Evaluation of gel electrophoresis techniques and laser ablation-inductively coupled plasma-mass spectrometry for screening analysis of Zn and Cu-binding proteins in plankton.
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.
Analytical Spectroscopy and Sensors Group, Institute of Environmental Sciences, University of Zaragoza, Pedro Cerbuna, 12, Zaragoza 50009, Spain. jimenezm@unizar.es
Maria S
MS
Jiménez
L
L
Rodriguez
Juan R
JR
Bertolin
Maria T
MT
Gomez
Juan R
JR
Castillo
D000818:Animals; D002021:Buffers; D002245:Carbon Dioxide / Q000737:chemistry; D002417:Cattle; D003300:Copper / Q000737:chemistry*; D017753:Ecosystem; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005998:Glycine / Q000031:analogs & derivatives / Q000737:chemistry; D053685:Laser Therapy / Q000379:methods*; D007834:Lasers; D013058:Mass Spectrometry / Q000379:methods*; D008670:Metals / Q000737:chemistry; D008970:Molecular Weight; D010756:Phosphoric Acids / Q000737:chemistry; D010933:Plankton / Q000737:chemistry; D011506:Proteins / Q000737:chemistry; D012709:Serum Albumin / Q000737:chemistry; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D015032:Zinc / Q000737:chemistry*
D002021:Buffers; D008670:Metals; D010756:Phosphoric Acids; D011506:Proteins; D012709:Serum Albumin; D002245:Carbon Dioxide; D012967:Sodium Dodecyl Sulfate; D003300:Copper; C030242:phosphoric acid; D015032:Zinc; D005998:Glycine; C100184:tricine