MiProChip: A Scalable Microfluidic Platform for Multiplexed Single-Cell Proteomics via Isobaric Labeling.

Single-cell proteomics (SCP) platforms are widely sought-after biomedical tools to complement existing omics technologies. Here, we present MiProChip, a microfluidic platform that integrates cell capture, lysis, protein digestion, tandem mass tag (TMT) labeling, on-chip pooling, and desalting a streamlined workflow for multiplexed SCP profiling. We optimized a chip-compatible TMT multiplexing protocol with a carrier-boosting strategy, enabling high-throughput operation and deep proteome coverage. MiProChip was designed to effectively reduce the mass spectrometry (MS) operation time, minimize adsorptive losses, enhance mixing, and stabilize flow for on-chip pooling, leading to a superior performance in recovery. Using PC9 and H1975 cells with a 100-cell carrier, a total of 3362 protein groups with 2775 ± 36 proteins were confidently identified across TMT-10-plex single-cell channels. Demonstration on murine colon adenocarcinoma cells identified 3199 proteins with 1669 ± 261 proteins per single cell to characterize galectin-8- and TGF-β-specific responses. Single-cell principal component analysis (PCA) showed separation of the control from treated groups, partial overlap between galectin-8 and TGF-β, and close clustering of TGF-β with the combination treatment, supporting a dominant TGF-β effect. Pathway enrichment analysis reveals their responsive pathway and distinguishes galectin-8- and TGF-β-specific responses, revealing downregulation of metastasis-related markers to support antimetastasis potential of galectin-8, which was not detected by bulk proteomic analysis. Collectively, MiProChip captured subtle proteomic heterogeneity and treatment-dependent single-cell responses, establishing a sensitive and robust platform for high-throughput SCP analysis.