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Quantitative Proteomic Analysis for High-Throughput Screening of Differential Glycoproteins in Hepatocellular Carcinoma Serum

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Quantitative Proteomic Analysis for High-Throughput Screening of Differential Glycoproteins in Hepatocellular Carcinoma Serum Cancer Biol Med 2015;12:246-254. doi: 10.7497/j.issn.2095-3941.2015.0010 ORIGINAL ARTICLE Quantitative proteomic analysis for high-throughput screening of differential glycoproteins in hepatocellular carcinoma serum Hua-Jun Gao1, Ya-Jing Chen1, Duo Zuo2, Ming-Ming Xiao1, Ying Li1, Hua Guo2, Ning Zhang1,2, Rui-Bing Chen1 1Research Center of Basic Medical Sciences & School of Medical Laboratory, Tianjin Medical University, Tianjin 300070, China; 2Laboratory of Cancer Cell Biology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin Key Laboratory of Cancer Prevention and Therapy, Tianjin 300060, China ABSTRACT Objective: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths. Novel serum biomarkers are required to increase the sensitivity and specificity of serum screening for early HCC diagnosis. This study employed a quantitative proteomic strategy to analyze the differential expression of serum glycoproteins between HCC and normal control serum samples. Methods: Lectin affinity chromatography (LAC) was used to enrich glycoproteins from the serum samples. Quantitative mass spectrometric analysis combined with stable isotope dimethyl labeling and 2D liquid chromatography (LC) separations were performed to examine the differential levels of the detected proteins between HCC and control serum samples. Western blot was used to analyze the differential expression levels of the three serum proteins. Results: A total of 2,280 protein groups were identified in the serum samples from HCC patients by using the 2D LC-MS/MS method. Up to 36 proteins were up-regulated in the HCC serum, whereas 19 proteins were down-regulated. Three differential glycoproteins, namely, fibrinogen gamma chain (FGG), FOS-like antigen 2 (FOSL2), and α-1, 6-mannosylglycoprotein 6-β-N-acetylglucosaminyltransferase B (MGAT5B) were validated by Western blot. All these three proteins were up-regulated in the HCC serum samples. Conclusion: A quantitative glycoproteomic method was established and proven useful to determine potential novel biomarkers for HCC. KEYWORDS Glycoprotein; hepatocellular carcinoma (HCC); mass spectrometry; proteomics; serum Introduction benign ones. Other methods such as computed tomography and nuclear magnetic resonance are usually very expensive for Hepatocellular carcinoma (HCC) ranks the second leading routine screening3. Serum is an ideal sample source for HCC cause of cancer-related deaths worldwide1. HCC is usually detection. The known serum biomarkers for HCC include AFP, associated with viral infections, such as hepatitis C and hepatitis des-γ-carboxyprothrombin (DCP), fucosylated AFP (AFP-L3), B. HCC diagnosis is generally based on ultrasound scanning Golgi protein 73 (GP73), isozyme of alkaline phosphatase and serum alpha-fetoprotein (AFP) estimation every 6 months2. (variant ALP), and isozyme of gamma-glutamyl transpeptidase However, ultrasound can only detect tumors larger than 3 cm, (novel γ-GTP)4,5. AFP is a classical biomarker, and an increase in and this method cannot distinguish malignant tumors from AFP serum level correlates with tumor size6. However, in patients with chronic liver diseases, AFP concentration is also slightly increased. DCP is an abnormal prothrombin that has been used Correspondence to: Rui-Bing Chen for clinical HCC screening. High DCP level reflects a poor E-mail: [email protected] 7 Received January 30, 2015; accepted May 8, 2015. prognosis . DCP sensitivity for early and small HCC (<2 cm) 8 Available at www.cancerbiomed.org is only 56.5% . AFP-L3 is a fucosylated species of AFP, and the Copyright © 2015 by Cancer Biology & Medicine serum concentration of AFP-L3 is associated with poor HCC Cancer Biol Med Vol 12, No 2 June 2015 247 differentiation9. Novel serum biomarkers are urgently required matched normal control cohort, was pooled for quantitative to increase the sensitivity and specificity of serum biomarker glycoproteomic analysis. HCC diagnoses were confirmed screening for early HCC diagnosis. through histopathologic study. Mass spectrometry-based proteomic approaches have evolved as powerful tools to discover novel biomarkers10,11. However, Reagents and materials identification of potential protein biomarkers from biofluid samples, such as serum and plasma, remains challenging because Iodoacetamide, N-acetyl-D-glucosamine, methyl-R-D- of their large protein concentration range. Efforts have been mannopyranoside, methyl-R-D-glucopyranoside, manganese made to simplify serum samples via affinity chromatography, chloride tetrahydrate, formaldehyde, deuterated formaldehyde, either by removing abundant proteins from the serum or and sodium cyanoborohydride were purchased from Sigma- enriching a subproteome with a common chemical structural Aldrich (St. Louis, MO). Agarose-bound Concanavalin A feature12-14, e.g., affinity depletion using antibody-conjugated (Con A, 6 mg lectin/mL gel) and wheat germ agglutinin materials12,13. The interest in abnormal protein glycosylation (WGA, 7 mg lectin/mL gel) were purchased from Vector research is increasing. The currently known HCC biomarkers, Laboratories (Burlingame, CA). Dithiothreitol (DTT) and namely, AFP, AFP-L3, DCP, and GP73, are all glycoproteins. sequencing grade modified trypsin were purchased from Many biomarkers clinically used for cancer diagnosis are also Promega (Madison, WI). Antibodies used in Western blot were glycoproteins, such as prostate-specific antigen in prostate all obtained from Santa Cruz Biotechnology (Dallas, TX). cancer15; Her2/neu in breast cancer16; CA-125 in ovarian cancer17; and CEA in colorectal, breast, pancreatic, and lung Lectin affinity chromatography (LAC) cancer18. Therefore, targeting glycoproteins in the serum can enrich the potential biomarkers while reducing the serum sample Lectin affinity columns were prepared by adding 400 μL each complexity for in-depth proteome analysis. of Con A and WGA slurry to empty Micro Bio-Spin columns In this study, lectin affinity chromatography (LAC) was used to (Bio-Rad Laboratories, Hercules, CA), as reported by Wei et al.19. enrich glycoproteins from blood samples of 40 healthy volunteers Con A exhibited a high affinity to high-mannose type N-glycans, and 40 HCC patients. Stable isotope dimethyl labeling and 2D whereas WGA was selective for N-acetyl-glucosamine (GlcNAc). liquid chromatography (LC) separation were used for quantitative Up to 40 mL of pooled serum was diluted 10 times with the mass spectrometric analysis to examine the differential levels of the binding buffer (20 mM Tris, 0.15 M NaCl, 1 mM CaCl2, 1 mM detected proteins. More than 2,000 proteins were characterized MnCl2, pH 7.4) and loaded onto the lectin affinity columns. in the serum. A panel of proteins exhibited significant changes in After shaking for 6 h, unretained proteins were discarded, and relative abundances between the HCC and control samples. The lectin beads were washed with 2.5 mL of binding buffer. The expression patterns of fibrinogen gamma chain (FGG), FOS-like captured glycoproteins were eluted with 2 mL of elution buffer antigen 2 (FOSL2), and α-1,6-mannosylglycoprotein 6-β-N- (10 mM Tris, 0.075 M NaCl, 0.25 M Nacetyl-D-glucosamine, acetylglucosaminyltransferase B (MGAT5B) were validated by 0.17 M methyl-R-D-mannopyranoside, and 0.17 M methyl-R- Western blot. D-glucopyranoside). The eluted fraction was concentrated using a 10 kDa Centricon Ultracel YM-10 filter (Millipore, Billerica, Materials and methods MA). BCA assays were performed to measure the protein concentration. Serum collection Protein digest and stable isotopic labeling The study was approved by the Ethics Committee of Tianjin Medical University. Serum samples were processed from each Concentrated samples were denatured with 6 M urea in 0.2 M individual by using a 12G BD Vacutainer Safety-LokTM blood sodium acetate buffer (pH 8) and reduced by incubation with collection system. After collection, samples were immediately 10 mM DTT at 37 for 1 h. Reduced proteins were alkylated placed on ice and allowed to stand for 30 min. Samples were for 1 h in darkness with 40 mM iodoacetamide. Alkylation then centrifuged at 3,000 rpm for 15 min and stored at −80 reaction was quenched℃ by adding DTT to a final concentration until analysis. A total of 40 HCC serum samples were collected of 50 mM. Samples were diluted to a final concentration of and divided into two groups. Each serum cohort, which℃ 1 M urea. Trypsin was added at a 50:1 protein-to-trypsin mass consisted of 20 HCC samples and 20 cases of age- and gender- ratio, and samples were incubated at 37 overnight. Sodium ℃ 248 Gao et al. Quantitative glycoproteomic analysis of HCC serum cyanoborohydride was added to the protein digest to a final between 1.8 and 2.0 kV, and the mass resolution used for MS concentration of 50 mM. Samples were labeled with either scan was 30,000. For tandem mass analysis, the eight most 0.2 mM formaldehyde or 0.2 mM deuterated-formaldehyde. The abundant ions were automatically selected for collisionally mixed peptides were vortexed and incubated at 37 for 1 h. activated dissociation. The mass window for precursor ion Up to 2 M NH4OH was added to quench the reaction, and the selection was m/z ±1, and the normalized collision energy was mixture was immediately dried using SpeedVac. Finally,℃ samples set at 35% for MS/MS. Dynamic exclusion parameters were set were reconstituted in water. as follows: exclusion duration was 60 s, and exclusion mass width was 0.01% relative to
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