Features of the ESI mechanism that affect the observation of multiply charged noncovalent protein complexes and the determination of the association constant by the titration method.
Several factors, attributable to the ESIMS mechanism, that can affect the assumptions of the titration method are examined: (1) The assumption that the concentrations in solution of the protein P, the ligand L, and the complex PL are proportional to the respective ion intensities observed with ESIMS, is examined with experiments in which ion intensities of two non-interacting proteins are compared with the respective concentrations. The intensities are found to be approximately proportional to the concentrations. The proportionality factors are found to increase as the mass of the protein is decreased. Very small proteins have much higher intensities. The results suggest that it is preferable to use only the intensity ratio of PL and P, whose masses are very close to each other when L is small, to determine the association constant KA in solution. (2) From the charge residue model (CRM) one expects that the solution will experience a very large increase of concentration due to evaporation of the precursor droplets, before the proteins P and PL are produced in the gas phase. This can shift the equilibrium in the droplets: P + L = PL, towards PL. Analysis of the droplet evaporation history shows that such a shift is not likely, because the time of droplet evolution is very short, only several micros, and the equilibrium relaxation time is much longer. (3) The droplet history shows that unreacted P and L can be often present together in the same droplet. On complete evaporation of such droplets L will land on P leading to PL and this effect will lead to values of KA that are too high. However, it is argued that mostly accidental, weakly bonded, complexes will form and these will dissociate in the clean up stages (heated transfer capillary and CAD region). Thus only very small errors are expected due to this cause. (4) Some PL complexes may have bonding that is too weak in the gas phase even though they have KA values in solution that predict high solution PL yields. In this case the PL complexes may decompose in the clean up stages and not be observed with sufficient intensity in the mass spectrum. This will lead to KA values that are too low. The effect is expected for complexes that involve significant hydrophobic interaction that leads to high stability of the complex in solution but low stability in the gas phase. The titration method is not suited for such systems.
Peschke
Michael
M
Department of Chemistry, E3-44, University of Alberta, T6G 2G2, Edmonton, Alberta, Canada.
Verkerk
Udo H
UH
Department of Chemistry, E3-44, University of Alberta, T6G 2G2, Edmonton, Alberta, Canada.
Kebarle
Paul
P
Department of Chemistry, E3-44, University of Alberta, T6G 2G2, Edmonton, Alberta, Canada. paul.kebarle@ualberta.ca.
D046912:Multiprotein Complexes / Q000737:chemistry*; D011506:Proteins / Q000032:analysis* / Q000737:chemistry*; D012107:Research Design; D021241:Spectrometry, Mass, Electrospray Ionization; D055672:Static Electricity; D017186:Titrimetry / Q000379:methods*
D046912:Multiprotein Complexes; D011506:Proteins