Ultra-sensitive mass spectrometry-based immunopeptidomics using digital microfluidics.

The identification of Tumor-Specific Antigens (TSAs) is critical for developing effective cancer immunotherapies. Mass spectrometry-based immunopeptidomics has emerged as a powerful tool for unbiased and systematic identification of 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 immunopeptides with higher sensitivity. For example, recent advancements in microchip pillar arrays have reported a two-fold increase in the yield of detected immunopeptides, clearly demonstrating the benefit of microfluidics in immunopeptidomics. However, state-of-the-art microfluidics methods in immunopeptidomics rely on complex tubing networks, pump systems, and demanding antibody requirements. These complexities present significant barriers to the widespread adoption of this technology within the cancer research community. Alternatively, digital microfluidics is fundamentally different and enables precise manipulation of micro- or nano-droplets using an electrode array and the principle of electrowetting on dielectric. Key advantages of digital microfluidics are the ability to control individual micro/nano-droplets of liquid without the need for micropipettes, pumps or complex tubing networks. To date, digital microfluidics has been widely used in biomedical research and has demonstrated success in various applications, but its potential for immunopeptide profiling and TSA discovery remains untapped. The overall objective is to enhance the efficiency of immunopeptide isolation from small cell populations by leveraging digital microfluidics. We hypothesize that miniaturization and automation of the immunopeptide isolation procedure by digital microfluidics will enable TSA discovery from as few as 10,000 to 100,000 tumor cells using a timsTOF Ultra mass spectrometer. Such advancement will lay the cornerstone for pioneering single-cell immunopeptidomics. In Aim 1, we will create the first “all-in-one” digital chip for ultra-efficient and scalable isolation of immunopeptides for TSA discovery at unprecedented sensitivity. The method will be rapid and unbiased by applying and integrating relevant surfactant, immunopeptide elution process, clean-up on the digital platform, and a direct interface with the liquid chromatography system. Importantly, our digital approach will be antibody-free, thereby drastically enhancing its scalability for wide adoption by the community. We will apply the “all-in-one” digital chip on low numbers of lung tumor cells isolated from patients to identify actionable TSAs. The project will establish a highly innovative measurement capability for personalized cancer immunotherapy. Project Number: 1R61CA297867-01A1 | Fiscal Year: 2025 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Etienne Caron | Institution: YALE UNIVERSITY, NEW HAVEN, CT | Award Amount: $672,371 | Activity Code: R61 | Study Section: Special Emphasis Panel[ZCA1 TCRB-D (M2)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11191806