Proteomic-based identification of haptoglobin-1 precursor as a novel circulating biomarker of ovarian cancer

Abstract

Screening for specific biomarkers of early-stage detection of ovarian cancer is a major health priority due to the asymptomatic nature and poor survival characteristic of the disease. We utilised two-dimensional gel electrophoresis (2DE) to identify differentially expressed proteins in the serum of ovarian cancer patients that may be useful as biomarkers of this disease. In this study, 38 ovarian cancer patients at different pathological grades (grade 1 (n=6), grade 2 (n=8) and grade 3 (n=24)) were compared to a control group of eight healthy women. Serum samples were treated with a mixture of Affigel-Blue and protein A (5 : 1) for 1 h to remove high abundance protein (e.g. immunoglobulin and albumin) and were displayed using 11 cm, pH 4-7 isoelectric focusing strips for the first dimension and 10% acrylamide gel electrophoresis for the second dimension. Protein spots were visualised by SYPRO-Ruby staining, imaged by FX-imager and compared and analysed by PDQuest software. A total of 24 serum proteins were differentially expressed in grade 1 (P<0.05), 31 in grade 2 (P<0.05) and 25 in grade 3 (P<0.05) ovarian cancer patients. Six of the protein spots that were significantly upregulated in all groups of ovarian cancer patients were identified by nano-electrospray quadrupole quadrupole time-of-flight mass spectrometry (n-ESIQ(q)TOFMS) and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOFMS) as isoforms of haptoglobin-1 precursor (HAP1), a liver glycoprotein present in human serum. Further identification of the spots at different pathological grades was confirmed by Western blotting using monoclonal antibody against a haptoglobin epitope contained within HAP1. Immunohistochemical localisation of HAP1-like activity was present in malignant ovarian epithelium and stroma but strong immunostaining was present in blood vessels, areas with myxomatous stroma and vascular spaces. No tissue localisation of HAP1-like immunoreactivity was observed in normal ovarian surface epithelium. These data highlight the need to assess circulating concentration of HAP1 in the serum of ovarian cancer patients and evaluate its potential as a biomarker in the early diagnosis of ovarian cancer.

Figure 1

Two-dimensional gel electrophoresis profile of (A) untreated serum and (B) Affi-Gel Blue and protein A-treated human serum. Human serum was treated with Affi-Gel Blue and protein A (5 : 1) on a column for 1 h before analysis by 2-DE. Protein (50 μg) was loaded on each gel. Results are representative of three independent experiments.

Figure 2

Depiction of the reference profile of all protein spots identified by 2-DE. Profile of proteins differentially expressed (in red) in the serum of (A) grade 1 (n=6), (B) grade 2 (n=8) and (C) grade 3 (n=24) ovarian cancer patients. Comparison between normal (n=8) and each pathological grade of cancer patients’ serum was made on replicate sets using PDQuest software.

Figure 3

Elevated expression of protein spots 1–6 in the serum of ovarian cancer patients compared to serum from healthy volunteers. Image analysis was performed by using PDQuest software. (A) normal serum, (B) grade 1, (C) grade 2 and (D) grade 3 ovarian cancer patients.

Figure 4

TOFMS spectra obtained for the six HAP1 protein spots. After trypsin digestion, peptide fragments were analysed by n-ESIQ(q)TOFMS in TOFMS and tandem MS mode. (A–F) Demonstrates the TOFMS spectra of protein spots 1–6. Database searching using the Pep Sea software allowed the identification of the six spots as isoforms of HAP1 (Swissprot accession number P00737). Sequence data are presented in Table 2.

Figure 4

TOFMS spectra obtained for the six HAP1 protein spots. After trypsin digestion, peptide fragments were analysed by n-ESIQ(q)TOFMS in TOFMS and tandem MS mode. (A–F) Demonstrates the TOFMS spectra of protein spots 1–6. Database searching using the Pep Sea software allowed the identification of the six spots as isoforms of HAP1 (Swissprot accession number P00737). Sequence data are presented in Table 2.

Figure 5

2-DE Western profile of protein spots 1–6 in (A) normal, (B) grade 1 and (C) grade 3 ovarian cancer patients by using monoclonal-anti haptoglobin. Serum samples were resolved for first and second dimensions as described in the Materials and Methods section.

Figure 6

Expression of HAP1-like immunoreactivity in ovarian tissues. Sections of the normal and malignant ovarian tissues were stained by the immunoperoxidase method for the expression of HAP1-like activity as described in the Materials and Methods. (A) HAP1-like activity in normal ovary; (B) in endometriod grade 1 ovarian tumour, (C) serous grade 2 ovarian tumour and (D) serous grade 3 ovarian tumour. Arrows indicate the expression of HAP1-like activity in epithelial cells, myxomatous stroma and ovarian vessels.