Detected early enough, patients with ovarian cancer have a 90% five-year survival rate. However, reliable and accurate circulating biomarkers that identify the early-stage disease are currently not available, condemning patients to a less than 30% chance of living beyond five years post-diagnosis.
Tang et al. report a targeted proteomics approach developed to quantify protein isoforms identified as candidate biomarkers in an earlier mouse xenograft model of epithelial ovarian cancer.1,2 Their results show that chloride intracellular channel (CLIC) proteins and tropomyosin (TPM) family members warrant further investigation as biomarkers for early detection.
The researchers measured peptides specific to the CLIC and TPM family of proteins using a targeted liquid chromatography–tandem mass spectrometry (LC-MS/MS) approach. They measured levels of the different isoforms present in pooled serum samples from patients with benign ovarian masses, comparing them to levels of patients with confirmed ovarian cancer.
Because serum is an extremely complex proteome to analyze, researchers first immunodepleted the pooled samples so that the low-abundance potential biomarker isoforms were not masked. They then separated proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Gel slices were removed and digested with trypsin, then analyzed using a Thermo Scientific LTQ Orbitrap XL mass spectrometer. MS data were analyzed using the SEQUEST algorithm (Thermo Scientific) in BioWorks v.3.3.1 (Thermo Scientific).
Using the pooled serum samples, Tang and co-workers established that the proteins identified in their earlier mouse xenograft model, CLIC1 and TPM1, were also present in patient samples. In addition, they found elevated levels of isoforms CLIC4, TPM2, TPM3 and TPM4, even though these had not been identified in the mouse study. The authors rationalize this is due to the close homology between human and mouse peptide fragments; the human peptides had been eliminated from earlier consideration because it was not possible to distinguish them from the background mouse proteome.
The researchers used the peptide fragments identified by the LTQ Orbitrap analysis to develop a label-free, multiple reaction monitoring (MRM) assay to measure levels of the protein isoforms of interest. Serum samples from 18 late-stage ovarian cancer patients and 15 non-cancer controls were immunodepleted and then separated by 1D-gel electrophoresis. The researchers assayed trypsin-digested samples using a hybrid triple quadrupole–linear ion trap mass spectrometer to quantify levels of the different protein isoforms.
Using this gel-based, label-free MRM (GeLC-MRM) technique, the researchers found that the CLIC and TPM isoforms’ levels correctly predicted the presence of cancer in late-stage cases (Mann-Whitney test, p<0.05), with CLIC4 being the most promising candidate. Tang et al. advise that further testing in larger cohorts, in addition to testing in patients with early-stage disease, is advisable before recommending these candidate biomarkers for diagnosis of ovarian cancer.
1. Tang, H-Y., et al. (2013) “Protein isoform-specific validation defines multiple chloride intracellular channel and tropomyosin isoforms as serological biomarkers of ovarian cancer,” Journal of Proteomics, 89 (pp. 165–78).
2. Tang, H-Y., et al. (2012) “A xenograft mouse model coupled with in-depth plasma proteome analysis facilitates identification of novel serum biomarkers for human ovarian cancer,” Journal of Proteome Research, 11(2) (pp. 678–91).
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