Electrophoresis 2013, 34, 1637–1648 1637 Lenka Vyslouzilova 1 Sona Krizkova 2,3 Jiri Anyz 1 David Hynek 2,3 Jan Hrabeta 4 Jarmila Kruseova 4 Tomas Eckschlager 4 Vojtech Adam 2,3 Olga Stepankova 1 Rene Kizek 2,3 1 Department of Cybernetics, Faculty of Electrical Engineering, Czech Technical University, Prague, Czech Republic 2 Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Brno, Czech Republic 3 Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic 4 Department of Paediatric Haematology and Oncology, 2nd Medical Faculty, Charles University in Prague and University Hospital Motol, Prague, Czech Republic Received October 14, 2012 Revised December 3, 2012 Accepted December 27, 2012 Research Article Use of brightness wavelet transformation for automated analysis of serum metallothioneins- and zinc-containing proteins by Western blots to subclassify childhood solid tumours In this study, we determined serum levels of metallothioneins (MTs) and zinc in children with solid tumours (neuroblastoma, Hodgkin lymphoma, medulloblastoma, osteosar- coma, Ewing sarcoma and nephroblastoma) by differential pulse voltammetry Brdicka reaction and ELISA. Zn(II) level in patients sera was 40% compared to controls, con- trariwise, MT level was 4.2 × higher in patients. No significant differences among single diagnoses were found both for Zn(II) and MT. When determined Zn(II)/MT ratio, in controls its value was 24.6, but it was 2.6 in patients. After Western-blotting with anti-MT and anti-Zn chicken antibodies, variable intensities of the bands within the samples were observed. The brightness curve obtained for each sample both for MT- and Zn blots was further analysed to produce a list of band positions together with some complementary information related to the intensity of the observed bands by the optimised algorithm. We constructed from those profiles decision trees that enable to distinguish different groups of tumours. The blood samples were heat-treated, in which we supposed mainly MT, but samples contained other thermostable Zn-containing proteins that were helpful for identification of embryonal tumours with 88% accuracy and for identification of sarcomas with 78% accuracy. In MT blots the accuracies were 53 and 45%, respectively. Simulta- neous analysis of MT and Zn blots did not increased accuracy of identification neither in embryonal tumours (80%) nor in sarcomas. Those results are promising not only from diagnostic point of view but particularly in the area of studying of individual MT isoforms and their aggregates in malignant tumours. Keywords: Cancer / Metallothionein / Signal processing / Zinc DOI 10.1002/elps.201200561 1 Introduction Metallothioneins (MTs) are evolutionary highly conserved ubiquitous metal-binding proteins, which were discovered by Margoshes and Valee as a cadmium-binding proteins in horse kidney in 1957 [1]. Later on, their involving in heavy metal homeostasis, oxidative stress coping, gene expression and transcription regulation, enzymes activation, apoptosis and cell proliferation have been found [2, 3]. Till now, four major isoforms (MT-1 through MT-4) have been identified in mammals [4, 5]. MTs genes are tightly linked, and at a mini- Correspondence: Dr. Rene Kizek, Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno, Czech Republic E-mail: kizek@sci.muni.cz Fax: +420-5-4521-2044 Abbreviation: MT, metallothionein mum they consist of eleven MT-1 genes (MT-1A, -B, -E, -F, -G, -H, -I, -J, -K, -L and -X) encoding functional or non-functional RNAs, and one gene for each of the other MTs isoforms (the MT-2 A gene, MT-3 gene and MT-4 gene) [6]. Concerning their primary structure, they are rich in cysteine and have no aromatic amino acids [7]. Current knowledge of MTs is juxta- posed with our understanding of the pathogenesis of disease [8]. MT is known to modulate three fundamental processes: (i) the release of gaseous mediators such as hydroxyl radical or nitric oxide; (ii) apoptosis; (iii) the binding and exchange of metals such as zinc and copper on one side, and cadmium and platinum on the other side. Associations among MTs and several diseases, including cancer, circulatory and septic shock, coronary artery disease and Alzheimer’s disease have been found. Furthermore, strong evidence exists that MTs modulate the immune system [5]. Colour Online: See the article online to view Figs. 1–5 in colour. C  2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com