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

## TL;DR

MiProChip is a microfluidic platform that enables high-throughput, multiplexed single-cell proteomics with improved efficiency and sensitivity.

## Contribution

MiProChip introduces a streamlined microfluidic workflow for isobaric labeling and single-cell proteomics with enhanced throughput and proteome coverage.

## Key findings

- MiProChip identified 3362 protein groups with high confidence in single-cell TMT-10-plex experiments.
- The platform detected 3199 proteins in murine colon adenocarcinoma cells, revealing treatment-specific proteomic responses.
- PCA and pathway analysis showed galectin-8's antimetastasis potential not observed in bulk proteomics.

## Abstract

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.