Thyroid
tumorigenesis involves qualitative and quantitative changes in
protein expression, which can be comprehensively studied by
proteome analysis. However, one of the technical bottlenecks of proteomics remains a reliable, sensitive and inexpensive method for quantification of differentially expressed
proteins. This is due to the limited linear range of most available
protein stains, i.e.
silver and
Coomassie blue, and high costs of commercially available fluorescent stains. In this paper we describe our experience with a lab-made
ruthenium based fluorescent
stain (
ruthenium II tris(bathophenanthroline disulfonate) (RuBPs)) to perform
proteome analysis of nodular
thyroid disease. We first compared the properties of RuBPs with two highly sensitive
protein stains: (1)
silver staining and (2) the commercially available
fluorescent dye Sypro Ruby. We show that in addition to its highly sensitive staining capabilities similar to
Sypro Ruby and
silver (2 ng), RuBPs offers several advantages such as a broad dynamic range (similar to
Sypro Ruby and 500 times broader than the dynamic range of
silver stain), low costs ( 0.03 per gel) and excellent compatibility with mass spectrometry. We then applied the inexpensive RuBPs
stain to 2D
gels (pH 4-7) of four benign
thyroid nodules and normal thyroid tissue. We were able to detect approximately 1800
protein spots/gel in our thyroid samples. Quantitative changes in
protein expression levels of at least 20-42
proteins were noted in the benign nodules compared with the normal thyroid tissue of the same patient. Differentially expressed spots were further characterised by nano-LC-FTICR and MALDI-TOF mass spectrometry. In summary we demonstrate, that the novel fluorescent
ruthenium II tris(bathophenanthroline disulfonate)
stain is a highly sensitive, reliable and inexpensive tool for quantitative
proteome analysis in thyroid nodular disease.