title: Sample preparation of serum to allow capillary electrophoresis analysis of prostate specific antigen isoforms.
abstract: 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.
authors.affiliation: Institute of Organic Chemistry (IQOG-CSIC), Madrid, Spain.
authors.forename: Noemi
authors.initials: N
authors.lastname: Farina-Gomez
authors.affiliation: Unit of Biochemistry, Faculty of Sciences, University of Girona, Girona, Spain.
authors.forename: Silvia
authors.initials: S
authors.lastname: Barrabes
authors.affiliation: Institute of Organic Chemistry (IQOG-CSIC), Madrid, Spain; Department of Chemistry, Universidad del Valle, Cali, Colombia.
authors.forename: Jorge E
authors.initials: JE
authors.lastname: Gomez-Lopez
authors.affiliation: Institute of Organic Chemistry (IQOG-CSIC), Madrid, Spain.
authors.forename: Monica
authors.initials: M
authors.lastname: Gonzalez
authors.affiliation: Institute of Organic Chemistry (IQOG-CSIC), Madrid, Spain.
authors.forename: Angel
authors.initials: A
authors.lastname: Puerta
authors.affiliation: Institute of Organic Chemistry (IQOG-CSIC), Madrid, Spain.
authors.forename: Diana
authors.initials: D
authors.lastname: Navarro-Calderon
authors.affiliation: Department of Urology, Hospital La Princesa, Madrid, Spain.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Albers-Acosta
authors.affiliation: Department of Urology, Hospital La Princesa, Madrid, Spain.
authors.forename: Carlos
authors.initials: C
authors.lastname: Olivier
authors.affiliation: Institute of Organic Chemistry (IQOG-CSIC), Madrid, Spain.
authors.forename: Jose C
authors.initials: JC
authors.lastname: Diez-Masa
authors.affiliation: Unit of Biochemistry, Faculty of Sciences, University of Girona, Girona, Spain. Electronic address: rosa.peracaula@udg.edu.
authors.forename: Rosa
authors.initials: R
authors.lastname: Peracaula
authors.affiliation: Institute of Organic Chemistry (IQOG-CSIC), Madrid, Spain. Electronic address: mfrutos@iqog.csic.es.
authors.forename: Mercedes
authors.initials: M
authors.lastname: de Frutos
mesh_terms: D015415:Biomarkers / Q000032:analysis / Q000097:blood; D019075:Electrophoresis, Capillary / Q000379:methods; D006801:Humans; D008297:Male; D017430:Prostate-Specific Antigen / Q000032:analysis / Q000097:blood*; D011471:Prostatic Neoplasms / Q000097:blood* / Q000175:diagnosis; D020033:Protein Isoforms / Q000032:analysis / Q000097:blood; D057825:Two-Dimensional Difference Gel Electrophoresis / Q000379:methods*
chemical_list: D015415:Biomarkers; D020033:Protein Isoforms; D017430:Prostate-Specific Antigen
keywords: Capillary electrophoresis; Prostate cancer biomarker; Prostate specific antigen; Sample treatment; Serum; Two-dimensional electrophoresis