Diabetes mellitus is an incurable disease, so it is necessary to establish a model to screen biomarkers for early warning in order to minimize the likelihood of long-term complications. Current- ly, advanced glycation...Diabetes mellitus is an incurable disease, so it is necessary to establish a model to screen biomarkers for early warning in order to minimize the likelihood of long-term complications. Current- ly, advanced glycation end products （AGEs） are considered to be biomarkers of many diseases, such as diabetes and its complications. In this study, a model for further proteomics study was es- tablished to analyze the glycation of HSA with 18 O-labeling strategy. 30 peptides were randomly se- lected to optimize tryptic digestion and 18O-labeling condition by HPLC-ESI/TOF. The best tryptic di- gestion condition was： HSA： Trypsin = 50： 1, w/w for 20 h. The best t8 O-labeling condition was to di- lute urea to 1 M and adjust KH2 POa--K2 HPO4 buffer pH to 6.0 to give a final labeling efficiency of 98.5 ± 0.7%. The inter- and intra-day precisions and stability were satisfactory. This model was es- tablished and optimized for further quantitative proteomics study.展开更多
Objective To compare quantitative proteomic analysis of bromotetrandrine (W198) which was a Class I new antitumor drug in China and tetrandrine (Tet) in K562 cell line using 18O-labeling method. Methods To illustrate ...Objective To compare quantitative proteomic analysis of bromotetrandrine (W198) which was a Class I new antitumor drug in China and tetrandrine (Tet) in K562 cell line using 18O-labeling method. Methods To illustrate its mechanism, a shotgun quantitative proteomic strategy employing 2D LC-MS-MS and trypsin catalyzed 18O-labeling quantification was carried out in this study. Compared to normal chronic leukemia cell line K562 and K562 induced by Tet, the proteomic changes of K562 induced by W198 were investigated. In order to validate the quantitation by the 18O-labeling, the analysis was done on an equivalent sample composed of the same amount of labeled and unlabeled proteins from normally cultured cells to act as a reference to the comparative sample. Results A threshold of ± 2-fold change for deciding whether a protein concentration was changed was settled for the following experiments. Comparing the 105 identified soluble proteins’ expression levels of the apoptosis starting up K562 cells after W198 induction with the normally cultured cells, 16 proteins were found with significantly altered expression levels after W198 treatment. Eight proteins were up-expressed including HMGB2, peroxiredoxin-2, and eIF4A-I, etc. Eight proteins were down-expressed including TCP-1, GRP94, GST-π, and SFGHs, etc. Compared to K562 induced by Tet, eight proteins of K562 were found with significantly altered expression levels after W198 treatment. Five proteins were up-expressed including HSP 90-β and 40S ribosomal protein S15a, etc. Three proteins were down-expressed including phosphoglycerate kinase 1, isoform 5 of interleukin enhancer-binding factor 3, etc. Conclusion The 18O-labeling MS-MS-based method is ideal as a discovery tool, but it is not suitable for validation using a large number of samples. Other more effective methods, such as Western blotting should be used for further validation of candidate cancer proteins discovered from 18O-labeling samples. In total, 105 soluble proteins were discovered, and 16 proteins w展开更多
Aging refers to a multidimensional process that all changes were accumulated in a person over time.These aging changes are associated with progressive increases in the chance of disease and death. Thus,it is necessary...Aging refers to a multidimensional process that all changes were accumulated in a person over time.These aging changes are associated with progressive increases in the chance of disease and death. Thus,it is necessary to establish a model to screen biomarkers to characterize and evaluate aging degree. In this study,an in vitro aging model was set up by formaldehdye and human serum albumin（ HSA）,the most abundant protein in human plasma,based on Maillard Reaction. The liquid chromatography tandem mass spectrometry（ LC-MS /MS） method with ^18O-labeling technique was employed to quantify modification degree of peptides cleaved from HSA. This model was established and optimized for further quantitative biomarker study.展开更多
The CP4-EPSPS gene is widely used in herbicide-tolerant plants/crops all over the world. In this study, a method was developed by coupling liquid chromatography with high sensitivity to tandem mass spectrometry to qua...The CP4-EPSPS gene is widely used in herbicide-tolerant plants/crops all over the world. In this study, a method was developed by coupling liquid chromatography with high sensitivity to tandem mass spectrometry to quantify the amount of CP4-EPSPS expression in Nicotiana tabacum leaves. The quantification of protein was converted to measure the unique peptide of CP4-EPSPS protein. One peptide unique to CP4-EPSPS was synthesized and labeled with.H2^18O to get 180 stable isotope labeled peptide. The peptide served as the internal standard. The validated method had good specificity and linearity. The intra-and inter-day precisions and accuracy for all samples were satisfactory. The results demonstrated that the novel method was sensitive and selective to quantify CP4- EPSPS in the crude extract without time-consuming pre-separation or.the purification procedures.展开更多
目的:采用定量蛋白质组学技术筛选鼻咽癌转移相关的膜蛋白质。方法:在富集高转移鼻咽癌细胞株5–8F和不转移鼻咽癌细胞株6–10B膜蛋白质的基础上,采用稳定同位素18O标记结合串联质谱技术比较两株细胞膜蛋白质组的差异,采用Western印迹...目的:采用定量蛋白质组学技术筛选鼻咽癌转移相关的膜蛋白质。方法:在富集高转移鼻咽癌细胞株5–8F和不转移鼻咽癌细胞株6–10B膜蛋白质的基础上,采用稳定同位素18O标记结合串联质谱技术比较两株细胞膜蛋白质组的差异,采用Western印迹对差异表达膜蛋白质EphA2的表达水平进行验证,采用生物信息学方法对差异蛋白质进行GO(gene oncology)功能聚类和全基因组及代谢途径数据库(Kyoto encyclopedia of genes and genomes,KEGG)信号通路分析。结果:鉴定了31个5–8F与6–10B细胞的差异膜蛋白,其中25个蛋白质在5–8F中表达上调、6个蛋白质表达下调,Western印迹技术验证了差异膜蛋白质EphA2的表达。生物信息分析显示,差异膜蛋白的功能主要涉及细胞黏附、受体再循环和细胞连接等生物学过程,并参与了14个KEGG通路,其中许多通路涉及细胞黏附、细胞连接以及细胞运动。结论:31个差异膜蛋白质可能在鼻咽癌转移过程中发挥重要作用,为进一步研究鼻咽癌转移的分子机制提供了新线索。展开更多
Recently, magnetic nanopartides （NPs） have been extensively used in food industry and biomedical treatments. However, the biocompatibility mechanism on expression proteomics, before consideration of magnetic NPs for...Recently, magnetic nanopartides （NPs） have been extensively used in food industry and biomedical treatments. However, the biocompatibility mechanism on expression proteomics, before consideration of magnetic NPs for clinical application, has not yet been fully elucidated. Therefore, this study was undertaken to identify potential biomarkers of metal ion signaling proteins in human cervical cancer cell line （HeLa） cells. Here, we report the in vitro investigations of the cell cycle response and significant changes in protein abundance of HeLa cells when exposed to self-tailored hydrophilic Fe2C NPs. The comparative proteomic approach based on 180 labeling coupled with high performance liquid chromatography/ electrospray ionization with ion trap mass analyzer （HPLC/ESI-Orbitrap） was applied, and 394 proteins were identified. There were 46 significantly differentiated proteins based on the specific metal ion signaling response. Among them, 60S ribosomal protein L37a, serine/arginine-rich splicing factor 7, calmodulin, and calumenin were downregulated, whereas transketolase was overexpressed. Functional interaction network of Fe2C-regulated proteins was successfully created by the STRING algorithm to show the strong interactions between proteins. This work will not only help to understand the molecular mechanism of metal ion signaling proteins that can potentially be used to develop therapeutic protocols for diagnosis of diseases but also give direction for tailoring biocompatible magnetic NPs.展开更多
建立了定量肽段串联体蛋白质(concatamers of Q peptides,QconCATs)结合18O同位素标记-多反应监测质谱的蛋白质绝对定量新方法。首先对QconCAT重组蛋白质进行了纯度表征,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)表征结果表明重组...建立了定量肽段串联体蛋白质(concatamers of Q peptides,QconCATs)结合18O同位素标记-多反应监测质谱的蛋白质绝对定量新方法。首先对QconCAT重组蛋白质进行了纯度表征,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)表征结果表明重组蛋白质的纯度在99%以上,相对分子质量约为63.4 kDa。对QconCAT重组蛋白质酶切后的肽段混合物进行质谱分析,并经pFind和pLabel软件处理,验证了目标肽段。还考察了QconCAT重组蛋白质的酶切效率和18O标记效率,并对QconCAT蛋白质结合18O标记-同位素稀释-多反应监测质谱方法进行了评价。实验结果表明,采用该方法对腾冲嗜热厌氧菌(Thermoanaerobacter tengcongensis,TTE)中选定蛋白质的肽段进行绝对含量测定时,相对标准偏差小于20%,准确度较高,说明该方法可用于复杂生物样本中蛋白质的绝对定量。更重要的是所建方法不仅解决了细胞培养氨基酸稳定同位素标记(SILAC)技术的重标试剂价格昂贵的问题,也为定量蛋白质组学提供了一种新的方法。展开更多
Detecting protein expression levels in organisms exposed to environmental pollutants can help us understand the action mechanism of toxicity. In the present study, adult female Sprague-Dawley (SD) rats received a cons...Detecting protein expression levels in organisms exposed to environmental pollutants can help us understand the action mechanism of toxicity. In the present study, adult female Sprague-Dawley (SD) rats received a consecutive intraperitoneal injection of tris(2,3-dibromopropyl) isocyanurate (TBC) for seven days, and the ^16O/^18O labeling comparative proteomic approach was used to study the change of liver proteome in these rats. Thirty six differential proteins were identified between the control group and the high-dose-exposed group (8 mg/kg), of which, twenty eight proteins were down-regulated and eight proteins were up-regulated. Bioinformatics analysis revealed that most of the differential proteins were related with the metabolic and cellular processes. In addition, the contents of malondialdehyde (MDA), catalase (CAT), and glutathione (GSH) in liver were measured to evaluate the oxidative stress status induced by TBC exposure. Together, these findings showed that TBC might be toxic to liver by disrupting metabolic process and apoptosis. The results might provide a better insight into the mechanism of toxicity induced by TBC.展开更多
采用胰蛋白酶催化的^(18)O标记联合高效液相色谱-高分辨率串联质谱技术(high performance liquid chromatography-linear ion trap/Orbitrap high-resolution mass spectrometry,HPLC-LTQ/Orbitrap MS/MS)研究阿胶的特征性多肽的^(18)O...采用胰蛋白酶催化的^(18)O标记联合高效液相色谱-高分辨率串联质谱技术(high performance liquid chromatography-linear ion trap/Orbitrap high-resolution mass spectrometry,HPLC-LTQ/Orbitrap MS/MS)研究阿胶的特征性多肽的^(18)O标记情况和明胶混合物中阿胶、牛皮明胶的定量情况。结果表明,阿胶的特征性多肽均能被2个^(18)O原子标记,当^(18)O和^(16)O标记多肽以20∶1、10∶1、5∶1、1∶1和1∶5的质量比例混合时,检测到的^(18)O与^(16)O标记多肽比值分别为19.66、10.24、5.01、1.00和0.20。当阿胶和牛皮明胶质量比以1∶10、1∶1和10∶1混合,以^(18)O标记物为内标,检测到的阿胶与明胶比值分别为0.10、1.02和9.72。因此,胰蛋白酶催化的^(18)O标记联合HPLC-LTQ/Orbitrap MS/MS适用于阿胶产品中目标明胶的含量检测,可为阿胶产品的质量控制提供技术支撑。展开更多
基金Supported by the National Key Technology R&D Program of China(2012YQ040140,2012CB91060)the National Natural Science Foundation of China(21205005)
文摘Diabetes mellitus is an incurable disease, so it is necessary to establish a model to screen biomarkers for early warning in order to minimize the likelihood of long-term complications. Current- ly, advanced glycation end products （AGEs） are considered to be biomarkers of many diseases, such as diabetes and its complications. In this study, a model for further proteomics study was es- tablished to analyze the glycation of HSA with 18 O-labeling strategy. 30 peptides were randomly se- lected to optimize tryptic digestion and 18O-labeling condition by HPLC-ESI/TOF. The best tryptic di- gestion condition was： HSA： Trypsin = 50： 1, w/w for 20 h. The best t8 O-labeling condition was to di- lute urea to 1 M and adjust KH2 POa--K2 HPO4 buffer pH to 6.0 to give a final labeling efficiency of 98.5 ± 0.7%. The inter- and intra-day precisions and stability were satisfactory. This model was es- tablished and optimized for further quantitative proteomics study.
文摘Objective To compare quantitative proteomic analysis of bromotetrandrine (W198) which was a Class I new antitumor drug in China and tetrandrine (Tet) in K562 cell line using 18O-labeling method. Methods To illustrate its mechanism, a shotgun quantitative proteomic strategy employing 2D LC-MS-MS and trypsin catalyzed 18O-labeling quantification was carried out in this study. Compared to normal chronic leukemia cell line K562 and K562 induced by Tet, the proteomic changes of K562 induced by W198 were investigated. In order to validate the quantitation by the 18O-labeling, the analysis was done on an equivalent sample composed of the same amount of labeled and unlabeled proteins from normally cultured cells to act as a reference to the comparative sample. Results A threshold of ± 2-fold change for deciding whether a protein concentration was changed was settled for the following experiments. Comparing the 105 identified soluble proteins’ expression levels of the apoptosis starting up K562 cells after W198 induction with the normally cultured cells, 16 proteins were found with significantly altered expression levels after W198 treatment. Eight proteins were up-expressed including HMGB2, peroxiredoxin-2, and eIF4A-I, etc. Eight proteins were down-expressed including TCP-1, GRP94, GST-π, and SFGHs, etc. Compared to K562 induced by Tet, eight proteins of K562 were found with significantly altered expression levels after W198 treatment. Five proteins were up-expressed including HSP 90-β and 40S ribosomal protein S15a, etc. Three proteins were down-expressed including phosphoglycerate kinase 1, isoform 5 of interleukin enhancer-binding factor 3, etc. Conclusion The 18O-labeling MS-MS-based method is ideal as a discovery tool, but it is not suitable for validation using a large number of samples. Other more effective methods, such as Western blotting should be used for further validation of candidate cancer proteins discovered from 18O-labeling samples. In total, 105 soluble proteins were discovered, and 16 proteins w
基金Supported by the National Natural Science Foundation of China(21205005,81471919)MOST China(2011YQ0900502)1000 Plan
文摘Aging refers to a multidimensional process that all changes were accumulated in a person over time.These aging changes are associated with progressive increases in the chance of disease and death. Thus,it is necessary to establish a model to screen biomarkers to characterize and evaluate aging degree. In this study,an in vitro aging model was set up by formaldehdye and human serum albumin（ HSA）,the most abundant protein in human plasma,based on Maillard Reaction. The liquid chromatography tandem mass spectrometry（ LC-MS /MS） method with ^18O-labeling technique was employed to quantify modification degree of peptides cleaved from HSA. This model was established and optimized for further quantitative biomarker study.
基金Supported by National Natural Science Foundation of China(21205005,81471919,21475010)MOST China(2011YQ0900502)+1 种基金1000 PlanResearch Foundation of China CDC(2014A101)
文摘The CP4-EPSPS gene is widely used in herbicide-tolerant plants/crops all over the world. In this study, a method was developed by coupling liquid chromatography with high sensitivity to tandem mass spectrometry to quantify the amount of CP4-EPSPS expression in Nicotiana tabacum leaves. The quantification of protein was converted to measure the unique peptide of CP4-EPSPS protein. One peptide unique to CP4-EPSPS was synthesized and labeled with.H2^18O to get 180 stable isotope labeled peptide. The peptide served as the internal standard. The validated method had good specificity and linearity. The intra-and inter-day precisions and accuracy for all samples were satisfactory. The results demonstrated that the novel method was sensitive and selective to quantify CP4- EPSPS in the crude extract without time-consuming pre-separation or.the purification procedures.
文摘目的:采用定量蛋白质组学技术筛选鼻咽癌转移相关的膜蛋白质。方法:在富集高转移鼻咽癌细胞株5–8F和不转移鼻咽癌细胞株6–10B膜蛋白质的基础上,采用稳定同位素18O标记结合串联质谱技术比较两株细胞膜蛋白质组的差异,采用Western印迹对差异表达膜蛋白质EphA2的表达水平进行验证,采用生物信息学方法对差异蛋白质进行GO(gene oncology)功能聚类和全基因组及代谢途径数据库(Kyoto encyclopedia of genes and genomes,KEGG)信号通路分析。结果:鉴定了31个5–8F与6–10B细胞的差异膜蛋白,其中25个蛋白质在5–8F中表达上调、6个蛋白质表达下调,Western印迹技术验证了差异膜蛋白质EphA2的表达。生物信息分析显示,差异膜蛋白的功能主要涉及细胞黏附、受体再循环和细胞连接等生物学过程,并参与了14个KEGG通路,其中许多通路涉及细胞黏附、细胞连接以及细胞运动。结论:31个差异膜蛋白质可能在鼻咽癌转移过程中发挥重要作用,为进一步研究鼻咽癌转移的分子机制提供了新线索。
文摘Recently, magnetic nanopartides （NPs） have been extensively used in food industry and biomedical treatments. However, the biocompatibility mechanism on expression proteomics, before consideration of magnetic NPs for clinical application, has not yet been fully elucidated. Therefore, this study was undertaken to identify potential biomarkers of metal ion signaling proteins in human cervical cancer cell line （HeLa） cells. Here, we report the in vitro investigations of the cell cycle response and significant changes in protein abundance of HeLa cells when exposed to self-tailored hydrophilic Fe2C NPs. The comparative proteomic approach based on 180 labeling coupled with high performance liquid chromatography/ electrospray ionization with ion trap mass analyzer （HPLC/ESI-Orbitrap） was applied, and 394 proteins were identified. There were 46 significantly differentiated proteins based on the specific metal ion signaling response. Among them, 60S ribosomal protein L37a, serine/arginine-rich splicing factor 7, calmodulin, and calumenin were downregulated, whereas transketolase was overexpressed. Functional interaction network of Fe2C-regulated proteins was successfully created by the STRING algorithm to show the strong interactions between proteins. This work will not only help to understand the molecular mechanism of metal ion signaling proteins that can potentially be used to develop therapeutic protocols for diagnosis of diseases but also give direction for tailoring biocompatible magnetic NPs.
基金Supported by the Basic Research Foundation of Beijing Institute of Technology(20141642002)
文摘Detecting protein expression levels in organisms exposed to environmental pollutants can help us understand the action mechanism of toxicity. In the present study, adult female Sprague-Dawley (SD) rats received a consecutive intraperitoneal injection of tris(2,3-dibromopropyl) isocyanurate (TBC) for seven days, and the ^16O/^18O labeling comparative proteomic approach was used to study the change of liver proteome in these rats. Thirty six differential proteins were identified between the control group and the high-dose-exposed group (8 mg/kg), of which, twenty eight proteins were down-regulated and eight proteins were up-regulated. Bioinformatics analysis revealed that most of the differential proteins were related with the metabolic and cellular processes. In addition, the contents of malondialdehyde (MDA), catalase (CAT), and glutathione (GSH) in liver were measured to evaluate the oxidative stress status induced by TBC exposure. Together, these findings showed that TBC might be toxic to liver by disrupting metabolic process and apoptosis. The results might provide a better insight into the mechanism of toxicity induced by TBC.
