Capillary Electrophoresis Analysis of Prostate-Specific Antigen (PSA).

The Capillary Electrophoresis (CE) profile of isoforms (peaks) of a glycoprotein can be useful to show alterations in its posttranslational modifications (PTMs) linked to diseases. These changes can modify the electrophoretic mobility of these isoforms in a minor extent and, therefore, very reproducible CE methods are needed to detect them. In this chapter, a method for the analysis of prostate-specific antigen (PSA) by Capillary Zone Electrophoresis (CZE) with UV detection is detailed. High reproducibility in the separation of a large number of PSA isoforms is achieved by performing capillary conditioning in acid media and by using a background electrolyte (BGE) at pH 8.0 formulated with decamethonium bromide and urea.

Sample preparation of serum to allow capillary electrophoresis analysis of prostate specific antigen isoforms.

Prostate cancer is the second most frequently diagnosed cancer in men worldwide. Currently prostate specific antigen (PSA) serum concentration is the most used prostate cancer marker, but it only shows limited specificity. Because PSA glycosylation is altered by prostate cancer, detecting glycosylation changes could increase PSA specificity as a prostate cancer marker. Changes in PSA glycosylation can modify its electrophoretic- behavior and techniques such as capillary zone electrophoresis (CZE) and two-dimensional electrophoresis (2-DE) could be applied to detect changes in PSA glycosylation. Most serum PSA is complexed with alpha-1 antichymotrypsin (ACT). To have access to most of the PSA, the complexed PSA has to be released as free PSA (fPSA); in addition, this total fPSA must be purified from the serum matrix so that it can be analyzed using CZE. In this work a methodology for isolating PSA from serum for its CZE analysis was established. By using PSA standard, the effect of this methodology, which combines conditions for dissociating complexed PSA and immunoaffinity chromatographic purification, was studied. It was seen that this highly repeatable sample treatment did not noticeably alter the circular dichroism (CD) spectrum or the CZE pattern of PSA standard. Therefore, as a proof-of-concept, the developed sample treatment was applied to serum from a cancer patient with a high PSA content. The following observations can be made from these experiments: first of all, the 2-DE pattern of serum PSA remained unchanged after sample treatment; second, as hypothesized, the established sample preparation methodology made it possible to obtain the CZE pattern of PSA from serum; and third, the CZE pattern of serum PSA and of PSA standard from seminal plasma of healthy individuals, both submitted to the sample treatment method, showed some differences regarding the proportion of CZE peaks of the glycoprotein. These differences could be related to possible changes in the linkages of peptide backbone, in glycosylation or in other post-translational modifications between samples from both origins.