A Simple and Flexible Strategy for Single-Cell Proteomic Analysis Based on Protein Immobilization and Digestion Tube Reactor

Mass spectrometry-based single-cell proteomics (SCP) analysis has witnessed rapid development over the past 10 years. However, the current preprocessing methodologies face several challenges: multiple time-consuming steps, reliance on costly consumables and advanced instrumentation, and the necessity for specialized expertise and training, which hinder the widespread application of deep SCP analysis. Here, we develop a simple and flexible strategy that seamlessly integrates single-cell sampling, preprocessing, and liquid chromatography tandem mass spectrometry (LC-MS/MS) injection by constructing a microliter single-cell protein immobilization and digestion tube reactor (SPIDR), which remains free from additional transfer steps. The reactor, made through inner surface functionalization of a commercially available insert tube, achieves the end-to-end single-cell rapid preprocessing within 1 h at a low cost. The microliter reactor with a relatively large volume, instead of the popular nanoliter/picoliter volume, significantly reduces operational difficulty and facilitates process automation. Using the SPIDR workflow, an average of 4186, 3171, and 4018 protein groups are quantified from single A549 cells (<i>n</i> = 16), HeLa cells (<i>n</i> = 16), and MCF-7 cells (<i>n</i> = 16), respectively. Furthermore, we investigate the proteomic heterogeneity of cervical cancer cells at different apoptotic stages following paclitaxel treatment at the single-cell level, demonstrating the potential of single-cell proteomics in addressing biological problems.