Exploring the Mechanism of Salt-Induced Signal Suppression in Protein Electrospray Mass Spectrometry Using Experiments and Molecular Dynamics Simulations

Protein analyses by electrospray ionization (ESI) mass spectrometry can suffer from interferences caused by nonvolatile salts. The mechanistic basis of this effect remains to be fully investigated. In the current work we explore the behavior of proteins under native and denaturing conditions in the presence of NaCl, CsCl, and tetrabutyl ammonium chloride (NBu₄Cl). All three salts interfere with the formation of “clean” [M + zH]^z+ protein ions by progressively deteriorating spectral S/N ratios. We propose that salt interferences can be dissected into two independent aspects, i.e., (i) peak splitting by adduct formation and (ii) protein ion suppression. NaCl degrades the spectral quality by forming heterogeneous [M + zH + n(Na – H) + m(Cl + H)]^z+ ions, while the integrated protein ion intensity remains surprisingly robust. Conversely, NBu₄Cl does not cause any adduction, while dramatically reducing the protein ion yield. These findings demonstrate that adduct formation and protein ion suppression are indeed unrelated effects that may occur independently of one another. Other salts, such as CsCl, can give rise to a combination of the two scenarios. Molecular dynamics simulations of water droplets charged with either Na⁺ or NBu₄⁺ provide insights into the mechanism underlying the observed effects. Na⁺ containing droplets evolve relatively close to the Rayleigh limit (z/z_R ≈ 0.74), whereas the z/z_R values of NBu₄⁺ charged droplets are considerably lower (∼0.59). This difference is due to the high surface affinity of NBu₄⁺, which facilitates charge ejection from the droplet. We propose that the low z/z_R values encountered in the presence of NBu₄⁺ suppress the Rayleigh fission of parent droplets in the ESI plume, thereby reducing the yield of progeny droplets that represent the precursors of gaseous protein ions. In addition, the rate of solvent evaporation is reduced in the presence of NBu₄⁺. Both of these factors lower the protein signal intensity. NaCl does not interfere with droplet fission, such that protein ions continue to form with high yieldalbeit in heavily adducted form. Our findings expand on earlier proposals of charge competition as a key factor during the ESI process for salt-contaminated solutions.