Solutions composed of single proteins and mixtures of proteins are subjected to electrospray ionization to study the influence of protein components on the responses of one another. Protein matrix effects in electrospray ionization are particularly relevant to the development of top-down protein identification methodologies involving protein mixtures, whereby whole protein ions are subjected to tandem mass spectrometry. Emphasis is placed largely on solutions composed of equal parts methanol and water and 1% acetic acid. The results, therefore, are relevant to low-pH solutions with significant organic content, a commonly used set of conditions in electrospray ionization mass spectrometry that tends to denature proteins. Under these conditions, very similar response curves are measured for a variety of proteins after charge normalization. That is, when the data are plotted in terms of the concentration of charge sites, rather than in terms of the concentration of protein molecules, the slopes of the response curves as well as the point at which response becomes less than linear with concentration are similar. Charge normalization is made on the basis of the weighted average charge of a protein, as reflected in the electrospray ionization mass spectrum. When proteins can be regarded as a collection of equivalent charge sites, the signal response from one protein can be used to predict the responses for other proteins. Furthermore, it is also possible to predict the dependence of the signal response for a particular protein in a mixture on the concentration of other proteins in the mixture. Examining signal response on a weighted average charge basis appears to be an effective means for identifying situations in which the protein does not behave as a collection of equivalent charge sites.

Solutions consisting of protein and small molecule mixtures have been subjected to electrospray ionization to study the influence of small molecule/cation components at high concentrations on the electrospray responses of proteins. Emphasis was placed on solutions consisting of equal parts methanol and water and containing 1 vol % acetic acid. The results, therefore, are relevant to low pH solutions with significant organic content, a commonly used set of conditions in electrospray ionization mass spectrometry that tends to denature proteins. A variety of small cations/molecules were selected to sample a range of chemical characteristics. For example, sodium and cesium cations were studied to represent metal ions, tetrabutylammonium and tetramethylammonium cations were studied to represent quaternary ammonium compounds with different surface activities, and octadecylamine and glycine were studied to represent species that compete for protons but have different surface activities. A methodology for measuring relative ion suppression efficiencies was developed and applied for protein ions derived from bovine cytochrome c. The form of the small cation (i.e., metal ion, quaternary ammonium ion, or protonated molecule) did not appear to be a factor in determining the efficiency with which protein ion signals were suppressed. The extent to which ions are expected to concentrate on the surface, however, was the major factor in determining the ion suppression efficiency. Itwas found that the ion suppression efficiency of the most surface active species in this study was comparable to that of a protein on another protein after normalization by charge. These results are particularly relevant to the development of mixture analysis strategies based on ionization and tandem mass spectrometry applied to mixtures of whole proteins.

Solutions consisting of single proteins and mixtures of proteins at different pH values have been subjected to both positive ion and negative ion nanoelectrospray ionization to study the influence of solvent pH and protein pI on the ionization responses of proteins. As has been noted previously, it is possible to form protein ions of one polarity despite the fact that the proteins are present as the opposite polarity in solution. However, total response under this condition tends to be at least an order of magnitude less than the condition in which the nanoelectrospray ionization polarity is the same as the net charge of the proteins in solution. Furthermore, maximum signals in positive ion mode were noted when the pH value of the solution was 4-5 units lower than the protein pI. In the negative ion mode, maximum protein anion signals were observed when the pH was roughly 5 units higher than the protein pI. While only small changes in the abundance-weighted average charge were noted as a function of solution conditions, the extent of sodium ion incorporation was seen to depend strongly on the relationship between net protein charge in solution and gas-phase ion polarity. Sodium ion incorporation was minimized under conditions of maximum signal (i.e., low pH positive ion mode and high pH negative ion mode). Sodium ion incorporation was highest when the protein ion polarities in solution and the gas phase were opposite. These observations are consistent with the charged residue model for electrospray ionization and suggest that a degree of selectivity for electrospray ionization applied to protein mixtures can be realized via judicious selection of solution pH and ionization polarity. Furthermore, the relative extent of sodium ion incorporation under a given set of conditions appears to correlate, at least qualitatively, with protein pI.

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In the elastic regime, GIFAD is equivalent to TEAS with an effective energy E⊥ between 1 meV and 1 eV providing a high sensitivity to topology and to attractive forces. The inelastic regime merges to the classical limit and is still in development.