Cytokine profiling in COVID-19 patients by using ion mobility-mass spectrometry-based parallel reaction monitoring

Cytokine therapy, a non-antigen-specific strategy, has led to several FDA-approved drugs. Given the role of dysregulated cytokine expression in diseases such as COVID-19, accurate quantification is critical in both clinical and research settings. While antibody-based assays offer high sensitivity, their reliance on specific antibodies limits multiplexing and increases analytical complexity. Conversely, mass spectrometry methods like multiplexed reaction monitoring provide higher throughput but lack the sensitivity to detect physiological cytokine levels and the resolution to distinguish structural isomers. Thus, a new MS-based approach is needed that integrates high sensitivity with the ability to resolve structurally similar cytokines. We developed an ion mobility-mass spectrometry (IM-MS)-based parallel reaction monitoring (PRM) method to establish the first Cytokine Ion Mobility Peptide (CIMP) databank and enable high-throughput cytokine profiling in serum samples from COVID-19 patients. By introducing ion mobility as an additional gas-phase separation dimension alongside liquid chromatography, the method enhances analyte resolution based on structural differences, facilitating the separation of isomers within the ion mobility trap. The incorporation of ion mobility as a complementary separation parameter enables the distinction of homologous cytokines and structural isomers (e.g., IFNA1/IFNA2, IFNL1/IFNL3, and peptide isomers), which remains challenging for conventional antibody-based assays. The method achieved a limit of detection of 62.9 fmol/L and a limit of quantification of 210 fmol/L across 31 cytokines, demonstrating greater sensitivity than traditional multiple reaction monitoring (MRM) approaches and enabling quantification at physiological concentration levels, assuming comparable background signal across platforms. The IM-MS-PRM method offers a multiplexed, high-throughput, and adaptable platform that eliminates the need for multiple assays while delivering excellent reproducibility. It enables accurate and sensitive cytokine quantification from minimal volumes of COVID-19 patient serum. Combined with the CIMP databank, this approach allows precise differentiation between early and late severe COVID-19 cases, supporting improved diagnostic and therapeutic decision-making.