RESEARCH ARTICLE Enrichment of low molecular weight fraction of serum for MS analysis of peptides associated with hepatocellular carcinoma Eduard Orvisky 1 , Steven K. Drake 2 , Brian M. Martin 3 , Mohamed Abdel-Hamid 4 , Habtom W. Ressom 1 , Rency S. Varghese 1 , Yanming An 1 , Daniel Saha 1 , Glen L. Hortin 2 , Christopher A. Loffredo 1 and Radoslav Goldman 1 1 Georgetown University, Department of Oncology, Lombardi Comprehensive Cancer Center, Washington, DC, USA 2 Clinical Chemistry Service, Department of Laboratory Medicine, NIH, Bethesda, MD, USA 3 Unit of Molecular Structures, LNT, NIMH, NIH, Bethesda, MD, USA 4 Viral Hepatitis Research Laboratory, NHTMRI, Cairo, Egypt A challenging aspect of biomarker discovery in serum is the interference of abundant proteins with identification of disease-related proteins and peptides. This study describes enrichment of serum by denaturing ultrafiltration, which enables an efficient profiling and identification of peptides up to 5 kDa. We consistently detect several hundred peptide-peaks in MALDI-TOF and SELDI-TOF spectra of enriched serum. The sample preparation is fast and reproducible with an average CV for all 276 peaks in the MALDI-TOF spectrum of 11%. Compared to unenriched serum, the number of peaks in enriched spectra is 4 times higher at an S/N ratio of 5 and 20 times higher at an S/N ratio of 10. To demonstrate utility of the methods, we compared 20 enriched sera of patients with hepatocellular carcinoma (HCC) and 20 age-matched controls using MALDI-TOF. The comparison of 332 peaks at p , 0.001 identified 45 differentially abun- dant peaks that classified HCCwith 90% accuracy in this small pilot study. Direct TOF/TOF se- quencing of the most abundant peptide matches with high probability des-Ala-fibrinopeptide A. This study shows that enrichment of the low molecular weight fraction of serum facilitates an efficient discovery of peptides that could serve as biomarkers for detection of HCC as well as other diseases. Received: June 14, 2005 Revised: October 18, 2005 Accepted: November 21, 2005 Keywords: Biomarker / Fibrinopeptide A / Hepatocellular carcinoma / MALDI-TOF MS / Serum Proteomics 2006, 6, 2895–2902 2895 1 Introduction Discovery of biomarkers for clinical use typically requires comparison of a large number of samples, which limits the applicability of many elegant proteomic methods [1, 2]. SELDI-TOF and SELDI-QqTOF analyses of serum were optimized for high-throughput comparison of biological samples as small as a few microliters [3]. Advanced statistical Correspondence: Radoslav Goldman, Georgetown University, LCCC Room S183, 3970 Reservoir Rd NW, Washington, DC 20057-1469, USA E-mail: rg26@georgetown.edu Fax: 11-202-6871988 Abbreviations: HCC, hepatocellular carcinoma; HMW, high mo- lecular weight; LMW, low molecular weight; SVM, support vector machine DOI 10.1002/pmic.200500443  2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com