Liquid Chromatographic Determination of Acidic β-Aspartyl and γ-Glutamyl Peptides in Extracts of Rat Brain

Abstract

This work describes further development of our previously presented method for determination of acidic sulfur/phosphor-containing amino acids, γ-glutamyl di/tripeptides, and β-aspartyl dipeptides. Automated precolumn fluorogenic derivatization was performed with o-phthaldialdehyde/β-mercaptoethanol and the derivatives were separated by reversed-phase liquid chromatography. The method was optimized for the analysis of brain tissue extracts. Due to the complex sample matrix, three separation schemes with complementary selectivities were developed. Different extraction protocols were evaluated and sonication of frozen tissue powder in methanol-H2O (9:1, v/v) yielded the highest recoveries and precision. β-Mercaphtoethanol and EDTA were added to the extraction media to inhibit spontaneous oxidation of thiol-containing amino compounds. Analyte identification was based on retention times and recovery of standards added to extracts. The following compounds were identified in rat cerebral cortex (mean tissue concentration ± SD, n = 6): γ-glutamylglutamine (38.5 ± 12.6 μM), γ-glutamylglutamate (14.4 ± 6.0 μM), γ-glutamyltaurine (4.9 ± 2.2 μM), β-aspartylglycine (4.0 ± 0.4 μM), β-aspartyltaurine (3.7 ± 0.6μM), O-phosphoserine (3.2 ± 0.8 μM), γ-glutamylcysteine (1.9 ± 0.3 μM), γ-glutamylglycine (1.1 ± 0.1 μM), and γ-glutamylcysteateglycine (0.8 ± 0.1μM). In addition over 15 unidentified components were found. Cysteate, cysteine sulfinate, homocysteate, homocysteine sulfinate, O-Sulfoserine, γ-glutamylaspartate, γ-glutamylcysteate, γ-glutamylhistidine, and β-aspartylalanine were not present at concentrations above 1 μM.