Unlocking the potential of microfluidics in mass spectrometry-based immunopeptidomics for tumor antigen discovery

The identification of tumor-specific antigens (TSAs) is critical for developing effective cancer immunother-apies. Mass spectrometry (MS)-based immunopeptidomics has emerged as a powerful tool for identifying TSAs as physical molecules. However, current immunopeptidomics platforms face challenges in measuring low-abundance TSAs in a precise, sensitive, and reproducible manner from small needle-tissue biopsies (<1 mg). Inspired by recent advances in single-cell proteomics, microfluidics technology offers a promising solution to these limitations by providing improved isolation of human leukocyte antigen (HLA)-associated peptides with higher sensitivity. In this context, we highlight the challenges in sample preparation and the rationale for developing microfluidics technology in immunopeptidomics. Additionally, we provide an over-view of promising microfluidic methods, including microchip pillar arrays, valved-based systems, droplet microfluidics, and digital microfluidics, and discuss the latest research on their application in MS-based im-munopeptidomics and single-cell proteomics.