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
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 were found with significantly altered expression levels after W198 treatment. These repressed or activated proteins are the potential drug targets of W198, which may provide novel targets for future development of biomarkers for cancer therapy.展开更多
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
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
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 were found with significantly altered expression levels after W198 treatment. These repressed or activated proteins are the potential drug targets of W198, which may provide novel targets for future development of biomarkers for cancer therapy.
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.
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.
