Tandem mass tag 10 plex kit

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.

Prior to tandem mass tag labelling, samples were randomized by co-variates [age, sex, post-mortem interval (PMI), diagnosis, etc.], into 50 total batches (8 samples per batch).^{35 (link)} Peptides from each individual (n = 400) and the GIS pooled standard (n = 100) were labelled using the tandem mass tag 10-plex kit (ThermoFisher 90406). Peptide eluents were separated on a self-packed C18 (1.9 μm, Dr. Maisch) fused silica column (25 cm × 75 μM internal diameter) by a Dionex UltiMate 3000 RSLCnano liquid chromatography system (Thermo Fisher Scientific).^{35 (link),36 (link)} Peptides were monitored on an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific). The mass spectrometer was set to acquire data in positive ion mode using data-dependent acquisition. Dynamic exclusion was set to exclude previously sequenced peaks for 20 s within a 10-ppm isolation window.^{35 (link),36 (link)} In this study, we only include peptides and participants with a missing rate less than 20% followed by random forest imputation,^{37 (link)} resulting in 7737 proteins and 386 individuals. Full details on proteomics data acquisition and processing can be found on synapse (https://www.synapse.org/#!Synapse:syn17015098).