质谱分析法在“先兆子痫”研究中的应用
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摘要
胎盘是母体与胎儿之间的重要器官,具有物质交换、新陈代谢及屏障作用等重要生物功能,胎盘功能异常会导致先兆子痫等妊娠疾病的产生。许多与胎盘功能有关的生理生化过程都是由胎盘中构成复杂网络的各种蛋白相互协调完成。因此,深入研究胎盘相关蛋白、蛋白间相互作用以及蛋白在不同生理病理条件下的差异表达以及翻译后修饰研究,有助于进一步了解胎盘的结构与功能,寻找疾病标志物、药物靶标,疾病的早期诊断、治疗、预后判断、早期预防等方面具有重要临床意义。
本研究通过BN/SDS-PAGE结合LC-MS/MS研究胎盘蛋白及蛋白质复合物,共鉴定了733个非冗余蛋白以及34个蛋白复合物,其中1个为新的未发现有相互作用的复合物。通过生物信息学分析以及结合文献报道的数据,发现这些已知的复合物主要是线粒体呼吸链复合物、整合素复合物、蛋白酶体复合物以及热休克蛋白复合物等。发现的新复合物为clathrin和small conductance calcium-activated potassium (SK) channel protein2。这两个蛋白的相互作用进一步通过antibody based gel shift assay、免疫共沉淀以及免疫荧光进行了验证。这些结果表明BN/SDS-PAGE结合LC-MS/MS是一种很好的分析胎盘蛋白质复合物的方法。
胎盘蛋白的磷酸化是目前胎盘蛋白质组学研究热点之一。从复杂样本中有效富集磷酸化蛋白以及磷酸化肽段是具有挑战性的工作。本研究合成了一系列形态可控、成分可调的钛掺杂介孔硅材料来有效的富集磷酸化肽段以及磷酸化蛋白。通过改变前体中钛/硅摩尔比来调节钛掺杂介孔硅材料的表面积、直径以及形状等,利用这一系列的材料来富集酶解后的标准磷酸化蛋白。研究结果表明,当钛/硅摩尔比为8：1时,该材料富集磷酸化肽段的效果最好,β-酪蛋白的检测极限能达到1Ofmol, a-酪蛋白有15个磷酸化肽段被检测到,优于商业化的二氧化钛珠子。我们将该材料用于胎盘线粒体磷酸化蛋白的富集,共鉴定了396个磷酸化肽段以及298个磷酸化蛋白,结果表明该材料能用于复杂样本的磷酸化肽段富集。此外,本研究还提供了一种合成成分可调的磷酸化肽段富集材料的方法。
由于先兆子痫是一种与胎盘功能发生异常有关的重要妊娠疾病,本研究通过基于质谱的二甲基标记定量蛋白质组学方法对正常孕妇以及先兆子痫孕妇胎盘蛋白质组进行了分析。鉴定出2636种蛋白,其中171种蛋白在先兆子痫胎盘中有差异表达。通过对差异蛋白生物信息学分析,发现这些蛋白主要参与习惯性流产、颅内HADHA高血压、胎儿生长缓慢、胎儿死亡、妊娠期高血压以及治病流产等信号通路。同时随机挑选五种差异蛋白进行免疫印迹以及免疫组化实验,对这些蛋白的表达情况进行验证,结果表明质谱鉴定结果与免疫学结果一致。综上所述,通过比较蛋白质组分析正常妊娠和先兆子痫的胎盘,确定了一些可能参与胎盘病理变化的重要蛋白,对这些蛋白质进行进一步的功能分析有助于了解先兆子痫胎盘以及与胎盘有关的疾病发生、发展和转型的关键生物学过程。
在上述研究基础上,本论文对先兆子痫胎盘质膜糖基化和磷酸化蛋白质组学进行了研究。通过二甲基标记结合糖基化和磷酸化肽段的富集方法,研究了先兆子痫胎盘质膜中的糖基化以及磷酸化蛋白表达谱,通过严格的数据筛选,共鉴定了1027条N-糖基化以及2094条磷酸化肽。7个糖基化肽段以及42个磷酸化肽段具有差异表达。这些差异蛋白进行分析表明这些蛋白参与胎儿死亡以及原发性高血压信号通路。通过分析正常以及先兆子痫妊娠的人胎盘质膜蛋白质组,我们构建了胎盘蛋白质相互作用图谱、先兆子痫胎盘蛋白质表达谱以及N-糖基化和磷酸化的蛋白质表达谱,鉴定了在先兆子痫发生过程中的关键蛋白,建立了磷酸化蛋白富集的新方法,这些研究为深入了解先兆子痫胎盘和胎盘相关的疾病提供了基础。
The placenta plays a pivotal role of promoting the exchange of nutrients and waste products between the maternal and fetal circulatory systems. In addition, it is a natural barrier against numerous bacterial and viral infections during pregnancy. Maternal preeclampsia (PE) and fetal intrauterine growth restriction(IUGR) are two of the most common and serious complications of human pregnancy associated with placental abnormalities. Identification and characterization of placental multi-protein complexes is an important step to integratedly understand the protein-protein interaction networks in placenta which determine placental function. Therefore, elucidation of the placental protein interaction, the expression levels as well as post-translational modifications in various physiological and pathological conditions would provide researchers with a better understanding of the structure and function of the placenta
In this study, blue native/sodium dodecyl sulfate polyacrylamide gel electrophoresis (BN/SDS-PAGE) and Liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to screen the multi-protein complexes in placenta.733unique proteins and34known and novel heterooligomeric multi-protein complexes including mitochondrial respiratory chain complexes, integrin complexes, proteasome complexes, histone complex, and heat shock protein complexes were identified. A novel protein complex, which involves clathrin and small conductance calcium-activated potassium (SK) channel protein2, was identified and validated by antibody based gel shift assay, co-immunoprecipitation and immunouorescence staining. These results suggest that BN/SDS-PAGE, when integrated with LC-MS/MS, is a very powerful and versatile tool for the investigation of placental protein complexes.
Extraction of phosphopeptides from rather complex biological samples has been a tough issue for deep and comprehensive investigation into phosphoproteomes. In this paper, we present a series of Ti-doped mesoporous silica (Ti-MPS) materials with tunable composition and controllable morphology for highly efficient enrichment of phosphopeptides. By altering the molar ratio of silicon to titanium (Si/Ti) in the precursor, the external morphology, Ti content,internal long-rang order, and surface area of Ti-MPS were all modulated accordingly with certain regularity. Tryptic digests of standard phosphoprotein α-and β-casein were employed to assess the phosphopeptide enrichment capability of Ti-MPS series. At the Si/Ti molar ratio of8:1, the optimum enrichment performance with admirable sensitivity and capacity was achieved. The detection limit for β-casein could reach10frnol, and15phosphopeptides from the digest of α-casein were resolved in the spectrum after enrichment, both superior to the behavior of commercial TiO2materials. More significantly, for the digest of human placenta mitochondria,396phosphopeptides and298phosphoproteins were definitely detected and identified after enrichment with optimized Ti-MPS material, demonstrating its remarkable applicability for untouched phosphoproteomes.In addition, this research also opened up a universal pathway to construct a composition-tunable functional material in pursuit of the maximum performance in applications.
Preeclampsia is a specific disorder known to promote maternal or perinatal mortality and morbidity during pregnancy. A large amount of evidences suggest that preeclampsia could be associated with placenta, although its etiology and pathogenesis have not been extensively investigated. To better understand the molecular mechanisms involved in pathological development of placenta in preeclampsia, LC-MS/MS technologies were used to construct a large-scale comparative proteome profile of human placentas in normal and preeclamptic pregnancies. A total of2636proteins were detected in human placentas, and171different proteins were definitively identified between control and preeclamptic placentas. Further bio informatics analysis indicated that these differentially expressed proteins correlate with several specific cellular processes which occur during pathological changes of preeclamptic placenta. Several proteins were randomly selected to verify their expression patterns with Western blotting, and their cellular localizations with immunohistochemistry. Elucidation of how the changes of protein expression coordinate pathological development would provide researchers with a better understanding of these critical biological processes at an early stage of preeclamptic development, and potential therapeutic molecular targets to regulate placenta function and treat preeclampsia.
To better understand the molecular mechanisms involved in pathological development of placenta in preeclampsia, high-resolution LC-MS/MS technologies were used to construct a comparative N-glycoproteomic and phosphoproteomic profiling of human placental plasma membrane in normal and preeclamptic pregnancies. A total of1027N-glyco-and2094phospho-sites were detected in human placental plasma membrane, and5N-glyco-and38phospho-proteins, respectively, with differentially expression were definitively identified between control and preeclamptic placental plasma membrane. Further bio informatics analysis indicated that these differentially expressed proteins correlate with several specific cellular processes which occur during pathological changes of preeclamptic placenta. Further bio informatics analysis indicated that these differentially expressed proteins correlate with several specific cellular processes which occur during pathological changes of preeclamptic placental plasma membrane.
In summary, through proteome analysis of human placenta protein complexes and proteome from normal and preeclamptic pregnancies, we constructed the protein complexes profile, the differentially expressed protein profile and the N-glyco-and phospho-protein expression profile, and identified a number of factors potentially involved in the pathological changes of placental Further functional analyses of these proteins will be conducive to understanding the critical biological processes in placenta of PE and placental related diseases which are caused by disruption of placenta development and transition
本研究通过BN/SDS-PAGE结合LC-MS/MS研究胎盘蛋白及蛋白质复合物,共鉴定了733个非冗余蛋白以及34个蛋白复合物,其中1个为新的未发现有相互作用的复合物。通过生物信息学分析以及结合文献报道的数据,发现这些已知的复合物主要是线粒体呼吸链复合物、整合素复合物、蛋白酶体复合物以及热休克蛋白复合物等。发现的新复合物为clathrin和small conductance calcium-activated potassium (SK) channel protein2。这两个蛋白的相互作用进一步通过antibody based gel shift assay、免疫共沉淀以及免疫荧光进行了验证。这些结果表明BN/SDS-PAGE结合LC-MS/MS是一种很好的分析胎盘蛋白质复合物的方法。
胎盘蛋白的磷酸化是目前胎盘蛋白质组学研究热点之一。从复杂样本中有效富集磷酸化蛋白以及磷酸化肽段是具有挑战性的工作。本研究合成了一系列形态可控、成分可调的钛掺杂介孔硅材料来有效的富集磷酸化肽段以及磷酸化蛋白。通过改变前体中钛/硅摩尔比来调节钛掺杂介孔硅材料的表面积、直径以及形状等,利用这一系列的材料来富集酶解后的标准磷酸化蛋白。研究结果表明,当钛/硅摩尔比为8：1时,该材料富集磷酸化肽段的效果最好,β-酪蛋白的检测极限能达到1Ofmol, a-酪蛋白有15个磷酸化肽段被检测到,优于商业化的二氧化钛珠子。我们将该材料用于胎盘线粒体磷酸化蛋白的富集,共鉴定了396个磷酸化肽段以及298个磷酸化蛋白,结果表明该材料能用于复杂样本的磷酸化肽段富集。此外,本研究还提供了一种合成成分可调的磷酸化肽段富集材料的方法。
由于先兆子痫是一种与胎盘功能发生异常有关的重要妊娠疾病,本研究通过基于质谱的二甲基标记定量蛋白质组学方法对正常孕妇以及先兆子痫孕妇胎盘蛋白质组进行了分析。鉴定出2636种蛋白,其中171种蛋白在先兆子痫胎盘中有差异表达。通过对差异蛋白生物信息学分析,发现这些蛋白主要参与习惯性流产、颅内HADHA高血压、胎儿生长缓慢、胎儿死亡、妊娠期高血压以及治病流产等信号通路。同时随机挑选五种差异蛋白进行免疫印迹以及免疫组化实验,对这些蛋白的表达情况进行验证,结果表明质谱鉴定结果与免疫学结果一致。综上所述,通过比较蛋白质组分析正常妊娠和先兆子痫的胎盘,确定了一些可能参与胎盘病理变化的重要蛋白,对这些蛋白质进行进一步的功能分析有助于了解先兆子痫胎盘以及与胎盘有关的疾病发生、发展和转型的关键生物学过程。
在上述研究基础上,本论文对先兆子痫胎盘质膜糖基化和磷酸化蛋白质组学进行了研究。通过二甲基标记结合糖基化和磷酸化肽段的富集方法,研究了先兆子痫胎盘质膜中的糖基化以及磷酸化蛋白表达谱,通过严格的数据筛选,共鉴定了1027条N-糖基化以及2094条磷酸化肽。7个糖基化肽段以及42个磷酸化肽段具有差异表达。这些差异蛋白进行分析表明这些蛋白参与胎儿死亡以及原发性高血压信号通路。通过分析正常以及先兆子痫妊娠的人胎盘质膜蛋白质组,我们构建了胎盘蛋白质相互作用图谱、先兆子痫胎盘蛋白质表达谱以及N-糖基化和磷酸化的蛋白质表达谱,鉴定了在先兆子痫发生过程中的关键蛋白,建立了磷酸化蛋白富集的新方法,这些研究为深入了解先兆子痫胎盘和胎盘相关的疾病提供了基础。
The placenta plays a pivotal role of promoting the exchange of nutrients and waste products between the maternal and fetal circulatory systems. In addition, it is a natural barrier against numerous bacterial and viral infections during pregnancy. Maternal preeclampsia (PE) and fetal intrauterine growth restriction(IUGR) are two of the most common and serious complications of human pregnancy associated with placental abnormalities. Identification and characterization of placental multi-protein complexes is an important step to integratedly understand the protein-protein interaction networks in placenta which determine placental function. Therefore, elucidation of the placental protein interaction, the expression levels as well as post-translational modifications in various physiological and pathological conditions would provide researchers with a better understanding of the structure and function of the placenta
In this study, blue native/sodium dodecyl sulfate polyacrylamide gel electrophoresis (BN/SDS-PAGE) and Liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to screen the multi-protein complexes in placenta.733unique proteins and34known and novel heterooligomeric multi-protein complexes including mitochondrial respiratory chain complexes, integrin complexes, proteasome complexes, histone complex, and heat shock protein complexes were identified. A novel protein complex, which involves clathrin and small conductance calcium-activated potassium (SK) channel protein2, was identified and validated by antibody based gel shift assay, co-immunoprecipitation and immunouorescence staining. These results suggest that BN/SDS-PAGE, when integrated with LC-MS/MS, is a very powerful and versatile tool for the investigation of placental protein complexes.
Extraction of phosphopeptides from rather complex biological samples has been a tough issue for deep and comprehensive investigation into phosphoproteomes. In this paper, we present a series of Ti-doped mesoporous silica (Ti-MPS) materials with tunable composition and controllable morphology for highly efficient enrichment of phosphopeptides. By altering the molar ratio of silicon to titanium (Si/Ti) in the precursor, the external morphology, Ti content,internal long-rang order, and surface area of Ti-MPS were all modulated accordingly with certain regularity. Tryptic digests of standard phosphoprotein α-and β-casein were employed to assess the phosphopeptide enrichment capability of Ti-MPS series. At the Si/Ti molar ratio of8:1, the optimum enrichment performance with admirable sensitivity and capacity was achieved. The detection limit for β-casein could reach10frnol, and15phosphopeptides from the digest of α-casein were resolved in the spectrum after enrichment, both superior to the behavior of commercial TiO2materials. More significantly, for the digest of human placenta mitochondria,396phosphopeptides and298phosphoproteins were definitely detected and identified after enrichment with optimized Ti-MPS material, demonstrating its remarkable applicability for untouched phosphoproteomes.In addition, this research also opened up a universal pathway to construct a composition-tunable functional material in pursuit of the maximum performance in applications.
Preeclampsia is a specific disorder known to promote maternal or perinatal mortality and morbidity during pregnancy. A large amount of evidences suggest that preeclampsia could be associated with placenta, although its etiology and pathogenesis have not been extensively investigated. To better understand the molecular mechanisms involved in pathological development of placenta in preeclampsia, LC-MS/MS technologies were used to construct a large-scale comparative proteome profile of human placentas in normal and preeclamptic pregnancies. A total of2636proteins were detected in human placentas, and171different proteins were definitively identified between control and preeclamptic placentas. Further bio informatics analysis indicated that these differentially expressed proteins correlate with several specific cellular processes which occur during pathological changes of preeclamptic placenta. Several proteins were randomly selected to verify their expression patterns with Western blotting, and their cellular localizations with immunohistochemistry. Elucidation of how the changes of protein expression coordinate pathological development would provide researchers with a better understanding of these critical biological processes at an early stage of preeclamptic development, and potential therapeutic molecular targets to regulate placenta function and treat preeclampsia.
To better understand the molecular mechanisms involved in pathological development of placenta in preeclampsia, high-resolution LC-MS/MS technologies were used to construct a comparative N-glycoproteomic and phosphoproteomic profiling of human placental plasma membrane in normal and preeclamptic pregnancies. A total of1027N-glyco-and2094phospho-sites were detected in human placental plasma membrane, and5N-glyco-and38phospho-proteins, respectively, with differentially expression were definitively identified between control and preeclamptic placental plasma membrane. Further bio informatics analysis indicated that these differentially expressed proteins correlate with several specific cellular processes which occur during pathological changes of preeclamptic placenta. Further bio informatics analysis indicated that these differentially expressed proteins correlate with several specific cellular processes which occur during pathological changes of preeclamptic placental plasma membrane.
In summary, through proteome analysis of human placenta protein complexes and proteome from normal and preeclamptic pregnancies, we constructed the protein complexes profile, the differentially expressed protein profile and the N-glyco-and phospho-protein expression profile, and identified a number of factors potentially involved in the pathological changes of placental Further functional analyses of these proteins will be conducive to understanding the critical biological processes in placenta of PE and placental related diseases which are caused by disruption of placenta development and transition
引文
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