RESEARCH ARTICLE Diagnostic biomarkers differentiating metastatic melanoma patients from healthy controls identified by an integrated MALDI-TOF mass spectrometry/bioinformatic approach Balwir Matharoo-Ball 1 * , Lucy Ratcliffe 1 * , Lee Lancashire 2 * , Selma Ugurel 3, 4 , Amanda K. Miles 1 , Daniel J. Weston 1 , Robert Rees 1 , Dirk Schadendorf 3, 4 , Graham Ball 2 and Colin S. Creaser 1 1 Interdisciplinary Biomedical Research Centre, School of Biomedical and Natural Sciences, Nottingham Trent University, Clifton Lane, Nottingham, UK 2 Loreus Ltd., Nottingham Trent University, Clifton Lane, Nottingham, UK 3 Skin Cancer Unit, German Cancer Research Center, Heidelberg, Germany 4 Department of Dermatology, University Hospital Mannheim, Germany The prognosis of advanced metastatic melanoma (American Joint Committee on Cancer (AJCC) stage IV) remains dismal with a 5-year survival rate of 6–18%. In the present study, an integrated MALDI mass spectrometric approach combined with artificial neural networks (ANNs) analysis and modeling has been used for the identification of biomarker ions in serum from stage IV melanoma patients allowing the discrimination of metastatic disease from healthy status with high specificities of 92% for protein ions and 100% for peptide biomarkers. Our ANNs model also correctly classified 98% of a blind validation set of AJCC stage I melanoma samples as non- stage IV samples, emphasizing the power of the newly defined biomarkers to identify patients with late-stage metastatic melanoma. Sequence analysis identified peptides derived from metas- tasis-associated proteins; alpha 1-acid glycoprotein precursor-1/2 (AAG-1/2) and complement C3 component precursor-1 (CCCP-1). Furthermore, quantitation of serum AAG by an immunoassay showed a significant (p,0.001) increase in AAG serum concentration in stage IV patients in comparison with healthy volunteers; moreover; the quantity of AAG plotted against MALDI-MS peak intensity classified the groups into two distinct clusters. Ongoing studies of other disease stages will provide evidence whether our strategy is sufficiently robust to give rise to stage-spe- cific protein/peptide signatures in melanoma. Received: January 10, 2007 Revised: March 6, 2007 Accepted: April 11, 2007 Keywords: Biomarkers / MALDI / Mass spectrometry / Melanoma Proteomics Clin. Appl. 2007, 1, 605–620 605 Correspondence: Professor Colin Creaser, Interdisciplinary Bio- medical Research Centre, School of Biomedical and Natural Sciences, Nottingham Trent University, Clifton Lane, Notting- ham, NG11 8NS, UK E-mail: colin.creaser@ntu.ac.uk Fax: 144-1509-223925 Abbreviations: AAG, alpha 1-acid glycoprotein; AAG-1/2, alpha 1-acid glycoprotein precursor-1/2; AJCC, American Joint Com- mittee on Cancer; ANN, artificial neural network; CCCPP-1, com- plement C3 component precursor-1; LDH, lactate dehydrogen- ase; NIR, normalized intensity ratios; * These authors contributed equally to this work. DOI 10.1002/prca.200700022  2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.clinical.proteomics-journal.com