Differential Proteomic Analysis of Endometriosis

CHEM. RES. CHINESE UNIVERSITIES 2010, 26(1), 70- 74Differential Proteomic Analysis of EndometriosisZHANG Li-hui', LIU Hong-yan', LI He-lian', ZHANG Xing-yi2 and SUN Mei'*1. Department of Gynecology & Obstetrics, 2. Department of Thoracic Surgery, 3. Department of Pathologythe Second Hospital of Jilin Universit, Changchun 130041, P. R. ChinaAbstract Differential proteins expressing in ectopic and eutopic endometria were investigated by means of proteo-mic analysis. Five patients in secretary phase were diagnosed as endometriosis by laparoscopy. The five ectopic en-domctria(two at stage II, two at stage Ill and one at stage IV) and five cutopic endometria were surgically excised.Onc-dimensional electrophoresis coupled with liquid chromatography and mass spectrometry was used to screen andidentify differential proteins. Three differential bands in one-dimensional electrophoresis were resolved by liquidchromatography and mass spectrometry and 14 up-regulated proteins were identified, including collagen a-1, a-2, .a-3(VI), a-l(XIV) chain, actin, annexin A2, EMILIN-I, frritin light polypeptide variant, fucosyltransfcrase 10myosin-9, protin S100-A9, KIAA1783 protein, and two hypothetical proteins. Our data provides a list of potentialbiomarkers for endometriosis. The identifications may be used to develop new diagnoses for endometriosis.Keywords Endometriosis; Onc-dimensional electrophoresis; Liquid chromatography; Mass spectrometry; ProteomicsArticle ID 1005-9040(2010)-01-070-051 Introductionmetry. They have identified some differential proteinsEndometriosis is a gynecological disease andin endometriosis patients' tissues and sera.Studies'8] have shown that despite the high-presents endometrial-like glands and stroma outsideresolution capability, the 2DE approach has the draw-the uterine cavity. The ectopic endometrium resultsbacks of labor-intensive and irresolvable for very ba-from intermittent variation when it is affected by thesic or acidic, and very large or small proteins. Moreo-ovarian hormone, which also has the same effect onver, surface enhance laser desorption/ionizationnormal endometra. Endometriosis is characterized byhyperplasia, infiltrate, disseminate and transfuse ma-time-of-flight mass spectrometry can merely analyzelignant behavior, which is similar to malignant tumors.low molecular weight proteins and it is difficult for itThe typical sign and symptom of endometriosis areto make protein identification. To avoid technicalpersistent algomenorrhea, dysgenesia, pelvic cavitydisadvantages, in the study, a novel approach withcombination of one dimensional electrophoresis,purple nodules, and chocolate cyst of ovary. Althoughliquid chromatography, and mass spectrometrylaparoscopy has emerged as one of the specific and(IDE/LC/MS) was adopted for diferential proteomicsensitive methods for the diagnosis of endometriosis,analysis of patients with endometriosis. Such an ap-inevitably it brings surgical stress and risks. Therefore,proach has not been applied to the study of endome-it is the urgent need to develop non-invasive diagnosistriosis to date. However, there still exists a potential toapproaches such as biochemistry.Over the years, an advance in proteomics hasprovide useful tools for the study of endometriosisopened a new avenue for discovering disease bio-pathogenesis and its diagnosis.markers. Recently,related proteomic analysis2 Experimentalrepresents the most important development for thestudy of diseases- 0!. Researchers have accomplished2.1 Tissue Preparationthe investigation of endometriosis with proteomicFive women with endometriosis submitted to la-analysis approaches. Zhang et al,!" have successfullyparoscopy were selected. Endomtriosis was con-analyzed the differential proteins in patients withfirmed by histology, and the extent of the disease wasendometriosis versus normal controls using two-stagAmerican Fertilitydimensional electrophoresis(2DE) and mass spectro- Sc中国煤化工puts Iw weeTYHCNMHG*Corresponding author. E-mail: sun.mci55@yahoo.comReceived May 20, 2009; accepted June 28, 2009.Supported by the National Natural Science Foundation of China(Nos.30670301, 30870354).No.1ZHANG Li-hui et al.7classified as stage II, two as stage II, and one as stageexcised from the gel and cut into pieces of 1 mm' inIV, respectively. Five patients' menstrual cycles weresize. In-gel digestion was performed as described pre-all at proliferative stage. The eutopic and ectopic en-viouslyl'l. Brifly, the gel pieces were dehydrated bydometrium tissues(Chocolate cyst) were obtained viaacetonitrile, rehydrated by 25 mmol/L ammoniumlaparoscopy and stored at- -80 。C. Except endometri-bicarbonate. In-gel proteins were digested by 0.5osis, no other diseases have been diagnosed on physi-μmoIL trypsin and twice extracted from the gel undercal examination and biochemical tests for the fivemicrowave iradiation. Gel was microwaved for 5 minwomen. None had received any hormonal treatmentfor each digestion and extraction step. Extractedwithin three months prior to study. This study waspeptides were lyophilized before mass spectrometryapproved by the Ethics Committee on the Clinicalanalysis.Study of Second Hospital of Jjilin University, China.2.5 LC/MS/MS and Database Searching2.2 Protein Sample PreparationThe lyophilized samples were re-dissolved inThe frozen biopsies were washed with phos-0.1% formic acid(buffer A containing 0. 1% formicphate-buffered saline in sterile Petri dishes to removeacid and 99.9% :H2O) before LC/MS analysis. Pep-any adhered hemoglobin and other proteins from he-tides were loaded onto self-packed C18 RP capillarymolysis at the time of sample collction. Tissues werecolumns( 100 mmx0.17 mm, i.d. 5 μum) with buffer A,cut into 1 mm' pieces, ground into powders in liquidand eluted with 5%- -30%(volume fraction) buffer Bnitrogen and dissolved in lysis buffer containing 7for 180 min. Eluted peptides were detected in a surveymol/L urea, 2 mol/L thiourea, 2%(mass fraction)scan from 400 to 1500 amu(three microscans) fol-CHAPS, 50 mmol/L Tris, 50 mmol/L DTT, 1 mmol/Llowed by five data-dependent MS/MS scans in a com-phenylmethylsulphonyI fluoride, and 1 ug/mL pro-pletely automated fashion on an LCQ DECA XP+ESItease inhibitor cocktail(Sigma, USA). Benzonasemass spectrometer. AlI MS/MS spectra were, respec-DNase and RNase(Merck, Germany) were added totively, searched using SEQUEST algorithm-basedthe mixture to remove DNA and RNA. Samples wereBioworks 3.1 SR1(Thermo Finnigan) against the In-centrifuged for 1 h at 10000g to remove any particu-ternational Protein Index human database(Versionlate materials. The protein concentration was deter-3.18) from the European Bioinformatics Institutemined with Bradford method. To avoid individualwebsite(www.ebi.ac.uk/IPI). Searches were per-proteome variation, the 5 eutopic endometria tssesformed with enzyme constraints.The stringentwere mixed and served as control group(CONT 1), .SEQUEST criteria were used for result analyis"!: (1)while the 5 ectopic endometrial tissues were mixed asDeltaCn score≥0.l; (2) Rsp score=l; (3) Xcorr .1.9tested group(ECTOPIC 2). The samples were kept atfor+1 charged peptides, with parially tryptic ends; (4)- 80 °C until use.Xcorr .2.2 for+2 charged peptides, with parially tryp-tic end; (5) Xcorr .2.7 for+2 charged peptides, without2.3 One- dimensional Electrophoresisregard to the end residues; (6) Xcorr .3.75 for+3A total tissue of 100 ug was mixed with glycinecharged peptides, without regard to the end residues.loading buffer, heated at 100。C for 5 min, cooled toTo avoid the false positive identification as lessroom temperature, and loaded onto the gel. Electro-as possible, IDE and LCMS analysis of each bandphoresis was performed on 12% polyacrylamide( 100from the two samples(eutopic and ectopic endometria)mmx80 mm slab gel with 0.75-mm spacers usingwas triply conducted. Only positive results are ac-Mini-Protean II followed by microwave-assistedceptable whenever the proteins can be identified atCoomassie Blue staining)9. The gel was incubated inleast in two runs.a microwave oven at 850 W for 1 min in the metha-nolacetic acid fixative followed by 1 min staining in2.6 Westerm Blot Analvsis中国煤化工the staining solution.。 study were sepa-ratedMYHCNMHGDS-PAGEwitha2.4 In-gel Digestionotal protein of 50 μg per lane and transferred ontoThe differential protein bands were manuallyImmobilon-P PVDF membrane(Millipore, Bedford,72CHEM. RES. CHINESE UNIVERSITIESVol.26MA) in CAPS transfer buffer for 45 min at 1.5from band 1. High abundant proteins were identifiedmA/cm' on a semidry electroblotter(BioRad, CA).each time and their spectrum numbers were similar.Western blot was performed via rapid immunodetec-This indicates a good reproducibility for IDE/LCMStion method. Having been soaked in 100% methanolanalysis.for 10 s and dried in air for 15 min, the membrane was100F(A)]probed with mouse anti-actin mAb(Cell Signal Tech-80 anology, MA, 1:300 dilution) then with horseradish0tperoxidase-conjugated horse anti-mouse Ig(AMSBiotechnology, Oxon, 1:3000 dilution). The blot was10 Fdeveloped with ECL detection reagent(Pierce Chemi-g20 wcal, Rockford, IL).30609120150I8Timc/min3 Results and Discussion)0 FB)|To assess reproducibility, the two mixed sam-80下ples(eutopic and ectopic endometria) from five pa-50 ttients were subjected to IDE/LC/MS analysis threetimes. As Fig.I shows in the IDE analysis, most bandsin the two groups were identical, but three differentialbands were found in the ectopic group. The three400 6008001000 12001400bands(bands 1- 3) were simultaneously excised and100 F(心)subjected to LCIMS analysis shown by Fig.2.y80 tb3b4Marker Control Experimant9700yI0.. ,be10 t4300163100400 600 800 1000 1200 1400 1600 1800 20002100Fig.2 Analysis of band 1 in ECTOPIC group by LCMS1400 - -(A) Base-peak chromatogram of three nuns during LCMS analysis. Peptideswere loaded and eluted in a 5%- -30% gradient of buffer B over 180 min.Fig.1 1DE separation of CONT and ECTOPIC groupsSelected pepides were subjected to MS/MS analysis; (B) MS survey scanArows indicaing three pairs of dfferential bands.during LCMS analysis at 138.73 min. Five pepides ions were scquniallTo evaluate the reproducibility and simplicity ofslcted on the mass spetrometer for the further sequence analysis of colsion-induced dissociation; (C) MS/MS scan of precursor ion at m/: 1097.84.1DE/LCMS analysis approach, band 1 was subject toIt is one of the five peptide ions chosen for analysis and identified asIDE analysis due to its abundance of proteins. Table 1YGGLHFSDQVEVFSPPGSDR Some b- and y-ions derived from thesummarizes the identification of the three analysespeptide ion were also indicated.Table 1 Proteins identifed in band 1 ECTOPIC sample for three runsSpectrum numberUnique/GroupIPI entryDescriptionRun 1Run 2Run3Group100022200a-3 Type VI collagen isoform 1 precursor62450IPI00291136Collagen a-1(VI) chain precursor4(1832P100304840Isofom 2C2 of cllagen a-2(VI) chain precursor392(IP1I00176193Isoform 1 of cllagen a- 1(XIV) chain precursorUniqueIP100013079EMILIN-I precursor100008603Actin, aortic smooth muscleIP100297646Collagen a-l(I) chain precursorIP100019502Myosin-9IP100440692FUT10 proteinIP100745872Serum albumin precursor中国煤化工IPI00306532KIAA1783 protein(fragment)MHCNMHG.IP100418169Annexin A2 isofom 1IP100740155Hypothetical protein DKFZp686J17211IP100021536Calmoduin-like protein 5No.1 .ZHANG Li-hui et al.73Overall, 24 unique/group proteins were identified.11 differential proteins were identified. In the secondSixteen proteins had theoretically-matched apparentpair of bands, only β crystallin B1 was identified inmolecule weight(Mw). Five proteins(actin, annexin A2,CONT and ECTOPIC groups. Thus three differentialcalmodulin-like protein 5, fucosyltransferase 10 pro-proteins were found in this pair of bands. Interestingly,tein and serum albumin precursor) were identified ashypothetical protein DKFZp686J17211 was alsohigher Mw in band 1. Two proteins(isoform 1 offound in the first pair of bands, thus confrmed its truedouble-stranded RNA-specific editase 1 andpositive identification. For the third pair of bands, theKIAA1783 protein) were found as lower Mw in band 3,three proteins(hemoglobin beta subunit, isoform 1 ofimplying that they might be fragments of intact pro-Double-stranded RNA-specific editase 1 and cytoch-teins. One hypothetical protein(DKFZp686J17211)rome c oxidase polypeptide Va, mitochondrial precur-was found at both higher(band 1) and lower Mw(bandsor) were found in CONT and ECTOPIC groups. .2), indicating its post-translational modification andAmong the five differential proteins in the third pairdegradation.of bands, it is likely that hemoglobin δ subunit, he-To find differential proteins, identifications frommoglobin a subunit and hemoglobin epsilon subunitthe two grouped samples were compared. In the firstwere contaminant proteins. It seems that KIAA1783pair of bands, two proteins and fourteen ones wereprotein in the first pair of bands was a fragment ofidentifed in CONT and ECTOPIC groups, respec-intact proteins. Taken together, 14 differentialtively. Collagen a-1(I) chain precursor was found inup-regulation proteins were identified in the threethe two grouped samples. Although calmodulin-likepairs of bands.protein 5 was only identified in one run in ECTOPICBased upon the identification of actin as differen-group, in view of the abundant suppression for LC/MStial protein by differential proteomic analysis, Westernanalysis'e, it is likely that protein 5 was a positiveblot analysis was conducted to further validate theidentification and was not considered as a differentialapproach of IDE/LCMS analysis. Actin expressionprotein.level in ECTOPIC was lower than that in CONTAmong the other 12 proteins, serum albumingroup, which is consistent with 1DE/LCMS analysisprecursor was considered as a contaminant protein, so(Fig.3). .CONT ECTOPIC CONT ECTOPIC CONT ECTOPIC CONT ECTOPIC CONT ECTOPICFig.3 Western blot detection of actin in CONT and ECTOPIC groupsProteomics provides a novel approach to disco-ble for anchoring smooth muscle cells to elastic flbers,ver more sensitive biomarkers for diagnosisl). In thisand involves the formation of elastic fiber and thestudy, we studied the differential proteomic expressionregulation of vessel assembly. Additionally, it has cellmaps of eutopic and ectopic endometria from five pa-adhesive ability. Based on the fact that ectopic endo-tients by 1DE/LC/MS approach. The 14 differentialmetrium is an invasive tissue, and cell attachment andunique/group proteins were identified, including col-cell movement are necessary for invasion of tissuel",lagen, a-1, a-2, a-3(VI), a-1(XIV) chain, actin, An-it is resalable that these proteins are involved in ec-nexin A2, EMILIN-1, Ferritin light polypeptide va-topic endometrium invasion.riant, fucosyltransferase 10, myosin-9, protein S100-In this study, another important differential pro-A9, KIAA1783 protein, and two hypothetical proteins.tein is actin that is a kind of cytoskeleton protein par-Functions of these proteins mainly involve cell-cellticipating in mitosis, signal transduction, and cell mo-adhesion, cell motility and signal transduction.tility. It has been shown that the abnormal expressionCollagen a-1, a-2, a-3(VI) chains are extracellu-of actin is related to the malignant proliferation andlar matrix structural constituents and mainly involvetumor invasion cells'4!. Studies have revealed thecell adhesion. Collagen a-l(XIV) chain is also anup-n中国煤化，工elated and actin-extracellular matrix structural constituent and plays anbindietriuml2.8,15. Theseadhesive role bintegrating collagen bundles.indic.CNMHGimportant role inEMILIN-1 is an extracellular matrix protein. There isendometriosis.evidence to suggest that EMILIN-1 may be responsi-Myosin-9 is a protein related to cell motility and74CHEM. RES. CHINESE UNIVERSITESVol.26plays a role in cytokinesis, cell shape and functions.difrentially expressed in eutopic and ectopic endo-KIAA1783 protein is the component of myosin com-metria. Despite the inentification of 14 diftrenialplex and also related to cell motility. As cell move-proteins in endometriosis, some of them are alsoment is necessary for ectopic endometrium tissue in-found in tumors and inflammation, suggesting thavasion!', it is plausible that myosin-9 and KIAA1783they are lack of specificity. Thus, combined determi-proteins might play an important role in ectopic en-nation of these proteins should be a better choice fordometrium invasion. Annexin 2 is a member of theclinical application.annexin family of Ca2+ and a membrane binding pro-In conclusion, this study represents a preliminarytein. The ability of annexin 2 to interact with actinproteomic analysis of endometriosis that provides acytoskeleton, and its potential to bind invasion-list of potential biomarkers for endometriosis. Ourassociated proteases suggest that annexin 2 could par-findings are helpful for understanding the nature ofticipate in invasion-associated processesll. Of note isendometriosis and eventually develop new diagnosticthat there exist controversies to this study. Indeed, theand therapeutic methods for endometriosis.down-regulation of annexin 2 has been shown in en-dometriosis via proteomic analysis'8].ReferencesProtein S100-A9, a member of S100 protein[川] Sangi-Haghpeykar H., Poindexter A. 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