Plasma Proteome and Inflammatory Profiling

Untargeted proteomics and targeted inflammatory profiling were performed on plasma collected at admission of 87 case-control pairs. Using mass spectrometry, proteins were identified and quantified as previously described (45 (link)). Briefly, 10 μl of plasma was depleted of the 12 most abundant proteins using spin columns (Thermo Fisher Scientific, Rockford, USA) following the manufacturer’s instructions. Labeled peptide pool samples were generated using the Tandem Mass Tag 10-plex kit (Thermo Fisher Scientific, Illinois, USA) and desalted using ZipTips (Millipore, Darmstadt, Germany) according to the manufacturer’s instructions. Chromatographic separation of peptides was carried out on the Dionex Ultimate 3000 nanoflow liquid chromatography system on a 75 μm by 50 cm C18 reverse-phase analytical column and measured using a Q Exactive Orbitrap mass spectrometer coupled to the chromatography system via a nanoelectrospray ion source (Thermo Fisher Scientific, Illinois, USA).
Concentrations of 29 inflammatory mediators were quantified using a human cytokine magnetic bead assay (EMD Millipore, Burlington, Massachusetts, USA) on the Magpix with Xponent software (version 4.2; Luminex Corp) and acquired median fluorescent intensity data analyzed using the Milliplex analyst software (version 3.5.5.0 standard). Table S5 lists all cytokines assessed.