Screening of selenium containing proteins in the Tris-buffer soluble fraction of African catfish (Clarias gariepinus) fillets by laser ablation-ICP-MS after SDS-PAGE and electroblotting onto membranes

Abstract

The anti-carcinogenic properties of selenium against certain types of cancer when present in organic forms justify the increasing interest in development of selenium fortified food. In this particular study, African catfish (Clarias gariepinus) were fed with a Se-enriched diet in order to enhance the selenium concentration in the fish fillet up to 0.85 ± 0.06 mg Se kg⁻¹ (0.26 ± 0.02 mg Se kg⁻¹ in control). Selenium distribution in proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was studied for better understanding of the metabolism of this element in the organism. Soluble proteins (Tris buffer) were investigated by conventional application of a glass homogenizer in comparison to application of ultrasound probe sonication, and the latter was convincing due to higher protein extraction efficiency and the ease and speed of application. Laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) was successfully applied for screening of selenium in proteins in fillets of African catfish separated by one-dimensional SDS-PAGE and after electroblotting onto nitrocellulose membranes, with a total Se amount below 0.4 ng loaded into a well. Selenium was detected in more than 11 protein spots. In order to improve the protein separation of one-dimensional SDS-PAGE, pre-fractionation of the Tris buffer soluble protein extract by size exclusion was carried out. Tryptic digestion of the Se-containing bands was performed for protein identification by nano-HPLC coupled to electrospray-MS/MS. A database search revealed several proteins which are located in muscle tissue. However, the very low Se concentration circumvents the detection of selenium containing amino acids in the tryptic peptides by electrospray-MS/MS.