A multiplexed LC-MS/MS method to reveal changes in inflammatory and coagulation cascades induced by host infection.

Abstract

Inflammation has been considered a pivotal player in the most severe forms of respiratory infections like COVID-19, which induces a significant alteration of inflammatory cytokines called cytokine storm. Above the immune system, abnormalities in coagulation parameters have been observed in COVID-19-affected subjects displaying an increased risk of a blood clot causing thromboembolic events. Currently, clinical methods for monitoring infections rely on serological tests that detect the antibody response using enzyme-linked immunosorbent assays (ELISA). While ELISA offers high sensitivity, it is limited by complex procedures, significant matrix interference, and antibody cross-reactivity. This study aims to develop a mass spectrometry (MS)-based analytical method to support clinical investigations into the host response to infection. The use of S-Trap column digestion in a short time overcomes the limitations of traditional bottom-up proteomics protocols by significantly reducing processing time and eliminating the need for a desalting step. A targeted tandem MS approach using multiple reaction monitoring (MRM) ion mode was established to simultaneously quantify a panel of approximately 60 proteins associated with the inflammatory response and coagulation cascade. Among the proteins analyzed, 90% exhibited a good instrumental response, with 63% showing significant dysregulation in COVID-19 patients compared to controls, enabling the identification of key protein changes linked to the host's infection response. The proposed method leverages the enhanced sensitivity and selectivity of the multiplexed LC-MRM/MS technique, made possible by triple quadrupole mass spectrometers. These instruments effectively filter precursor and product ions, allowing specific monitoring of fragmentation patterns unique to each peptide sequence. By combining a rapid digestion protocol with a multiplexed LC-MRM/MS approach, this method offers a valuable solution for clinical laboratories aiming for high-specificity, high-performance analyses within a shortened analysis time.