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

Abstract

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.

Ion-Ion and ion-molecule reactions at the surface of proteins produced by nanospray. Information on the number of acidic residues and control of the number of ionized acidic and basic residues

“…We are confident that this is not the case with the apparatus that we use. The nanospray droplets are very small, with an approximate radius of 0.15 m at the flow rates that we use, and they are expected to evaporate within less than 200°s° [7].°Therefore,°the°droplet°evaporation°will°be close to complete before the droplets enter the transfer capillary, and if not, then certainly complete by the time they exit the heated transfer capillary.…”

“…According to IEM the ions, instead of staying in the evaporating droplet until the droplet evaporates completely as is the case for CRM, escape form the droplet by "ion evaporation" at an earlier stage. The escape takes place only after the droplets have become very small (droplet diameter in the 8 -10 nm°range)° [24].°Therefore,°these°droplets°have°experi-enced extensive solvent evaporation and the concomitant very large increase of concentration of all solutes in the°droplet° [7].°The°high°concentration°will°promote°ion pairing between the ions present in the solution and result in reactions analogous to eqs 5 and 6 but involving the peptides rather than the protein. On escape from the droplet, the peptide will acquire charge from the surface of the droplets and when a sodium salt was used, that charge will be mostly due to Na ϩ ions.…”

“…The first generation offspring droplets also evaporate fast and can undergo fission leading to second generation droplets. Early graphic representations of such droplet histories [2] could be replaced by more accurate droplet histories [7] that are based on recent observations by Beauchamp and coworkers [8].…”

“…The bonds must be strong enough to survive the clean up stage in the interface to the mass analysis, i.e., the heated sampling capillary and the subsequent CAD stage [7, 10c]. The droplet history given in [7] indicates°that°the°observed°protein ions, when NSI is used, originate mostly from the first generation offspring droplets.…”

Stability of the homopentameric b subunits of shiga toxins 1 and 2 in solution and the gas phase as revealed by nanoelectrospray fourier transform ion cyclotron resonance mass spectrometry

“…It can be seen that there is little change in the width of the peaks, indicating that the adducts are kinetically quite stable. Consequently, it is unlikely that the adducts are solvent molecules or neutral buffer components, because these would be expected to readily dissociate from the protein ions in the heated ion cell [36]. With increasing spray duration, the relative abundance of the Stx2 B pentamer ions decreased and concomitantly, ions corresponding to monomer (B ϩ5 Figures 3b, c) appeared in the mass spectra.…”