All AIBL and KARVIAH samples were randomized before sample preparation. Albumin and immunoglobulin G immuno-depletion was achieved by a commercially available immunoaffinity column (ProteoPrep, Sigma-Aldrich) with a starting volume of 30 μl. For enzymatic digestion, 100 μg of immunodepleted sample was initially incubated with 100 mM triethylammonium bicarbonate (TEAB) and 0.1% (w/v) SDS. Reduction and alkylation were achieved in 1 mM tris (2-carboxyethyl) phosphine for 1 hour at 55°C, followed by incubation in 7.5 mM iodoacetamide. Protein samples were individually digested overnight in 4 μg of trypsin (sequencing grade, Roche) reconstituted in 100 mM TEAB. TMT10plex reagents (Thermo Fisher Scientific), reconstituted in acetonitrile (ACN), were added to the appropriate samples and incubated for 1 hour at room temperature (RT). Samples were treated with 5% hydroxylamine and incubated at RT for a further 15 min. For the 284 samples included in the study, a total of 32 “TMT10plex groups” were created by combining nine clinical samples (AIBL or KARVIAH) and one study reference. The study reference was an equal contribution of all samples examined in the study. After combining, TMT10plex groups were incubated at RT for a further 15 min.

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