MiProChip：アイソバリックラベリングによるマルチプレックス単一細胞プロテオミクス用のスケーラブルなマイクロ流体プラットフォーム

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.