Elevated Levels of Phosphorylated Fibrinogen-r-Isoforms and Differential Expression of Other Post-Translationally Modified Proteins in the Plasma of Ovarian Cancer Patients Yuko Ogata,† Carrie J. Hepplmann,‡ M. Cristine Charlesworth,‡ Benjamin J. Madden,‡ Melinda N. Miller,‡ Kimberly R. Kalli,§ William A. Cilby,§ H. Robert Bergen III,‡ Diana A. Saggese,| and David C. Muddiman*,| Seattle Biomedical Research Institute, Seattle, Washington 98109, Mayo Proteomics Research Center, Department of Biochemistry and Molecular Biology and Department of Obstetrics and Gynecology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, and W.M. Keck FT-ICR Mass Spectrometry Laboratory, Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695 Received July 12, 2006
We evaluated the differentially expressed proteins in the plasma of ovarian cancer (OVC) patients using 2-D SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with post-translational modification (PTM) specific stains after the removal of six high-abundance proteins. The pooled plasma from patients with stage III or IV OVC was compared to a pooled postmenopausal age-matched control. Several proteins were identified as differentially expressed in the plasma of OVC patients. Among them, the phosphorylated fibrinogen-R-chain isoform (containing fibrinopeptide-A) was found to be up-regulated. Previously in our laboratory, phosphorylated fibrinopeptide-A was found to be up-regulated in the low molecular weight fraction of serum derived from OVC patients. We examined the levels of phosphorylated fibrinogen-R-chain in each patient that constituted the pooled plasma using Western blot, mass spectrometry (MS), and PTM specific stains. Phosphoprotein bands containing fibrinogen-R-chain fragments showed up-regulation in all OVC patients. Keywords: Ovarian cancer • biomarker • fibrinogen • two-dimensional gel electrophoresis • mass spectrometry • glycosylation • phosphorylation • protein depletion • Western blot
1. Introduction Ovarian cancer (OVC) is the fifth most frequent cause of cancer death in women.1 The American Cancer Society estimates that 20 180 women will be diagnosed with OVC in 2006 and that 15 310 women will die from the disease.1 The overall 5-year survival rate for women who are newly diagnosed with ovarian cancer is 45%. If diagnosed in the advanced stage (III or IV), the average survival rate is 29%. In stark contrast, if OVC is diagnosed in the early stages (I or II), while it is still confined to the ovary or limited spread within the pelvis, respectively, there is a 94% 5-year survival rate. However, due to the heterogeneity of OVC and the lack of predictive biomarkers, only 19% are diagnosed in the early stages.1 Therefore, early detection presents the best hope for decreasing the mortality from this disease. OVC manifests few early symptoms and lacks an easily performed diagnostic test for women with early symptoms. * Corresponding author. Phone: (919) 513-0084. Fax: (919) 513-7993. E-mail:
[email protected]. † Seattle Biomedical Research Institute. ‡ Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine. § Department of Obstetrics and Gynecology, Mayo Clinic College of Medicine. | North Carolina State University.
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Several different diagnostic tumor markers have been studied, and the field has advanced tremendously, but tumor markers are still limited by their low sensitivity and specificity. For a biomarker to be approved for screening in the general population, it must have a 10% positive predictive value.2 Currently, the most widely used marker for OVC is carbohydrate (cancer) antigen 125 (CA125).2 Approximately 83% of patients with advanced OVC have CA 125 levels >35 U/mL, but assays of this protein only detect a small proportion of patients with early-stage OVC.3,4 Nonetheless, CA125 has limited clinical application for the screening of early-stage disease since its positive predictive value is only 0.83-0.99% due to the low incidence rate (0.01% for individuals 65), despite its specificity of nearly 99% and a sensitivity of 50-60%.5 In the past 20 years, many new tumor markers have been discovered. Some of the most promising biomarkers are the following: macrophage colony-stimulating factor 1 (M-CSF), which is most commonly expressed in the secreted proteoglycan form and the intermembrane surface form;6 Mucin 1 (MUC1), a glycoprotein that contains several O-linked glycosylation sites with 50-80% of its mass coming from oligosaccharides;7 prostasin, a protease whose activity is largely dependent on the catalytic triad of aspartic acid, histidine, and 10.1021/pr060344+ CCC: $33.50
2006 American Chemical Society
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Elevated Levels of Fibrinogen-r-Isoforms
serine;8 the human tissue kallikriens, a group of parallel overexpressed genes in ovarian cancer that may form a cascade that initiates and maintains certain immune responses;9,10 and mesothelin, a membrane anchored protein that may play a role in spreading ovarian cancer throughout the peritoneal cavity.11 Recently, Mor et al. found that the combination of four proteins (leptin, prolactin, osteopontin, and insulin-like growth factor II) may possibly be an alternative marker with a specificity of 95% and a sensitivity of 95% using cytokine rolling circle amplification microarrays and ELISA.12 Although this is a significant improvement in sensitivity, with the low OVC prevalence rate, any screening strategy for early detection must achieve a minimum of 99.6% specificity.13 New technologies have also been utilized in the discovery of biomarkers. For example, Zheng et al. identified apolipoprotein A1, a truncated form of transthyretin and a cleavage fragment of inter-R-trypsin inhibitor heavy-chain H4 using SELDI-TOF MS,14 while Ahmed et al. reported serotransferrin and haptoglobin using the same technology.15 The protein HE4 was shown to be a possible marker using a new bead-based ELISA and microarray technology.16 Recently, Lebrilla and coworkers used MALDI-FT-ICR MS to identify potential oligosaccharide biomarkers in ovarian cancer patients.17 There are also several tests in development such as lysophosphatidic acid (LPA),18-20 and the IgM cancer recognition (CARE) test.21 A variety of traditional proteomics approaches has been also utilized for OVC biomarker discovery including 2-D PAGE analysis of serum samples;15 however, an extensive validation process is still required for their utility as a screening tool. As one explores the serum/plasma proteome in search of biomarkers, certain characteristics of the biological matrix need to be addressed to best utilize the analytical techniques available. In this study, we have addressed two issues related to the plasma proteome: (1) the extremely large dynamic range in protein concentrations and (2) post-translational modifications (PTMs) of proteins, which are often crucial for protein function/dysfunction. In plasma, the 30 most abundant proteins make up 98% of the protein by mass, while the remaining few percent constitute thousands of proteins.22 Therefore, removal of more abundant proteins can dramatically improve the number of proteins to be identified by reducing the dynamic range of protein levels in plasma to better match that of the downstream analytical platform. Previously in our laboratory, we have utilized the multiple affinity removal system (MARS) HPLC column (Agilent Technologies) for removal of six abundant proteins (human serum albumin (HSA), transferrin, IgG, IgA, antitrypsin, and haptoglobin) prior to 2-D PAGE analysis of cerebrospinal fluid (CSF),23 which has a similar protein composition as serum/plasma. The technique was useful in increasing the number of proteins visualized in 2-D PAGE (∼50% increase in the number of spots identified) and helpful in studying low-abundance proteins including those harboring PTMs. PTMs, in particular, phosphorylation, are of great interest in cancer studies as numerous oncogenes encode overactive mutant forms of protein tyrosine kinases (PTKs), whose normal counterparts regulate cell division and differentiation in response to extracellular signals. In addition, PTKs are also involved in the regulation of cytoskeleton and cell adhesion systems, which determine whether a cell will remain in place or migrate.24-27 In this study, we used a MARS HPLC column for abundant protein depletion, and PTM specific stains to study the differ-
entially expressed total, glyco-, and phosphoproteins in the pooled plasma samples from ovarian cancer patients as compared to case controls using 2-D PAGE. Previously in our laboratory, we have used HPLC coupled with high resolving power mass analyzers for differential expression analysis of the low molecular fraction of serum.28,29 We found that a phosphorylated fibrinopeptide-A had a predictive value in differentiating late-stage ovarian cancer patients from agematched controls. In the current study, we identified several proteins that were up- or down-regulated in OVC plasma. One of the up-regulated proteins was a phosphorylated fibrinogenR-chain isoform (containing fibrinopeptide-A). We examined the levels of the phophorylated fibrinogen-R-chain in plasma of each individual patient constituting the plasma pooled using PTM specific stains, Western blot, and mass spectrometry (MS) to confirm that the up-regulation is observed in all OVC patients.
2. Experimental Procedures 2.1. Plasma Samples. The plasma samples were procured from five women with stage III or IV OVC and five age-matched controls and stored in EDTA preserved tubes at -80 °C until they were pooled. The average age of individuals at the blood draw date was 66 for both controls and OVC patients. CA-125 values for OVC patients varied from 37 to 8775 U/mL with an average value of 2200 U/mL. A total of 500 µL of pooled OVC and control samples was generated by mixing 100 µL of plasma from each patient. A total of 350 µL of pooled plasma samples was diluted 5-fold with buffer A (proprietary phosphate-salt solution, pH 7.4, containing 0.02% NaN3; Agilent, Wilmington, DE), and the six high-abundance proteins were removed using the MARS HPLC column (4.6 mm × 100 mm; Agilent). The protein removal was achieved in nine sequential runs for each pool at a flow rate of 0.5 mL/min during the flow-through collection and at 1 mL/min during elution using a BioLogic Pathfinder chromatography system (Bio-Rad, Hercules, CA). To ensure the complete removal of the six abundant proteins, the column flow-through (depleted plasma protein solution) underwent the removal process once more after volume reduction to ∼1/5 using 5 kDa MWCO Vivaspin 6 (Sartorius, Edgewood, NY). 1-D gels were used to evaluate the completeness of the protein removal process at each step (data not shown). The protein concentration of the depleted protein sample for each pool was measured using a BCA protein assay reagent (Pierce, Rockford, IL) with bovine serum albumin (BSA) as a standard. The protein samples were buffer-exchanged into 25 mM ammonium bicarbonate (pH 8.3) using 5 kDa MWCO Vivaspin 6, and the volume was reduced to ∼400 µL for both OVC and control samples. A total of 700 µg of protein each from OVC and control depleted plasma samples was transferred to Eppendorf tubes from both OVC and control samples and dried in a SpeedVac (Savant, Farmingdale, NY). The protein was redissolved in a dissociation solution (7 M urea, 2 M thiourea, 4% CHAPS, 100 mM DTT, 1% 3-10 Pharmalyte, Amersham Biosciences, Piscataway, NJ) and vortexed for 2 h. The samples were further diluted with rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 60 mM DTT, 0.5% Pharmalyte, 0.25% 3-10 nL of IPG buffer, Bio-Rad) to 100 µg/380 µL just prior to IEF. 2.2. 2-D Electrophoresis. A total of six gels were developed (triplicate each from OVC and control samples). For the first dimension isoelectric focusing, ReadyStrip IPG strips, 18 cm, nonlinear (pH 3-10; Bio-Rad) were used. The sample in Journal of Proteome Research • Vol. 5, No. 12, 2006 3319
research articles rehydration buffer were applied to the IPG strips (100 µg/380 µL/strip) and rehydrated overnight. The strips were focused using the PROTEAN IEF unit (Bio-Rad) for a total of 52 000 V h, maintaining a limiting current of 30 µA/strip. The 20 × 20 × 0.1 cm 4-15% gradient gels were cast using acrylamide/bis acrylamide (29:1), 0.375 M Tris/HCl (pH 8.8), 0.05% w/v ammonium persulfate, and 0.03% v/v N,N,N′,N′tetramethylethylenediamine (TEMED) in MilliQ water. The IEF strips were equilibrated with gentle agitation for 10 min with ∼5 mL each of 1% w/v DTT in equilibration buffer (20% v/v glycerol, 0.38 M Tris base, pH 8.8. 6 M urea, and 2% SDS), followed by 15 min with 2% w/v iodoacetamide in equilibration buffer. The IPG strips were positioned on top of the gels and sealed in place with agarose sealing solution (1% w/v agarose in SDS-PAGE running buffer containing a trace of bromophenol blue dye). Two protein standard mixtures were used: CandyCane glycoprotein molecular weight standards and PeppermintStick phosphoprotein molecular weight standards (Molecular Probes). Paper wicks were wetted with 2 µL of each standard and inserted in the agarose on either side of the IPG strips. The gels were electrophoresed for 12 min at 140 V and 6 h at 200 V in running buffer containing 25 mM Tris, 0.1% SDS, and 190 mM glycine. 2.3. Gel Staining and Image Analysis. The gels were stained with Pro-Q Diamond for phosphoproteins, Pro-Q Emerald 488 for glycoproteins, and Sypro Ruby for total proteins (Molecular Probes) in the order listed. Specific staining conditions and the image analysis were described elsewhere.23 The images were scanned using a Bio-Rad Molecular Imager FX (Pro Plus) and analyzed using PDQuest (v 7.3) gel analysis software (Bio-Rad). Match sets containing two groups (OVC and control) were created for total, phospho-, and glycoprotein gel images separately, and differentially expressed protein spots were evaluated between the two groups. To adjust for small variations in sample load and staining, the gel images were normalized prior to the analysis using the total densities in gel images. The criteria used to define differentially expressed protein spots were (1) the difference in spot quantity >3 and (2) Student’s t-test >99% confidence level for total proteins and >95% confidence level for phospho- and glycoproteins. The gels were silver stained for visualization before differentially expressed spots were excised for in-gel tryptic digestion following the same protocols described previously.23 2.4. Mass Spectrometry and Database Analysis. Mass spectra were obtained using a thermoFinnigan LTQ linear ion trap mass spectrometer (ThermoElectron, San Jose, CA) coupled to a Michrom Paradigm MS4 (Michrom Bioresources Inc., Auburn, CA) with a 75 µm × 5 cm ProteoPep C18 PicoFrit nanoflow column. Experimental conditions are described elsewhere.23 The MS/MS data were searched against tryptic peptide sequences from the SWISS--PROT database using Mascot (Matrix Sciences, London, UK) search algorithms. All searches were conducted with differential modifications allowing +16 for methionine sulfoxide and +57 for carboxamidomethyl-cysteine. The search was restricted to trypsin generated peptides allowing for up to three missed cleavages. The taxonomy was set to humans. Peptide mass tolerance was (1.5 Da, and fragment mass tolerance was set to (0.8 Da. In this study, proteins were identified using the following criteria: (1) the number of peptides used for identification >2 and (2) the probability based MOWSE scores of the peptides >45. At the previous search criteria, scores >45 indicated an error rate of 5% or less. 3320
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2.5. Membrane Staining and Western Blotting of Individual Samples. To confirm the increased level of phosphorylated fibrinogen-R-chains in individual patients in OVC pooled plasma, the 1-D PAGE of the individual samples was examined using PTM specific stains, Western blot, and mass spectrometry. First, 10 µL of each plasma sample underwent the abundant protein depletion using the same multiple affinity removal system described previously. After measuring the protein concentrations of the column flow-through with the Bradford method using bovine serum albumin (BSA) as a standard, the sample buffer was exchanged to 20 mM TrisHCl (pH 8.0) using 5 kDa MWCO Vivaspin 6. The samples were dried in a SpeedVac and redissolved in Laemmli sample buffer (Bio-Rad) with 5% mercaptoethanol (Bio-Rad) and boiled in a water bath for 3 min. A total of 12 µg of protein/well was loaded on 12.5% Tris-HCl Criterion gels (Bio-Rad) for Western blot analysis. Duplicate gels were made for band excision and MS analysis. Gels were electrophoresed for 12 min at 140 V and 1 h at 200 V. The protein was transferred to PVDF membranes (Immobilon-P, Millipore) for 1 h at 100V, and the membranes were dried. The membranes were sequentially stained with Pro-Q Diamond phosphoprotein blot stain (Molecular Probe) followed by a Sypro Ruby blot stain (Molecular Probe) according to the manufacturer’s procedure. Images were scanned using a BioRad Molecular Imager FX (Pro Plus) after each staining procedure. The membrane was then blocked with 5% dry milk in Tris-buffered saline (TBST: 20 mM Tris, 150 mM NaCl, pH 7.5, 0.1% Tween 20) over night at 4 °C. The membrane was incubated with a primary antibody against fibrinogen-R (goat polyclonal, Santa Cruz Biotechnology, Inc. Santa Cruz, CA) for 1 h at room temperature. After washing the membranes 3 times with TBST, they were incubated with a secondary antibody conjugated to horseradish peroxidase (HRP-anti-goat IgG) (BioRad) for 30 min at room temperature. After washing, the membranes were overlaid with Pierce SuperSignal West Femto Maximum Sensitivity Substrate for 4 min and allowed to drip off. The images were developed using Kodak BioMax Light Film. The 1-D band density analysis was done using Quantity One 1-D analysis software (v 4.4.1, Bio-Rad) for the images acquired using a Bio-Rad Molecular Imager FX. One of the duplicate gels was used for protein identification using mass spectrometry. After staining for phosphoproteins and total proteins, bands of interest were excised, digested with trypsin, and analyzed using LC-MS/MS as described previously.
3. Results and Discussion 3.1. 2-D PAGE Analysis. Six abundant proteins were depleted from pooled OVC and control plasma samples using MARS column prior to gel analysis. Triplicate gels (4-15% gradient) were made for both OVC and control samples, and all gels were stained for total, phospho-, and glycoprotein with Sypro Ruby, Pro-Q Diamond, and Pro-Q Emerald dyes, respectively. Representative gel images are shown in Figure 1 (total protein) and Figure 2 (glyco- and phosphoprotein). The differentially expressed protein spots were identified using PDQuest software (v. 7.3) (Bio-Rad) by creating match sets separately for different stains. A total of 50 total protein spots, 21 glycoprotein spots, and 18 phosphoprotein spots were identified as either up- or down-regulated (difference in spot quantity >3) with Student’s t-test >99% for total protein and >95% for glyco- and phosphoprotein. They are marked in Figure 1 with red, green, and blue circles for total, glyco-, and phosphoprotein, respectively.
Elevated Levels of Fibrinogen-r-Isoforms
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Figure 1. 2-D PAGE of OVC and control stained for total proteins. The differentially expressed proteins are marked in Figure 1 with red, green, and blue circles for total-, glyco-, and phosphoproteins, respectively. Those up-regulated proteins of OVC patients are marked in OVC gel, and down-regulated proteins are marked in the control gel. The numbers in the figure correspond to the protein numbers in Table 1, where protein identifications are listed. The enlarged gel images of the boxed areas are shown in Figure 3A for all gels.
Figure 2. 2-D PAGE of OVC and control stained for glyco- and phosphoproteins. The enlarged gel images of the boxed areas are shown in Figure 3B for all gels.
Among the 21 glycoprotein spots, seven spots were also identified as differentially expressed for total protein, and one
for phosphoprotein, marked with two overlapping red and green circles and red and blue circles, respectively. Journal of Proteome Research • Vol. 5, No. 12, 2006 3321
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Table 1. List of Differentially Expressed Proteins/Protein Fragments Identified Using Mass Spectrometrya protein ID
PTMb
protein no.
accession
1 2 3 4 5 6 7 8 9 10
P01024 P01028 P27169 P02748 P08697 P02649 P01023 P19827 P02679 P02671
Down-regulated proteins/protein fragments in OVC patients complement C3 precursor complement C4 precursor serum paraoxonase/arylesterase 1 complement component C9 precursor R-2-antiplasmin precursor apolipoprotein E precursor R-2-macroglobulin precursor inter-R-trypsin inhibitor heavy-chain H1 precursor fibrinogen γ-chain precursor Fibrinogen-R/R-E-chain precursor [contains fibrinopeptide-A]
T/G G T T T T T/G G/P T T
1 2 3 10 11 12
P01024 P01028 P27169 P02671 P02675 P02741
Up-regulated proteins/protein fragments in OVC patients complement C3 precursor complement C4 precursor serum paraoxonase/arylesterase 1 fibrinogen-R/R-E-chain precursor [contains fibrinopeptide-A] fibrinogen-β-chain precursor C-reactive protein precursor
T T/G T/G P T T
a Some proteins were identified as both up- and down-regulated, indicating that the involvement of enzymes leads to the novel cleavage products. b T ) total protein, G ) glycoprotein, and P ) phosphoprotein.
Figure 3. Enlarged images of CRP (A) and fibrinogen-R-chain region (B) of 2-D PAGE indicated in Figures 1 and 2 for all gels. Circled spots were identified as up-regulated in OVC plasma. CRP and fibrinogen-R-chain gel images are from gels stained for total and phosphoproteins, respectively.
Since the PTM specific dyes bind to the modifications themselves (i.e., carbohydrates or phosphate groups), differentially expressed glyco- or phosphoprotein spots that did not show differential expression in total protein indicate an increase in the level of modification while the amount of total protein remains unchanged. It is notable that none of the differentially expressed phosphorylation spots showed significant changes in total protein quantity, thus indicating hyper- (or hypo-) phosphorylation. 3.2. Mass Spectrometric Identification of Differentially Expressed Protein Spots. A total of 82 unique differentially expressed spots were excised, and their protein identifications were determined using mass spectrometry. A total of 56 spots with sufficient protein quantities yielded protein identifications with LC-MS/MS to a total of 12 proteins listed in Table 1. Many differentially expressed spots are part of the pearl necklacelike chains of spots that are isoforms of the same protein. In addition, some proteins (complement C3 and C4 precursors, fibrinogen, and serum praoxonase/arylesterase 1) were identified as both up- and down-regulated, indicating the involvement of enzymes that lead to the novel cleavage products. C-Reactive protein (CRP) (MW ) 25 023 Da, pI ) 5.45) was found to be up-regulated in OVC patients using the 3322
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total protein stain. Figure 3A shows the enlarged CRP spot areas for all gels. The location of this area in relation to the entire 2-D PAGE is also indicated in Figure 1. This result is consistent with the knowledge that CRP is an inflammatory response marker and in late-stage OVC, the tumor burden (mass) is significant and the cancer is metastatic. Thus, given that CRP is an inflammatory response marker, the specificity of this marker for OVC (or other diseases) is highly questionable. Some fibrinogen-R-chain (MW ) 94 973, pI ) 5.7 for unprocessed precursor including fibrinopeptide-A) spots were found to be down-regulated using gels stained for total protein and up-regulated using gels stained for phosphoprotein. Fibrinogen is a symmetrical dimer composed of six paired polypeptide chains (R-, β-, and γ-chains). Each molecule contains two outer D domains connected to a central E domain.30 Fibrin is formed after thrombin cleavage of fibrinopeptide-A from the fibrinogen-R-chain, which is the major component of blood clots. Down-regulated fibrinogen-R total protein spots were located in the ∼70 kDa MW region of the gel (Figure 1, protein no. 10), which is consistent with the previous annotation in plasma 2-D PAGE (http://www.expasy.org/ch2dothergifs/publi/plasma-
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Figure 4. MW 45-24 kDa region of the Western blot and the same membrane stained for phosphoprotein and total protein. The arrows indicate bands that contained fibrinogen-R-chain confirmed by either Western blot or MS. Quantity One 1-D analysis software (v 4.4.1) identified three bands in the top half of the region for both controls and OVC individuals.
basic.gif). In this region of plasma 2-D PAGE, the fibrinogenR/R-E-chain is generally present as a sequence of multiple spots due to different isoforms. The three down-regulated total protein spots were located toward the more acidic region of the gel from the rest of the fibrinogen-R-spot cluster, which may indicate down-regulation of enzymes contributing to the formation of these isoforms. Up-regulated fibrinogen-R-spots were found in a region of the 2-D gel where it has not been annotated previously. The spot locations are marked in Figure 1 with protein no. 10, and the region is noted in the OVC phosphoprotein gel image in Figure 2 (45-25 kDa area). The spot images from all gels for the region specified in Figure 2 are shown in Figure 3B. In total protein gel images, these spots are barely visible and did not show significant change in quantities between OVC patients and controls, indicating hyper-phosphorylation of these fibrinogen-R-isoforms. There are seven known phosphorylation sites within the fibrinogen-R/R-E-chain, which are amino acid locations 22, 364, 460, 576, 577, 578, and 618.31-34 Amino acid 22 is part of fibrinopeptide-A. Peptides used to identify the up-regulated fibrinogen-R-isoforms were matched to peptides from the locations near the N-terminus (amino acid locations from 1 to ∼300), and considering the MW region of the gel (
