The Development of a Double-Label Two-Dimensional Electrophoresis Procedure and Its Use in a Search For Blood Protein Abnormalities in Neurological Diseases

Abstract

A search for protein abnormalities in peripheral blood in patients with multiple sclerosis, depression, and Alzheimer's disease was made with the aim of discovering protein abnormalities suitable as protein markers for these diseases. A double-label two-dimensional electrophoresis procedure for the comparison of two complex protein mixtures was developed. This included the modification of a previously-described procedure (Jentoft and Dearborn (1979) J. Biol. Chem. 254, 4359) for labelling proteins by reductive methylation of amino groups with [3H]- or [14C] formaldehyde and sodium cyanoborohydride to enable the optimal labelling of plasma and serum samples. Formaldehyde (0.2 M) and 75 mM sodium cyanoborohydride were required for optimal labelling of 0.05 ml of serum diluted 1:2 at pH 7.5 at room temperature. The reaction was complete after 15 min when 23 mM formaldehyde was used. A fourfold increase in the sensitivity of autoradiography over existing double-label methods was obtained by performing autoradiography before processing the gel for fluorography. A spot in the electrophoretic gel that contained 17-28 ng of labelled protein (serum concentration 5-10 µg/ml) was detectable. A number of factors influencing the sensitivity and resolution of the procedure were investigated. The double-label procedure was modified to enable the analysis of proteins requiring detergent for aqueous solubility. Samples were disrupted by sonication, solubilised in sodium dodecyl sulphate, and concentrated, before being subjected to a modified labelling procedure. The modified double-labelling procedure was applied to the analysis of erythrocyte membranes, platelets, and isolated placental microvilli. The high resolving power and sensitivity of the procedure were retained. Several steps in the procedure were investigated for the possible introduction of artefactual protein differences. The double-label procedure was used to compare the proteins of plasma, serum, erythrocyte membranes, and platelet preparations pooled from a number of patients with a particular disease with pooled samples derived from healthy individuals. The analysis of serum, plasma, and erythrocyte membrane proteins from patients with multiple sclerosis revealed no significant differences, however, differences were found in platelet proteins. These were not consistently present in further investigations using individual samples and a second set of pooled samples. Analysis of plasma and erythrocyte membrane proteins from depressed patients, and plasma and platelet proteins from patients with Alzheimer's disease did not reveal any significant differences. Differences from normals were found in erythrocyte membrane proteins from patients with Alzheimer's disease, however, these were not consistently present in further investigations in which individual samples and a second set of pooled samples were analysed.