Rapid and Efficient Microfluidic Platform for Single-Cell Proteomic Sample Preparation and Profiling

Single-cell proteomics provides a precise insight into cellular heterogeneity and functional networks within biological processes, yet it is currently hindered by complex and time-consuming sample preprocessing, difficulties in single-cell isolation, significant protein loss, and insufficient depth of coverage. Here, we present a single-cell proteomics microfluidic platform (ScPMP) that integrates cell capture, lysis, protein reduction, alkylation, and online digestion, achieving rapid (∼22 min) and all-in-one sample preprocessing. Compared with conventional methods (bulk dilution and low-binding tube), ScPMP demonstrates significant advantages in protein coverage depth (2-fold) and dynamic range of protein abundance (1−2 orders of magnitude wider), especially for low-abundance proteins. ScPMP is applicable to different cell types, demonstrating broad applicability, with a total of 3356 and 2499 protein groups identified from individual adherent (HeLa) and suspension cells (Jurkat), respectively. Moreover, the integrated platform exhibited good quantitative repeatability, with average CV values below 25% across 100−800 pg of protein inputs, while individual-cell proteomic profiles showed high overall consistency (Pearson’s correlation coefficients of 0.71−0.96). We applied ScPMP to the single-cell proteomics differential analysis before and after cisplatin treatment, revealing the mechanism by which cisplatin inhibits cancer through DNA damage. Overall, ScPMP provides a rapid, efficient, and integrated tool for single-cell proteomics sample preprocessing and offers a new strategy for personalized medicine and drug resistance studies based on single cells.