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.