ClinProt技术在子宫内膜异位症研究中的应用
摘要
子宫内膜异位症(内异症)是常见的妇科疾患,发病率在育龄期妇女约10-15%,且有逐渐上升的趋势。该疾病的主要表现为慢性盆腔痛、痛经、不孕和附件包块,严重的影响了妇女的生活质量。同时虽然子宫内膜异位症是一种良性疾病,但具有侵袭、转移与复发等恶性行为。研究表明,早期的内异症病变主要集中在腹膜,对盆腔结构的影响不大,早期诊断和治疗有助于患者生活质量的提高。目前,关于内异症的早期诊断,尚且没有有效的标志物,没有标志物广泛应用于临床,用以评价疾病的严重程度判断、评估疗效和术后随访。本实验采用ClinProt技术联合MALDI-TOF,建立子宫内膜异位症患者腹腔液在分子量800-10000Da范围内的蛋白质/肽图谱,期望寻找该病的特异性蛋白,并鉴定蛋白性质,明确该蛋白在内异症中的作用,为早期诊断和发病机制的研究提供依据。同时使用ClinProt技术联合MALDI-TOF技术建立内异症患者尿中的低分子量蛋白质/肽图谱,建立高效率诊断模型。该实验通过联合MALDI-TOF/TOF和LC-MS/MS,鉴定出差异蛋白为胶原的前体,可能在内异症的发生发展过程中起到重要作用。本实验基于ClinProt技术建立的诊断模型效率高,将来该技术有可能广泛应用于临床,实现对该病的的无创诊断,为内异症的早期诊断和治疗随访提供依据,同时也为将来子宫内膜异位患者的腹腔液和尿中的蛋白质组学研究提供了经验和数据。
OBJECTIVES
This study aimed to to investigate the possibility of using the ClinProt technique to find diagnostic markers that are able to better discriminate healthy subjects from patients affected by endometriosis(EMs). Endometriosis is a common gynecological disease. Biomakers for early detection, prognosis, follow-up, and differential diagnosis of Ems are needed in daily clinical practice.
METHODS
Peritoneal fluid and urine samples were analyzed by the ClinProt and MALDI-ToF technique. We combined the automated high through put MALDI-TOF measurements and LC-MS/MS to identify the different proteins.
RESULTS
After comparing the peritoneal fluid protein mass spectra of39patients with34controls,18differently expressed potential biomarkers (P<0.01) were identified. Using ClinproTool software, we established GA algorithm.When using the a cluster of five signals (m/z=4458.23,2379.3,8519.94,6376.99,6190.27) to classify the samples, the sensitivity for diagnosing endometriosis was88.5%, specificity was88.5%.Urine samples from30patients and31controls were also analyszed by MALDI-TOF. Using GA algorithm, The combination of five markers (m/z=1433.98,1599.48,2085.61,6798.07,3217.29) lead to an improvement in diagnostic efficacy, with specificity and sensitivity of95.7%and96.7%, respectively.Com bind ing the MALDI-TOF/TOF with LC-MS/MS, we identified the proteins as collagen precursor.
CONCLUSIONS
These results may be useful to set up new diagnostic or prognostic tools.
OBJECTIVES
This study aimed to to investigate the possibility of using the ClinProt technique to find diagnostic markers that are able to better discriminate healthy subjects from patients affected by endometriosis(EMs). Endometriosis is a common gynecological disease. Biomakers for early detection, prognosis, follow-up, and differential diagnosis of Ems are needed in daily clinical practice.
METHODS
Peritoneal fluid and urine samples were analyzed by the ClinProt and MALDI-ToF technique. We combined the automated high through put MALDI-TOF measurements and LC-MS/MS to identify the different proteins.
RESULTS
After comparing the peritoneal fluid protein mass spectra of39patients with34controls,18differently expressed potential biomarkers (P<0.01) were identified. Using ClinproTool software, we established GA algorithm.When using the a cluster of five signals (m/z=4458.23,2379.3,8519.94,6376.99,6190.27) to classify the samples, the sensitivity for diagnosing endometriosis was88.5%, specificity was88.5%.Urine samples from30patients and31controls were also analyszed by MALDI-TOF. Using GA algorithm, The combination of five markers (m/z=1433.98,1599.48,2085.61,6798.07,3217.29) lead to an improvement in diagnostic efficacy, with specificity and sensitivity of95.7%and96.7%, respectively.Com bind ing the MALDI-TOF/TOF with LC-MS/MS, we identified the proteins as collagen precursor.
CONCLUSIONS
These results may be useful to set up new diagnostic or prognostic tools.
引文
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[1]王姝,郎景和.子宫内膜异位症—一种干细胞疾病?[J].现代妇产科进展,2008,17(10)：721-724.
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[20]Joshi SG.Zamah NM.Serum and peritoneal fluid proteins in women with and without endometriosis[J].J Fertil Steril,1986,46(6):1077-82.
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[27]胡芬,张炜,康晓楠等.应SELDI-TOF-MS技术初步建立子宫内膜异位症诊断模型[J].生殖与避孕,2008,28(3)：168-173.
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[2]中华医学会妇产科分会子宫内膜异位症协作组.子宫内膜异位症的诊断与治疗-规范[J].中华妇产科杂志,2007：42(9)：645-8.
[3]Hanah S. Disease proteomies[J]. Nature.2003,422(6928):226-232.
[4]Sau-Mei Leung, Samuel C. Mok. Quality management of ovarian cancer serum profiles using functionalizied magnetic beads, AnchorchipTM technology, MALDI-TOF MS and MALDI-TOF/TOF MS/MS. AACR Annual Meeting,2004.
[5]Villanueva J, Philip J, and Entenberg D, et al. Serum peptide profiling by magnetic particle-assisted, automated sample processing and MALDI-TOF mass spectrometry[J]. Anal, Chem,2004,76:1560-1570.
[6]Josep Villanueva, David R. Shaffer, and Paul Tempst. Differential exoprotease activities confer tumor-specific serum peptidome pattems[J]. J Clin Investigation 2006,116(1):271-284.
[7]Ketterlinus R, Hsieh SY, Teng SH, et al. Fishing for biomarkers:analyzing mass spectrometry data with the new ClinProTools software[J].Biotechniques.2005,6 Suppl:37-40.
[8]PaulA.Fowler,JennyTattum.Siladitya Bhattacharya.etc.An investigation of the effects of endometriosis on the proteome of human eutopic endometrium:A heterogeneous tissue with a complex disease[J].Proteomics,2007,7:1.
[9]ZhangH.FengJie et al.Effect of surface-enhanced laser desorption/ionization time-of-flightmass spectrometry on identifing biomarkers of endometriosis[J]. Chinese Medical Journal,2009,122(4):373-376.
[10]Liang Wang.Wei Zheng,Lin Mu,Su-Zhan Zhang.Identifying biomarkers of endometriosis using serum protein fingerprinting and artificial neural networks[J].International Journal of Gynecology and Obstetrics,2008,101:253-258.
[11]刘海元,郑艳华,张建中等.子宫内膜异位症患者血浆蛋白质指纹图谱诊断分类树模型的初步建立[J].中华妇产科杂志,2009,44(8)：601-604.
[12]胡芬,张炜,康晓楠等.应SELDI-TOF-MS技术初步建立子宫内膜异位症诊断模型[J].生殖与避孕,2008,28(3)：168-173.
[13]Juan Casado-Vela.Eva Rodriguez-Suarez,Ibon lloro.et al.Comprehensive Proteomic Analysis of Human Endometrial Fluid Aspirate[J].Journal of Proteome esearch,2009,8(10):4622-4632.
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