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

• Published in: Analytical and bioanalytical chemistry Vol. 417; no. 18; pp. 4015 - 4023
• Main Authors: Pinto, Gabriella, Illiano, Anna, Serpico, Stefania, Maurelli, Fabio, Scaglione, Elena, Colicchio, Roberta, Vitiello, Mariateresa, Varelli, Marco, Salvatore, Paola, Amoresano, Angela
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.07.2025; Springer Nature B.V
• Subjects: Abnormalities; Analytical Chemistry; Antibodies; Antibody response; Biochemistry; Blood Coagulation; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; Chemokines; Chromatography; Chromatography, Liquid - methods; Coagulation; Communication; Coronaviruses; COVID-19; COVID-19 - blood; Cross-reactivity; Cytokine storm; Cytokines; Digestion; Enzyme-linked immunosorbent assay; Enzymes; Food Science; Growth factors; Humans; Immune system; Immunoassay; Immunoassays; Infections; Inflammation; Inflammation - blood; Inflammatory response; Laboratory Medicine; Liquid Chromatography-Mass Spectrometry; Mass spectrometry; Mass spectroscopy; Monitoring; Monitoring methods; Monitoring/Environmental Analysis; Multiplexing; Peptides; Proteins; Proteomics; Proteomics - methods; Quadrupoles; Respiratory tract infection; SARS-CoV-2; Scientific imaging; Serological tests; Spectrometers; Tandem Mass Spectrometry - methods; Thromboembolism; Tumor necrosis factor-TNF; United States > US; Germany; Inflammation; Targeted proteomics; Viral infection; Mass spectrometry; Coagulation cascade
• ISSN: 1618-2642; 1618-2650; 1618-2650
• DOI: 10.1007/s00216-025-05950-9

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. Graphical Abstract