Matrix effects and selectivity in electrospray ionization of protein mixtures

Peng Pan, Purdue University

Abstract

Solutions comprised of single proteins, protein mixtures and mixtures of proteins and small molecules/ions were subjected to electrospray ionization to study the influence of mixture components on the responses of proteins (i.e. matrix effects). Emphasis was placed largely on solutions with significant organic content and at low pH. Under these conditions, very similar response curves were measured for a variety of proteins after charge normalization. When proteins can be regarded as a collection of equivalent charge sites, it is straightforward to predict both the signal response for the protein as a function of concentration from the response of another protein and the dependence of signal response on the concentration of another protein. The influences of small cation components on the positive electrospray responses of proteins were also studied. The results demonstrate that the form of the small cation is not a factor in determining the efficiency with which protein ion signals were suppressed. The extent to which cations are expected to concentrate on the surface, however, was the major factor in determining the ion suppression efficiency. The role of solution pH and protein pI in nanoelectrospray ionization of protein mixtures was investigated. 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 is at least an order of magnitude less than the condition in which the ionization polarity is the same as the net charge of the proteins in solution. Furthermore, maximum signals were noted when the solution pH was 4–5 units lower or higher than the protein pI for positive or negative ion mode, respectively. These observations 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.

Degree

Ph.D.

Advisors

McLuckey, Purdue University.

Subject Area

Analytical chemistry

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