MBP-lipid deimination assay

Thirty micrograms of either LPC 18:1 or LPC 18:0 was incubated in the presence of 10 μg of recombinant MBP protein in PBS and allowed to interact for 30 min at room temperature. After incubation, each reaction was adjusted to a final concentration of 1.66 mm CaCl2 and incubated overnight at 37°C in the presence of purified PAD (catalog #P1584, Sigma-Aldrich). Proteins were then precipitated using standard acetone precipitation protocols, evaporated using the CentriVap Concentrator system (catalog #64132–2696, Labconco), and processed for mass spectrometry analysis (see section Sample preparation for protein mass spectrometry).