Evaluation of gel electrophoresis conditions for the separation of metal-tagged proteins with subsequent laser ablation ICP-MS detection.
Although laser ablation (LA)-ICP-MS has been reported for the determination of metalloproteins separated by gel electrophoretic techniques (GE), systematic studies that define the conditions essential for successful measurements are still scarce. In this paper we present the results of our studies of basic conditions for the effective application of GE-LA-ICP-MS for the separation of metal-binding proteins, focusing on their stability during GE and post-separation gel treatment. The stability of metal-protein complexes (haemoglobin, myoglobin, superoxide dismutase, carbonic anhydrase, transferrin, albumin, cytochrome c) during GE is dependent on the nature of the metal-protein interaction and the principle of separation. We have observed that non-denaturing GE is a suitable separation technique for most metal-protein complexes (e.g. Zn in carbonic anhydrase and Fe in Tf and myoglobin were quantitatively recovered in a spiked liver cytosol), whereas separation by denaturing GE strongly impaired the stability of the complexes. Equally important is the post-separation treatment of the gel to enable successful detection of the metal. LA-ICP-MS requires drying of the gel without loss of protein-bound metal or cracking of the gel. This was successfully achieved using glycerol followed by heating. We demonstrate that staining of the gel prior to LA-ICP-MS using silver or Coomassie blue is not recommended, since most protein-bound metal is lost during the staining procedure. Furthermore it has been shown that only line scanning with a speed of less than 30 microm/s can reliably distinguish between lines 1 mm apart, while raster spot analysis carries the risk of misinterpretation due to contamination in/on inhomogeneous gels.
Raab
Andrea
A
Department of Chemistry, University of Aberdeen, UK.
Pioselli
Barbara
B
Munro
Caroline
C
Thomas-Oates
Jane
J
Feldmann
Jörg
J
D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005990:Glycerol / Q000737:chemistry; D053685:Laser Therapy; D013058:Mass Spectrometry; D008667:Metalloproteins / Q000032:analysis*; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D012680:Sensitivity and Specificity; D013997:Time Factors
D008667:Metalloproteins; D005990:Glycerol