Purpose: Proteomic profiling using surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF MS) enables the identification of biomarkers for cancer.We evaluated the sensitivity and specificity of SELDI-TOF MS for detection of established hepatocellular cancer (HCC) and compared it against a-fetoprotein (AFP), Lens culinaris agglutinin^reactive AFP (AFP-L3), and prothrombin induced by vitamin K absence-II (PIVKA-II). Experimental Design: Forty-one patients with HCC and 51 patients with hepatitis C cirrhosis were enrolled. Serum was analyzed by SELDI-TOF MS using three Ciphergen protein array types. Results: An 11-peak algorithm for HCC detection was identified. Using the AFP cutoff of 20 ng/mL, the sensitivity was 73% and the specificity was 71%. Using the AFP-L3 cutoff of 10% yielded a sensitivity of 63% and a specificity of 94%. Using the PIVKA-II cutoff of 125 milliabsorbance units (mAU), the sensitivity was 84% and the specificity was 69%. Overall, the sensitivity and specificity of SELDI-TOF MS for HCC were 79% and 86%, respectively. In multivariate analysis, the 11-peak SELDI profile was predictive of HCC independent of AFP, PIVKA, and AFP-L3. Among eight patients with the largest tumor size of <2 cm, SELDI-TOF MS correctly identified seven whereas AFP, AFP-L3, and PIVKA-II identified only three, one, and one, respectively. One of the 11 peaks in the SELDI-TOF MS 11-peak predictor from SELDI-TOF MS was identified as cystatin C. Conclusions: SELDI-TOF MS accurately distinguished patients with HCC from those with hepatitis C virus cirrhosis, was more accurate than traditional biomarkers in identifying small tumors, and should be further evaluated.The incidence of hepatocellular carcinoma (HCC) is on the rise in the United States (1 -3). Recently, El-Serag et al. showed that the incidence of HCC had increased from 1.8 per 100,000 to 2.5 per 100,000 over one decade and that nearly all of this increase was attributable to infection with hepatitis C virus (HCV; ref. 2). Once cirrhosis has developed, retrospective studies have suggested that patients will develop either hepatic decompensation or HCC at a rate of 2% to 7% per year (4 -8). The general practice among many physicians has been to screen for HCC using ultrasound and serum a-fetoprotein (AFP) levels at 3-month to 6-month intervals.However, even with this screening regimen, many patients still present with either large HCC (>5 cm) or multifocal HCC (more than three lesions) or HCC that has invaded the portal vein or other critical structures. The limitations of ultrasound, the primary radiologic screening modality under current use, include its operator dependence and its poor ability to differentiate malignant from benign nodules in the small cirrhotic liver. Although imaging with triphasic computed tomography scan and magnetic resonance imaging with i.v. gadolinium can improve the diagnostic accuracy, these techniques are time consuming and too expensive for widespread screening at the present time. Because outco...

Biomarker discovery approaches in urine have been hindered by concerns for reproducibility and inadequate standardization of proteomics protocols. In this study, we describe an optimized quantitative proteomics strategy for urine biomarker discovery, which is applicable to fresh or long frozen samples. We used urine from healthy controls to standardize iTRAQ (isobaric tags for relative and absolute quantitation) for variation induced by protease inhibitors, starting protein and iTRAQ label quantities, protein extraction methods, and depletion of albumin and immunoglobulin G (IgG). We observed the following: With ongoing advances in mass spectrometry (MS) and proteomics technology, proteomics analysis is progressively occupying a central position in biomarker discovery platforms. Biofluids such as urine and blood are the preferred media for proteomics analysis because of their ease of collection and extensive history of use in clinical laboratory practice. Urine, in particular, is an information-rich fluid that can be collected non-invasively and in large quantities. Many urine proteins are produced or shed in the kidney and urogenital tract (1), making urine a promising proximal source of biomarkers for diseases affecting these structures.However, proteomics-based biomarker discovery in urine faces multiple challenges. Urine proteomics is complicated by low urine protein concentration, variations in pH, and high concentrations of salts and urea or other urine components that interfere with sample processing. The urine proteome can also change with individual variables such as hydration, diurnal change, diet, and physical activity as well as variation in sample collection, processing, and storage. In addition, urine proteomics shares the usual challenges of biomarker discovery in other biofluids such as throughput, cost, and the need for a reproducible and quantitative work flow.Isotopic or isobaric labeling methods to reduce variation, increase throughput, and enable quantitative analysis have been developed to address some of these challenges. One such method, isobaric tags for relative and absolute quantitation (iTRAQ) 1 (2), combines relative and absolute peptide quantification with multiplexing ability to enable an increased throughput as well as simultaneous comparison of up to eight samples within one experimental run. Variations induced by urine sample processing have been systematically evaluated for proteomics analyses using two-dimensional gel electrophoresis (3-6), differential gel electrophoresis (7), and liquid chromatography-coupled mass spectrometry (LC-MS) (5,8,9). However, no systematic analyses of urine sample collection and processing have been reported for iTRAQ.Before utilizing iTRAQ-based quantitative proteomics for urine biomarker discovery, we evaluated the impact of variation in several processing steps (addition of protease inhibitors, the starting protein quantities, quantity of the iTRAQ label, protein extraction methods, and depletion of abundant proteins) on iTRAQ protein identifica...