Quantitative Proteomic Analysis of LPS-Activated Macrophages

Bone marrow-derived macrophages were primed with 1 ng/ml LPS for 3 days and stimulated with 100 ng/ml LPS for 7 h. Monensin-containing GolgiStop (BD Biosciences) was added for the last 6 h of culture. After washing, cells were lysed in lysis buffer (4% SDS, 50 mM TEAB pH 8.5, 10 mM TCEP), boiled and sonicated with a BioRuptor (30 cycles: 30 s on, 30 s off) before alkylation with 20 mM iodoacetamide for 1 h at room temperature in the dark. Lysates were subjected to the SP3 protein clean-up procedure (22 (link)), eluted into digestion buffer (0.1% SDS, 50 mM TEAB pH 8.5, 1 mM CaCl2) and digested with trypsin at a 1:50 (enzyme:protein) ratio. TMT labeling and peptide clean-up were performed according to the SP3 protocol. Samples were eluted into 2% DMSO, combined, and dried under vacuum. TMT samples were fractionated using offline high pH reverse-phase chromatography. Peptides were separated, concatenated to 22 fractions, dried and peptides redissolved in 5% formic acid and analyzed by LC-MS.