Quantitative assessment of serum heat shock protein 27 for the diagnosis of epithelial ovarian cancer using targeted proteomics coupled with immunoaffinity enrichment

Background: Heat shock protein 27 (HSP27) may take part in the epithelial ovarian cancer (EOC) malignant process because it is elevated in the serum of EOC patients, suggesting that HSP27 may serve as an EOC biomarker to complement the standard serum carbohydrate antigen 125 (CA125) test. Thus, accurate quantification of serum HSP27 would assist the diagnosis of EOC. Methods: Liquid chromatography-tandem mass spectrometry (LC MS/MS)-based targeted proteomics coupled with an immunoaffinity enrichment assay was developed and validated to monitor HSP27 concentrations in serum. Results: Tryptic peptide 80QLSSGVSEIR89 was selected as a surrogate analyte for quantification, and an immuno-depleted serum extract was used as a surrogate matrix. Immunoaffinity enrichment was effective for protein enrichment and sensitivity enhancement, and the resulting LOQ was 500 pg/ml ( > 10-fold increase). Then, serum HSP27 concentrations in EOC patients, benign ovarian tumors patients and healthy volunteers were accurately determined to be 4.95 +/- 0.37 ng/ml, 2.98 +/- 0.16 ng/ml and 2.82 +/- 0.15 ng/ml, respectively, suggesting that the EOC samples had significantly higher concentrations of HSP27 than a sample from benign ovarian tumor patients. The experimental values for the samples were compared with those obtained from enzyme-linked immune sorbem assays (ELISAs). The ROC curve analysis showed that the combined area under the curve (AUC) for CA125 and HSP27 was 0.88, which is significantly superior to that of CA125 alone. Conclusions: Targeted proteomics coupled with immunoaffinity enrichment may provide more accurate quantification of low-abundant proteins.