Endometrial Carcinoma Biomarker Discovery and Verification Using Differentially Tagged Clinical Samples with Multidimensional Liquid Chromatography and Tandem Mass Spectrometry

The utility of differentially expressed proteins discovered and identified in an earlier study (DeSouza, L., Diehl, G., Rodrigues, M. J., Guo, J., Romaschin, A. D., Colgan, T. J., and Siu, K. W. M. (2005) Search for cancer markers from endometrial tissues using differentially labeled tags iTRAQ and cleavable ICAT with multidimensional liquid chromatography and tandem mass spectrometry. J. Proteome Res. 4, 377–386) to discriminate malignant and benign endometrial tissue samples was verified in a 40-sample iTRAQ (isobaric tags for relative and absolute quantitation) labeling study involving normal proliferative and secretory samples and Types I and II endometrial cancer samples. None of these proteins had the sensitivity and specificity to be used individually to discriminate between normal and cancer samples. However, a panel of pyruvate kinase, chaperonin 10, and α1-antitrypsin achieved the best results with a sensitivity, specificity, predictive value, and positive predictive value of 0.95 each in a logistic regression analysis. In addition, three new potential markers were discovered, whereas two other proteins showed promising trends but were not detected in sufficient numbers of samples to permit statistical validation. Differential expressions of some of these candidate biomarkers were independently verified using immunohistochemistry. The utility of differentially expressed proteins discovered and identified in an earlier study (DeSouza, L., Diehl, G., Rodrigues, M. J., Guo, J., Romaschin, A. D., Colgan, T. J., and Siu, K. W. M. (2005) Search for cancer markers from endometrial tissues using differentially labeled tags iTRAQ and cleavable ICAT with multidimensional liquid chromatography and tandem mass spectrometry. J. Proteome Res. 4, 377–386) to discriminate malignant and benign endometrial tissue samples was verified in a 40-sample iTRAQ (isobaric tags for relative and absolute quantitation) labeling study involving normal proliferative and secretory samples and Types I and II endometrial cancer samples. None of these proteins had the sensitivity and specificity to be used individually to discriminate between normal and cancer samples. However, a panel of pyruvate kinase, chaperonin 10, and α1-antitrypsin achieved the best results with a sensitivity, specificity, predictive value, and positive predictive value of 0.95 each in a logistic regression analysis. In addition, three new potential markers were discovered, whereas two other proteins showed promising trends but were not detected in sufficient numbers of samples to permit statistical validation. Differential expressions of some of these candidate biomarkers were independently verified using immunohistochemistry. Differential tagging with isotopic reagents, such as ICAT (1Gygi S.P. Rist B. Gerber S.A. Turecek F. Gelb M.H. Aebersold R. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.Nat. Biotechnol. 1999; 17: 994-999Crossref PubMed Scopus (4293) Google Scholar) or the more recent variation that uses isobaric tagging reagents, iTRAQ 1The abbreviations used are: iTRAQ, isobaric tags for relative and absolute quantitation; PCM, potential cancer marker; EmCa, endometrial carcinoma; SCX, strong cation exchange; RP, reverse phase; PIGR, polymeric immunoglobulin receptor; PK, pyruvate kinase; Cpn 10, chaperonin 10; PV, predictive value; PPV, positive predictive value; AUC, area under the curve; MIF, macrophage migration-inhibitory factor; AAT, α1-antitrypsin; CKB, creatine kinase chain B; PEBP, phosphatidylethanolamine-binding protein; hnRNP D0, heterogeneous nuclear ribonucleoprotein D0; WFDC2, WAP four-disulfide core domain protein 2; PMN, polymorphonuclear neutrophil; MMP-2, metalloproteinase-2; RA, retinoic acid; P-LAP, placental leucine aminopeptidase; IL, interleukin; LAP3, leucine aminopeptidase 3; SLPI, secretory leucocyte protease inhibitor; NE, neutrophil elastase. 1The abbreviations used are: iTRAQ, isobaric tags for relative and absolute quantitation; PCM, potential cancer marker; EmCa, endometrial carcinoma; SCX, strong cation exchange; RP, reverse phase; PIGR, polymeric immunoglobulin receptor; PK, pyruvate kinase; Cpn 10, chaperonin 10; PV, predictive value; PPV, positive predictive value; AUC, area under the curve; MIF, macrophage migration-inhibitory factor; AAT, α1-antitrypsin; CKB, creatine kinase chain B; PEBP, phosphatidylethanolamine-binding protein; hnRNP D0, heterogeneous nuclear ribonucleoprotein D0; WFDC2, WAP four-disulfide core domain protein 2; PMN, polymorphonuclear neutrophil; MMP-2, metalloproteinase-2; RA, retinoic acid; P-LAP, placental leucine aminopeptidase; IL, interleukin; LAP3, leucine aminopeptidase 3; SLPI, secretory leucocyte protease inhibitor; NE, neutrophil elastase. (Applied Biosystems, Foster City, CA), followed by multidimensional LC and MS/MS analysis is quickly being recognized as one of the more powerful methodologies in the search for biomarkers for various disease states. Our recent studies using both ICAT and iTRAQ reagents as means to facilitate the and relative of proteins from endometrial tissue in some potential cancer markers J. analysis of the proliferative and secretory of the protein and protein PubMed Scopus Google J. Search for cancer markers from endometrial tissues using differentially labeled tags iTRAQ and with multidimensional liquid chromatography and tandem mass Proteome Res. 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