Pick-up Single-Cell Proteomic Analysis for Quantifying up to 3000 Proteins in a Tumor Cell

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 tumor 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, 1804-3349, 1778-3049 and 1074-2487 protein groups are quantified in single A549 cells ( n = 37), HeLa cells ( n = 44) and U2OS cells ( n = 27), respectively. Benefiting from the flexible cell picking-up ability, we study tumor cell migration at the single cell proteome level, demonstrating the potential in practical biological research from single-cell insight.