Collisional cooling enhances the ability to observe non-covalent interactions within the inducible nitric oxide synthase oxygenase domain: dimerization, complexation, and dissociation.

The investigation of protein quaternary structure, protein-cofactor, and protein-ligand interactions by mass spectrometry is often limited by the fragility of such interactions under experimental conditions. To develop more gentle conditions of perhaps general use, we used as a model for study the oxygenase domain of murine inducible nitric oxide synthase (iNOS), which is homodimeric, binds heme and tetrahydrobiopterin H(4)B cofactors, and the substrate L-arginine. The energetics of the collisions in q2 and in the lens region of the mass spectrometer were manipulated for varying the degree of solvation around the non-covalently bound ions. Furthermore, the number of low-energy collisions in the collision cell of the instrument was varied, focusing and dampening the ion beam. Under gentle source collision conditions, and using multiple low-energy collisions in the collision cell of the mass spectrometer, dimers of the iNOS oxygenase domain containing heme, H(4)B, and arginine were observed intact after electrospraying at pH values near neutrality; a mutant of this protein (Trp188 --> Phe) was monomeric and did not bind cofactors. The pH dependence of the iNOS oxygenase domain under acidic conditions was also studied; while heme remained bound to the protein between pH 2.5 and 4.0, the dimeric structure was disrupted. Our findings confirm that non-covalently bound macromolecular complexes are retained and observable using electrospray mass spectrometry under the appropriate experimental conditions.