REGULAR ARTICLE Proteomic analysis of the proliferative and secretory phases of the human endometrium: Protein identification and differential protein expression Leroi DeSouza 1, 2 , Georg Diehl 1 , Eric C.C. Yang 1, 2 , Jingzhong Guo 1 , Mary Joe Rodrigues 3 , Alexander D. Romaschin 4, 5 , Terence J. Colgan 3, 5 and K.W. Michael Siu 1 1 Department of Chemistry and Centre for Research in Mass Spectrometry, and 2 Department of Biology, York University, Toronto, Ontario, Canada 3 Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada 4 Biochemistry, Toronto General Hospital, Toronto, Ontario, Canada 5 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada Proteomic analyses of the proliferative and secretory phases of the human endometrium were carried out to identify proteins and discover differentially expressed proteins using isotope-coded affinity tags, three stages of chromatographic separation and online tandem mass spectrometry (MS/MS). From an initial list of 346 proteins identified by ProICAT, manual inspection of MS/ MS spectra and confirmatory searches pared the list down to 119 positively identified proteins. Only five of the proteins showed consistent differential expression. The utility of some of these proteins as indicators of true differential expression in the endometrium is open to discussion. The two proteins with unquestionable differential expressions in the secretory endometrium are: glutamate NMDA receptor subunit zeta 1 precursor and FRAT1. Some of the proteins that show no differential expression have previously been examined in gene-expression studies with simi- lar conclusions. Received: March 19, 2004 Revised: June 3, 2004 Accepted: June 15, 2004 Keywords: Differential protein expression / Isotope-coded affinity tag / Multidimensional liquid chromatography / Protein identification / Secretory and proliferative endometrium 270 Proteomics 2005, 5, 270–281 1 Introduction The endometrium is the muscosal lining of the uterus, the function of which is to provide a suitable site for implanta- tion and development of a fertilized ovum. Underneath the endometrium is a thick muscular wall, the myometrium, which expands considerably to accommodate, protect and nourish the fetus, and is responsible for expulsion of the fetus during birth. The endometrium comprises three histo- logically distinctive layers: The deepest layer adjacent to the myometrium is the stratum basalis; this is the persisting germative layer that undergoes minimal changes during the menstrual cycle. The intermediate layer, stratum spongio- sum, is characterized by the presence of a spongy stroma. The thinner, most superficial layer, is the stratum compac- tum. These last two layers undergo dramatic and character- istic changes during the menstrual cycle, culminating in menstrual sloughing, and are therefore often jointly referred to as the stratum functionalis. Following menses, the endometrium undergoes major phenotypic changes characterized by two major phases. The proliferative phase begins after menses and terminates at Correspondence: Professor K.W. Michael Siu, Department of Chemistry and Centre for Research in Mass Spectrometry, York University, 4700 Keele Street, Toronto, Ontario, Canada M3J 1P3 E-mail: kwmsiu@yorku.ca Fax: 11-416-736-5936 Abbreviations: GADPH, glyceraldehyde-3-phosphate dehydro- genase; ICAT , isotope-coded affinity tag; IDA, information-de- pendent acquisition  2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.de DOI 10.1002/pmic.200400920