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.