Evaluation of gel electrophoresis techniques and laser ablation–inductively coupled plasma-mass spectrometry for screening analysis of Zn and Cu-binding proteins in plankton

Abstract

The determination of metal-binding proteins in plankton is important because of their involvement in photosynthesis, which is fundamental to the biogeochemical cycle of the oceans and other ecosystems. We have elaborated a new strategy for screening of Cu and Zn-containing proteins in plankton on the basis of separation of proteins by use of Blue-Native PAGE (BN-PAGE), which entails use of a non-denaturing Tris–tricine system and detection of metals in the proteins by laser ablation inductively coupled plasma mass spectrometry (LA–ICP–MS). For comparison, denaturing PAGE based on Tris–glycine and Tris–tricine systems and Anodic-Native PAGE have also been investigated. A large number of protein bands with MW between 20 and 75 kDa were obtained by use of Tris–glycine PAGE but detection of metals by LA–ICP–MS was unsuccessful because of loss of metals from the proteins during the separation process. Different protein extraction, purification, and preconcentration methods were evaluated, focussing on both issues—achieving the best extraction and characterization of the proteins while maintaining the integrity of metal–protein binding in the plankton sample. Use of 25 mmol L⁻¹ Tris–HCl and a protease inhibitor as extraction buffer with subsequent ultrafiltration and acetone precipitation was the most efficient means of sample preparation. Two Cu and Zn proteins were detected, a protein band corresponding to a MW of 60 kDa and another poorly resolved band with a MW between 15 and 35 kDa.