Search for cancer markers from endometrial tissues using differentially labeled tags iTRAQ and clCAT with multidimensional liquid chromatography and tandem mass spectrometry

A total of nine potential markers for endometrial cancer (EmCa) have been discovered and identified from endometrial tissue homogenates using a combination of differentially labeled tags, iTRAQ and clCAT, with multidimensional liquid chromatography and tandem mass spectrometry. The tissues were snap frozen in liquid nitrogen within 15-20 min after devitalization. Samples for proteomic analysis were treated with protease inhibitors before processing. Marker proteins that were overexpressed in EmCa are chaperonin 10, pyruvate kinase M1 or M2 isozyme, calgizzarin, heterogeneous nuclear ribonucleoprotein DO, macrophage migratory inhibitory factor, and polymeric immunoglobulin receptor precursor; those that were underexpressed are alpha-l-antitrypsin precursor, creatine kinase B, and transgelin. The chaperonin 10 result confirms our earlier observation of overexpression in EmCa tissues using surface-enhanced laser desorption/ionization mass spectrometry, verified by Western analysis and immunohistochemistry [Yang, E. C. C. et al. J. Proteome Res. 2004, 3, 636-643]. Pyruvate kinase was observed to be overexpressed using both iTRAQ and clCAT labeling. All nine markers have been found to be associated with various forms of cancer. A panel of these plus other markers may confer sufficient selectivity for diagnosing and screening of EmCa. The use of clCAT led to identification of a higher proportion of lower-abundance signaling proteins; conversely, iTRAQ resulted in a higher percentage of the more abundant ribosomal proteins and transcription factors.