Nematic protein organisation technique (NPOT), heteroassemblies isolation, and proteomics

The NPOT proprietary technology is based on the Kirkwood-Buff molecular crowding and aggregation theory^15,16. THP-1 cell lysates were prepared under low temperature (4 °C) in the absence of any detergent, reducing agent or protease or phosphatase inhibitors. All probable dilutions and washes are performed in HBSS with equal osmolality, trace elements, vitamins and salts in concentrations as close as possible to those of the interstitial medium or cytoplasm. Evodiamine (10⁻⁶M) is put in contact with the total tissue material. The macromolecular assemblies related to the ligand are separated using a differential microdialysis system, based on a transitory pH gradient (5–10) wherein the macromolecules (protein groups) migrate in the liquid phase based on their physico-chemical properties. The use of pH gradient in NPOT is important in order to cover the range of existing physiological extracellular and intracellular pH values, to assure the detection of relevant interactions which might be pH-dependent. The migrating macromolecules growing gradually form nematic crystals to macromolecular heteroassemblies thanks to the molecular interactions between evodiamine and its targets. The heteroassemblies are trapped in mineral oil. The latter are isolated and identified by mass spectroscopy directly in liquid.

Under a Microscope, each heteroassembly formed was isolated by microdissection and washed in acetone prior to solubilisation in standard Hepes saline buffer solution (HBSS solution).

Prior to LC-MS/MS experiments heteroassemblies were solubilised directly in 10 µl of 2D buffer (7M Urea, 2M Thiourea, 4% CHAPS, 20 mM DTT, 1 mM PMSF). Proteins were precipitated in acetate buffer by centrifugation for 20 min at 7500xg. Thereafter pellets were digested for 1 h with trypsin Gold (Promega) at 37 °C. Trypsin Gold was resuspended at 1 µg/µL in 50 mM acetic acid, and then diluted in 40 mM NH₄HCO₃ to 20 µg/ml. The samples were dried in SpeedVac^® at room temperature. Peptides were purified and concentrated by using ZipTip^® pipette tips (Millipore Corporation) before proceeding for mass spectrometry analysis through 1 h LC-MS/MS analyses protocol in an ESI-QUAD-TOF machine. Proteins were identified using Mascot software.