Electrospray ionization of protein mixtures at low pH.

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.