The analysis of composition and abundance of the raft proteome of microglia using a tandem mass tag (TMT)-based quantitative proteomic analysis.

To determine the proteins in the membrane raft, we used the TMT-labeling and nano-liquid chromatography mass spectrometry (nano-LC-MS/MS) analysis by Creative Proteomics (NY, USA; https://www.creative-proteomics.com/). Rat primary microglia were treated with IL-6 (25 ng/ml) for 15 min. Membrane rafts were obtained by flotation assay. Samples were prepared from three independent experiments. Proteins in equal volumes of raft fractions were digested with trypsin, desalted, and labeled with a TMT reagent (Thermo Fisher Science). The TMT-labeled peptides were fractionated and analyzed by nano-LC-MS/MS. The resulting MS/MS data were analyzed and searched against the rat protein database using Proteome Discoverer 2.1.