cell proteomic analysis for quantifying up to 3000 proteins in a Mammalian cell.

Wang, Yu; Guan, Zhi-Ying; Shi, Shao-Wen; Jiang, Yi-Rong; Zhang, Jie; Yang, Yi; Wu, Qiong; Wu, Jie; Chen, Jian-Bo; Ying, Wei-Xin; Xu, Qin-Qin; Fan, Qian-Xi; Wang, Hui-Feng; Zhou, Li; Wang, Ling; Fang, Jin; Pan, Jian-Zhang; Fang, Qun

The shotgun proteomic analysis is currently the most promising single-cell protein sequencing technology, however its identification level of ~1000 proteins per cell is still insufficient for practical applications. Here, we develop a pick-up single-cell proteomic analysis (PiSPA) workflow to achieve a deep identification capable of quantifying up to 3000 protein groups in a mammalian cell using the label-free quantitative method. The PiSPA workflow is specially established for single-cell samples mainly based on a nanoliter-scale microfluidic liquid handling robot, capable of achieving single-cell capture, pretreatment and injection under the pick-up operation strategy. Using this customized workflow with remarkable improvement in protein identification, 2449–3500, 2278–3257 and 1621–2904 protein groups are quantified in single A549 cells (n = 37), HeLa cells (n = 44) and U2OS cells (n = 27) under the DIA (MBR) mode, respectively. Benefiting from the flexible cell picking-up ability, we study HeLa cell migration at the single cell proteome level, demonstrating the potential in practical biological research from single-cell insight. Single-cell proteomics is of fundamental importance to capture biological heterogeneity, while limited in proteome depth. Here, the authors develop a pick-up single-cell proteomic analysis (PiSPA) workflow to achieve a deep coverage of quantifying up to 3000 protein groups in a mammalian cell.

AMINO acid sequence; PROTEINS; HELA cells; CELL migration; WORKFLOW; PROTEOMICS

Nature Communications, 2024, Vol 15, Issue 1, p1

2041-1723

Academic Journal

10.1038/s41467-024-45659-4