Tandem Mass Tag (TMT) Labeling Strategy

We used a multiplexed isobaric labeling approach to allow for simultaneous identification and quantification of proteins from multiple biological samples. A 10-plex TMT labeling kit (ThermoScientific, NJ, USA) was used for analyses of protein samples from AD/controls and Aged/Young and an aged-matched control sample was used as a reference sample per plex for normalization of data and as a reference point for the different runs. This labeling strategy allowed for all different groups to be randomized and analyzed within the same batch. All samples and isobaric label tags were handled blind to the experimenter. Twenty microliter aliquots of each label (dissolved in 20 ul of acetonitrile solution) were dried down in the speed vacuum and re-suspended in 25 mM TEAB made up in acetonitrile solution. Re-suspended labels were subsequently added to 10 ul of dried digested protein samples, and allowed to incubate for 1 h at room temperature, after which 1 ul of formic acid solution was added to stop the reaction. Labeled samples were pooled together in entire batches and subsequently dried in the speed vacuum.