Tandem Mass Tag Proteomics Workflow

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).