Peptides in Low Molecular Weight Fraction of Serum Associated with Hepatocellular Carcinoma

11 Disease Markers 29 (2010) 11–20 DOI 10.3233/DMA-2010-0721 IOS Press Peptides in low molecular weight fraction of serum associated with hepatocellular carcinoma Yanming Ana , Slavka Bekesovaa, Nathan Edwardsb and Radoslav Goldmana,b,∗ a Georgetown University, Department of Oncology, Lombardi Comprehensive Cancer Center, Washington, DC, USA b Georgetown University, Department of Biochemistry and Molecular & Cellular Biology, Washington, DC, USA Abstract. The incidence of hepatocellular carcinoma (HCC) in the United States is increasing and the increase is projected to continue for several decades. The overall survival of HCC patients is poor and treatments are not effective in part because most of the diagnoses come at a late stage. The development of new markers for detection of HCC would significantly improve patient prognosis. This paper describes identification of candidate markers previously reported in our serologic study of an Egyptian population by quantitative comparison of matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectra. To identify these marker candidates, we performed LC-MS/MS sequencing that identified nine native peptides associated with HCC, including two reported previously. Four truncations of N terminus of complement C3f and a fibrinopeptide increased in control sera; two complement C4α peptides, a zyxin peptide, and a coagulation factor XIII peptide increased in cancer patient sera. We have also identified increased biliverdin diglucuronide in the sera of cancer patients. These peptides could potentially serve as markers of HCC following additional validation studies; however, association of similar peptides with other diseases and cancers dictates a very cautious approach. Keywords: Hepatocellular carcinoma, mass spectrometry, serum, complement, biliverdin diglucuronide 1. Introduction Hepatocellular carcinoma (HCC) is a highly fatal cancer that affects annually approximately half a million people worldwide [1,2]. Liver cancer ranks fifth in frequency of cancers in the world. Since the mid1980s, the incidence of HCC in the United States has been rising and this increase is predicted to continue in the next few decades primarily due to hepatitis C viral (HCV) infection [2–5]. The overall survival rate of HCC patients is poor which is in part related to the lack of reliable tools for early diagnosis. A diagnosis of HCC is often made at a late stage when the disease is too advanced for an effective treatment. Surveillance of patients at high risk for developing HCC is ∗ Corresponding author: Radoslav Goldman, Department of On- cology, Georgetown University Medical Center, 3970 Reservoir Rd NW, LCCC Room S183, Washington, DC 20057, USA. Tel.: +1 202 687 9868; Fax: +1 202 687 1988; E-mail: . an important strategy that can potentially decrease the cancer-related mortality. Chronic hepatitis C viral infection with subsequent liver cirrhosis is the major risk factor for development of HCC [2,6,7]. Patients with cirrhosis are recommended to undergo routine surveillance. Diagnosis is most often based on radiological imaging. Abdominal ultrasound is the most common imaging modality used in the surveillance of HCC, but it is operator dependent and limited in its ability to differentiate HCC from such non-neoplastic lesions as regenerative nodules [2,8]. The only serologic marker for HCC surveillance is alpha fetoprotein (AFP). AFP has reported sensitivity of 39 to 65%, specificity of 76 to 94%, and positive predictive values of 9% to 50% [2,9]. This is not sufficient for an efficient detection of HCC. New biomarkers for early detection and prevention of HCC are needed and a search is under way in several laboratories [10–13]. The management of the disease is predicted to benefit from identification of appropriate serologic markers ISSN 0278-0240/10/$27.50  2010 – IOS Press and the authors. All rights reserved 12 Y. An et al. / Peptides in hepatocellular carcinoma that would assist with the optimization of therapeutic decisions. It was shown that the presence of tumors is associated with changes in the composition of native peptides in serum [14]. Liver is a major source of circulating proteins and HCC is expected to induce significant perturbations. Proteases and peptidases are reportedly deregulated in HCC [15–18] and it is reasonable to expect that cancer-specific peptides and fragments of proteins will be found in the low molecular weight fraction of serum [19–21]. Our laboratory has developed a denaturing ultrafiltration method for enrichement of the LMW serum proteins and peptides for MALDI-TOF mass spectrometric analysis [19,20]. The method has allowed a fast comparison of hundreds of patient samples which is expected to decrease the gap between marker discovery and clinical validation [22]. In previous articles, we reported consistent differences associated with HCC in a large sample of patients from an Egyptian population (73 HCC cases, 72 cancer-free controls, and 52 chronic liver disease controls) [19,21]. Here we describe mass spectrometric identification of nine of the differentially abundant peptide peaks which will further facilitate examination of their association with HCC. 2. Experimental procedures 2.1. Materials AmiconUltra-4 Centrifugal Filter Devices with 30 kDa cut-off were purchased from Millipore (Bedford, MA, USA). Sep-Pak Vac RC C18 Cartridges were bought from Waters Corporation (Milford, MA). BCA protein assay was purchased from Pierce Biotechnology (Rockford, IL, USA). α-cyano-4-hydroxycinaminic acid (CHCA) was purchased from Bruker Daltonics (Billerica, MA, USA). Opti-TOF 384 well MALDI plate and mass calibration standards were purchased from Applied Biosystems (Framingham, MA). Other chemicals and solvents were purchased from SigmaAldrich (St. Louis, MO, USA); solvents were of HPLC grade. 3. Blood sample collection and preparation Serum samples of HCC cases and controls with(out) chronic liver disease were obtained in collaboration with the National Cancer Institute of Cairo University, Egypt from 2000 to 2002 as described previously [19, 21]. Blood samples were collected by a trained phlebotomist around 10 am and processed within a few hours according to a standard protocol. Aliquots of sera were frozen at −80◦ C immediately after processing until analysis; all mass spectrometric measurements were performed on twice-thawed sera. For sequencing of peptides, 0.5 ml of serum was diluted in 2.5 ml 8M guanidine hydrochloride and ultrafiltered at 3,000 x g for 90 minutes at 4◦C using 30-kDa AmiconUltra membrane (Millipore). The ultrafiltrate was desalted on a Sep-Pak C18 cartridge (Waters) according to the manufacture’s protocol and dried in a centrifugal vacuum evaporator. 4. Fractionation of proteins The dried ultrafiltrate was resuspended in 20 µL 0.1% TFA/water and separated on Chromolith Performance RP-18 Column (100 × 4.6 mm) (EMD Chemicals, NJ) at room temperature. A flow (1 m (...truncated)