Monogalactosyldiacylglycerols(MGDGs)have potential applications in food products,cosmetics and pharmaceuticals.MGDGs from microalgae with high amounts of polyunsaturated fatty acids(PUFAs)have potential functions,whic...Monogalactosyldiacylglycerols(MGDGs)have potential applications in food products,cosmetics and pharmaceuticals.MGDGs from microalgae with high amounts of polyunsaturated fatty acids(PUFAs)have potential functions,which arise the interest of the researchers.MGDGs were prepared by silica gel column chromatography with the appropriate mobile phase,while due to the similarity to molecular structure of MGDGs,digalactosyldiacylglycerols(DGDGs)were the most difficult impurities to separate during the extraction process of MGDGs from Nannochloropsis oceanica IMET1 and Arthrospira platensis.In order to obtain MGDGs from microalgae using low toxic solvent system,a novel material Click thiol-ene cysteine(Click TE-Cys)was employed to achieve its selective separation by differentiation of the hydrophilic interaction.The mixture of MGDGs and DGDGs standards were separated from each other by Click TE-Cys solid phase extraction(SPE),which was further confirmed by the result of LC-MS.The molecular interaction of MGDGs and DGDGs with Click TE-Cys demonstrated that DGDGs had more hydrogen bonds with Click TE-Cys material,which might cause a higher hydrophilic interaction.In this study,the Click TE-Cys material exhibited higher hydrophilicity with DGDGs and effectively separated MGDGs from 4 species microalgae by‘flow-through'mode using ethanol as mobile phase.展开更多
Complex systems exist widely,including medicines from natural products,functional foods,and biological samples.The biological activity of complex systems is often the result of the synergistic effect of multiple compo...Complex systems exist widely,including medicines from natural products,functional foods,and biological samples.The biological activity of complex systems is often the result of the synergistic effect of multiple components.In the quality evaluation of complex samples,multicomponent quantitative analysis(MCQA)is usually needed.To overcome the difficulty in obtaining standard products,scholars have proposed achieving MCQA through the“single standard to determine multiple components(SSDMC)”approach.This method has been used in the determination of multicomponent content in natural source drugs and the analysis of impurities in chemical drugs and has been included in the Chinese Pharmacopoeia.Depending on a convenient(ultra)high-performance liquid chromatography method,how can the repeatability and robustness of the MCQA method be improved?How can the chromatography conditions be optimized to improve the number of quantitative components?How can computer software technology be introduced to improve the efficiency of multicomponent analysis(MCA)?These are the key problems that remain to be solved in practical MCQA.First,this review article summarizes the calculation methods of relative correction factors in the SSDMC approach in the past five years,as well as the method robustness and accuracy evaluation.Second,it also summarizes methods to improve peak capacity and quantitative accuracy in MCA,including column selection and twodimensional chromatographic analysis technology.Finally,computer software technologies for predicting chromatographic conditions and analytical parameters are introduced,which provides an idea for intelligent method development in MCA.This paper aims to provide methodological ideas for the improvement of complex system analysis,especially MCQA.展开更多
Human serum albumin(HSA) has emerged as a pivotal biomarker and prognostic indicator for various human diseases. Real-time sensing and visual tracking of HSA in plasma or other biological systems will immensely facili...Human serum albumin(HSA) has emerged as a pivotal biomarker and prognostic indicator for various human diseases. Real-time sensing and visual tracking of HSA in plasma or other biological systems will immensely facilitate the basic researchers and clinicians to better understand HSA-associated biological processes. Herein, a novel near-infrared(NIR) fluorescent probe(7-HTCF) was rationally constructed for light-up sensing and in-situ imaging of HSA in real samples, based on the principle of twisted intramolecular charge transfer(TICT). Under physiological conditions, 7-HTCF could be efficiently trapped by HSA to form a stable complex via binding on a non-drug binding site, while the complex emitted strong fluoresce signals around 670 nm. Further investigations demonstrated that 7-HTCF displayed a great combination of excellent selectivity and good chemical stability, as well as rapid fluorescent response and ultra-high sensitivity for HSA detection. Particularly, the newly developed light-up probe has been successfully utilized for quantitative detection of HSA in diluted plasma samples, while its readouts are hardly affected by the addition of therapeutic agents and herbal medicines. 7-HTCF is also successfully used for in-situ imaging of the reabsorbed HSA in living renal cells, while this dye exhibits good cell permeability and high resolution for in-situ imaging in living cells. Collectively, a novel TICT-based near-infrared fluorescent probe was devised for highly selective and ultra-sensitive sensing of HSA in plasma samples or imaging HSA in living cells, which offered a practical tool for clinical tests and for exploring HSA-associated biological processes.展开更多
Background: Danshen is an important traditional Chinese medicine(TCM) used for the treatment of cardiovascular and cerebrovascular diseases. Separation and analysis of its components have been widely investigated. How...Background: Danshen is an important traditional Chinese medicine(TCM) used for the treatment of cardiovascular and cerebrovascular diseases. Separation and analysis of its components have been widely investigated. However, the systematical two dimensional liquid chromatography(2D-LC) methods have not been developed to comprehensively separate and characterize its components.Objective: In this work, double off-line 2D-LC methods were aimed to develop for the systematical separation of compounds from Danshen.Methods: Using solid phase extraction(SPE), the Danshen extract was divided into a medium-polar fraction(Sample I) and a weak-polar fraction(Sample Ⅱ) according to their polarities. Based on reversed-phase liquid chromatography(RPLC) and hydrophilic interaction liquid chromatography(HILIC) modes, a 2D-HILIC × RPLC system and a 2D-RPLC × RPLC system were designed for the separation of Sample Ⅰ and Sample Ⅱ, respectively. According to reversed-phase and HILIC columns selectivities characterized in our previous reports, ZIC-HILIC and XTerra C18 were employed to build the 2D-HILIC × RPLC system and Click TE-CD and XTerra C18 for the 2D-RPLC × RPLC system,respectively.Results: The 2D-HILIC × RPLC and 2D-RPLC × RPLC systems exhibited excellent orthogonality for the separation of Sample Ⅰ and Sample Ⅱ,respectively. Their orthogonalities were 88.42% and 63.24%. Based on these double 2D-LC systems combined with mass spectrometry, at least 200 compounds were found and 33 compounds of them were identified, including 16 phenolic acids and 17 diterpenoid quinines.Conclusion: These results suggest that these two off-line 2D-LC methods are effective for the separation and characterization of components in Danshen.展开更多
In the intrinsic pathway of apoptosis, stresses of mitochondrial reactive oxygen species （mitoROS） might be sensed as more effective signals than those in cytosol, as mitochondria are the major sources of reactive o...In the intrinsic pathway of apoptosis, stresses of mitochondrial reactive oxygen species （mitoROS） might be sensed as more effective signals than those in cytosol, as mitochondria are the major sources of reactive oxygen species （ROS） and pivotal components during cell apoptosis. Mitochondrial superoxide dismutase （SOD2） takes the leading role in eliminating mitoROS, and inhibition of SOD2 might induce severe disturbances overwhelming the mitochondrial oxidative equilibrium, which would elevate the intracellular oxidative stresses and drive cells to death. Herein, we report a general strategy to kill cancer cells by targeted inhibition of SOD2 using 2-methoxyestradiol （2-ME, an inhibitor for the SOD family） via a robust mitochondria-targeted mesoporous silica nanocarrier （mtMSN）, with the expected elevation of mitoROS and activation of apoptosis in HeLa cells. Fe304@MSN was employed in the mitochondria-targeted drug delivery and selective inhibition of mitochondrial enzymes, and was shown to be stable with good biocompatibility and high loading capacity. Due to the selective inhibition of SOD2 by 2-ME/mtMSN, enhanced elevation of mitoROS （132% of that with free 2-ME） was obtained, coupled with higher efficiency in initiating cell apoptosis （395% of that with free 2-ME in 4 h）. Finally, the 2-ME/mtMSN exhibited powerful efficacy in targeted killing of HeLa cells by taking advantage of both biological recognition and magnetic guiding, causing 97.0% cell death with only 2 Dg/mL 2-ME/mtMSN, hinting at its great potential in cancer therapy through manipulation of the delicate mitochondrial oxidative balance.展开更多
Prostate cancer(PCa)is the second most prevalent malignancy in males across the world.A greater knowledge of the relationship between protein abundance and drug responses would benefit precision treatment for PCa.Here...Prostate cancer(PCa)is the second most prevalent malignancy in males across the world.A greater knowledge of the relationship between protein abundance and drug responses would benefit precision treatment for PCa.Herein,we establish 35 Chinese PCa primary cell models to capture specific characteristics among PCa patients,including gene mutations,mRNA/protein/surface protein distributions,and pharmaceutical responses.The multi-omics analyses identify Anterior Gradient 2(AGR2)as a pre-operative prognostic biomarker in PCa.Through the drug library screening,we describe crizotinib as a selective compound for malignant PCa primary cells.We further perform the pharmacoproteome analysis and identify 14,372 significant protein-drug correlations.Surprisingly,the diminished AGR2 enhances the inhibition activity of crizotinib via ALK/c-MET-AKT axis activation which is validated by PC3 and xenograft model.Our integrated multi-omics approach yields a comprehensive understanding of PCa biomarkers and pharmacological responses,allowing for more precise diagnosis and therapies.展开更多
Detection and determination of multi-phosphopeptides from protein digestion products are difficult due to the low abundance and ion-suppression effect arised from the existence of their high-abundance counterparts. Cl...Detection and determination of multi-phosphopeptides from protein digestion products are difficult due to the low abundance and ion-suppression effect arised from the existence of their high-abundance counterparts. Click OEG-CD[olio（ethylene glycol）（OEG） linked β-cyclodextrin（CD）] matrix has shown excellent performance in phosphopeptide enrichment. However, few multi-phosphopeptides were detected previously. In this investigation, an improved method aiming at enhancing the enrichment selectivity and mass spectrometry（MS） detection of multi-phosphopeptide was developed via optimizing the sample loading amount on per mg of matrix. Mass spectra were obtained on a Nano liquid chromatography-electrospray ionization-quadrupole time of flight-mass spectrometry （LC-ESI-qTOF-MS）. The enrichment selectivity of double-phosphopeptide could be enhanced with the increase of loading amount on per mg of matrix when taking the mixture of mono-, double-, and non-phosphopeptides as probe. Furthermore, the multi-phosphopeptide enrichment selectivity was enhanced under the condition of optimized loading amount when taking the product of typtic digestion a-casein as test sample. When the loading amount was 573 pmol/mg matrix, up to 20 a-casein phosphopeptide signals（including 15 multi-phosphopeptides） were detected. The result is much better than that of our previous report. The reduction of ion-suppression effect arised from the existence of high-abundance non- or mono-phosphopeptides and the stronger interactions between multiphosphopepides and the matrix were contributed to the result. The study could be helpful to the better utilization of Click OEG-CD matrix in the enrichment of multi-phosphopeptide from complex biosamples in the subsequent investigation.展开更多
The human serum proteome is closely associated with the state of the body.Endogenous peptides derived from proteolytic enzymes cleaving on serum proteins are widely studied due to their potential application in diseas...The human serum proteome is closely associated with the state of the body.Endogenous peptides derived from proteolytic enzymes cleaving on serum proteins are widely studied due to their potential application in disease-specific marker discovery.However,the reproducibility of peptidome analysis of endogenous peptides is significantly influenced by the proteolytic enzymes within body fluids,thereby limiting the clinical use of the endogenous peptides.We comprehensively investigated the N and C terminus of endogenous peptides using peptidomics.The cleavage site patterns of the N and C terminus and adjacent sites from all the identified endogenous peptides were highly conserved under different sample preparation conditions,including long-term incubation at 37℃ and pretreatment with repeated freeze-thaw cycles.Furthermore,a distinguishable cleavage site pattern was obtained when a different disease serum was analyzed.The conserved cleavage site pattern derived from proteolytic enzymes holds potential in highly specific disease diagnosis.展开更多
基金supported by the National High Technology Research and Development Program‘863’(No.14AA022004)the Key Research and Development Plan in Shandong Province(No.YYSP016)the National Natural Science Foundation of China(No.21505131)。
文摘Monogalactosyldiacylglycerols(MGDGs)have potential applications in food products,cosmetics and pharmaceuticals.MGDGs from microalgae with high amounts of polyunsaturated fatty acids(PUFAs)have potential functions,which arise the interest of the researchers.MGDGs were prepared by silica gel column chromatography with the appropriate mobile phase,while due to the similarity to molecular structure of MGDGs,digalactosyldiacylglycerols(DGDGs)were the most difficult impurities to separate during the extraction process of MGDGs from Nannochloropsis oceanica IMET1 and Arthrospira platensis.In order to obtain MGDGs from microalgae using low toxic solvent system,a novel material Click thiol-ene cysteine(Click TE-Cys)was employed to achieve its selective separation by differentiation of the hydrophilic interaction.The mixture of MGDGs and DGDGs standards were separated from each other by Click TE-Cys solid phase extraction(SPE),which was further confirmed by the result of LC-MS.The molecular interaction of MGDGs and DGDGs with Click TE-Cys demonstrated that DGDGs had more hydrogen bonds with Click TE-Cys material,which might cause a higher hydrophilic interaction.In this study,the Click TE-Cys material exhibited higher hydrophilicity with DGDGs and effectively separated MGDGs from 4 species microalgae by‘flow-through'mode using ethanol as mobile phase.
基金the National Natural Science Foundation of China(Grant No.:81803734)National S&T Major Special Project for New Innovative Drugs Sponsored(Grant No.:2019ZX09201005).
文摘Complex systems exist widely,including medicines from natural products,functional foods,and biological samples.The biological activity of complex systems is often the result of the synergistic effect of multiple components.In the quality evaluation of complex samples,multicomponent quantitative analysis(MCQA)is usually needed.To overcome the difficulty in obtaining standard products,scholars have proposed achieving MCQA through the“single standard to determine multiple components(SSDMC)”approach.This method has been used in the determination of multicomponent content in natural source drugs and the analysis of impurities in chemical drugs and has been included in the Chinese Pharmacopoeia.Depending on a convenient(ultra)high-performance liquid chromatography method,how can the repeatability and robustness of the MCQA method be improved?How can the chromatography conditions be optimized to improve the number of quantitative components?How can computer software technology be introduced to improve the efficiency of multicomponent analysis(MCA)?These are the key problems that remain to be solved in practical MCQA.First,this review article summarizes the calculation methods of relative correction factors in the SSDMC approach in the past five years,as well as the method robustness and accuracy evaluation.Second,it also summarizes methods to improve peak capacity and quantitative accuracy in MCA,including column selection and twodimensional chromatographic analysis technology.Finally,computer software technologies for predicting chromatographic conditions and analytical parameters are introduced,which provides an idea for intelligent method development in MCA.This paper aims to provide methodological ideas for the improvement of complex system analysis,especially MCQA.
基金financially supported by the National Key Research and Development Program of China (No. 2021YFE0200900)National Natural Science Foundation of China (Nos. 81922070, 81973286, 82003847, 81703604)+3 种基金Shanghai Science and Technology Innovation Action Plans (Nos. 20S21901500 and 20S21900900)supported by Shanghai Science and Technology Committee, Innovation Team and Talents Cultivation Program of National Administration of Traditional Chinese Medicine (No. ZYYCXTD-D-202004)Shanghai Talent Development Fund (No. 2019093)Shanghai University of Traditional Chinese Medicine Postgraduate Innovation Training Special (No. Y2021034)。
文摘Human serum albumin(HSA) has emerged as a pivotal biomarker and prognostic indicator for various human diseases. Real-time sensing and visual tracking of HSA in plasma or other biological systems will immensely facilitate the basic researchers and clinicians to better understand HSA-associated biological processes. Herein, a novel near-infrared(NIR) fluorescent probe(7-HTCF) was rationally constructed for light-up sensing and in-situ imaging of HSA in real samples, based on the principle of twisted intramolecular charge transfer(TICT). Under physiological conditions, 7-HTCF could be efficiently trapped by HSA to form a stable complex via binding on a non-drug binding site, while the complex emitted strong fluoresce signals around 670 nm. Further investigations demonstrated that 7-HTCF displayed a great combination of excellent selectivity and good chemical stability, as well as rapid fluorescent response and ultra-high sensitivity for HSA detection. Particularly, the newly developed light-up probe has been successfully utilized for quantitative detection of HSA in diluted plasma samples, while its readouts are hardly affected by the addition of therapeutic agents and herbal medicines. 7-HTCF is also successfully used for in-situ imaging of the reabsorbed HSA in living renal cells, while this dye exhibits good cell permeability and high resolution for in-situ imaging in living cells. Collectively, a novel TICT-based near-infrared fluorescent probe was devised for highly selective and ultra-sensitive sensing of HSA in plasma samples or imaging HSA in living cells, which offered a practical tool for clinical tests and for exploring HSA-associated biological processes.
基金funded by Project of National Science Foundation of China(81473436,81274077 and 81403100)
文摘Background: Danshen is an important traditional Chinese medicine(TCM) used for the treatment of cardiovascular and cerebrovascular diseases. Separation and analysis of its components have been widely investigated. However, the systematical two dimensional liquid chromatography(2D-LC) methods have not been developed to comprehensively separate and characterize its components.Objective: In this work, double off-line 2D-LC methods were aimed to develop for the systematical separation of compounds from Danshen.Methods: Using solid phase extraction(SPE), the Danshen extract was divided into a medium-polar fraction(Sample I) and a weak-polar fraction(Sample Ⅱ) according to their polarities. Based on reversed-phase liquid chromatography(RPLC) and hydrophilic interaction liquid chromatography(HILIC) modes, a 2D-HILIC × RPLC system and a 2D-RPLC × RPLC system were designed for the separation of Sample Ⅰ and Sample Ⅱ, respectively. According to reversed-phase and HILIC columns selectivities characterized in our previous reports, ZIC-HILIC and XTerra C18 were employed to build the 2D-HILIC × RPLC system and Click TE-CD and XTerra C18 for the 2D-RPLC × RPLC system,respectively.Results: The 2D-HILIC × RPLC and 2D-RPLC × RPLC systems exhibited excellent orthogonality for the separation of Sample Ⅰ and Sample Ⅱ,respectively. Their orthogonalities were 88.42% and 63.24%. Based on these double 2D-LC systems combined with mass spectrometry, at least 200 compounds were found and 33 compounds of them were identified, including 16 phenolic acids and 17 diterpenoid quinines.Conclusion: These results suggest that these two off-line 2D-LC methods are effective for the separation and characterization of components in Danshen.
文摘In the intrinsic pathway of apoptosis, stresses of mitochondrial reactive oxygen species （mitoROS） might be sensed as more effective signals than those in cytosol, as mitochondria are the major sources of reactive oxygen species （ROS） and pivotal components during cell apoptosis. Mitochondrial superoxide dismutase （SOD2） takes the leading role in eliminating mitoROS, and inhibition of SOD2 might induce severe disturbances overwhelming the mitochondrial oxidative equilibrium, which would elevate the intracellular oxidative stresses and drive cells to death. Herein, we report a general strategy to kill cancer cells by targeted inhibition of SOD2 using 2-methoxyestradiol （2-ME, an inhibitor for the SOD family） via a robust mitochondria-targeted mesoporous silica nanocarrier （mtMSN）, with the expected elevation of mitoROS and activation of apoptosis in HeLa cells. Fe304@MSN was employed in the mitochondria-targeted drug delivery and selective inhibition of mitochondrial enzymes, and was shown to be stable with good biocompatibility and high loading capacity. Due to the selective inhibition of SOD2 by 2-ME/mtMSN, enhanced elevation of mitoROS （132% of that with free 2-ME） was obtained, coupled with higher efficiency in initiating cell apoptosis （395% of that with free 2-ME in 4 h）. Finally, the 2-ME/mtMSN exhibited powerful efficacy in targeted killing of HeLa cells by taking advantage of both biological recognition and magnetic guiding, causing 97.0% cell death with only 2 Dg/mL 2-ME/mtMSN, hinting at its great potential in cancer therapy through manipulation of the delicate mitochondrial oxidative balance.
基金supported by National Natural Science Foundation of China(22137002[Y.D.],32071432[M.T.],81872102[H.J.])University Innovation Research Group Project of Chongqing(CXQT21016[Y.D.])+3 种基金High-Level Innovation Platform Project of Chongqing[Y.D.]Talent Program of Chongqing(CQYC202003053[Y.D.])The collaborative project of Chinese Academy of Sciences institutions and universities in Chongqing(HZ2021006[Y.D.])Innovative Research Team of High-Level Local Universities in Shanghai(SSMU-ZDCX20181202[M.T.]).
文摘Prostate cancer(PCa)is the second most prevalent malignancy in males across the world.A greater knowledge of the relationship between protein abundance and drug responses would benefit precision treatment for PCa.Herein,we establish 35 Chinese PCa primary cell models to capture specific characteristics among PCa patients,including gene mutations,mRNA/protein/surface protein distributions,and pharmaceutical responses.The multi-omics analyses identify Anterior Gradient 2(AGR2)as a pre-operative prognostic biomarker in PCa.Through the drug library screening,we describe crizotinib as a selective compound for malignant PCa primary cells.We further perform the pharmacoproteome analysis and identify 14,372 significant protein-drug correlations.Surprisingly,the diminished AGR2 enhances the inhibition activity of crizotinib via ALK/c-MET-AKT axis activation which is validated by PC3 and xenograft model.Our integrated multi-omics approach yields a comprehensive understanding of PCa biomarkers and pharmacological responses,allowing for more precise diagnosis and therapies.
基金Supported by the National Natural Science Foundation of China（Nos.21105100, 21105007, 21475129）, the Dalian Science and Technology Project, China（No.2012E15SF145） and the Natural Science Foundation of Liaoning Province, China （No.201202054）.
文摘Detection and determination of multi-phosphopeptides from protein digestion products are difficult due to the low abundance and ion-suppression effect arised from the existence of their high-abundance counterparts. Click OEG-CD[olio（ethylene glycol）（OEG） linked β-cyclodextrin（CD）] matrix has shown excellent performance in phosphopeptide enrichment. However, few multi-phosphopeptides were detected previously. In this investigation, an improved method aiming at enhancing the enrichment selectivity and mass spectrometry（MS） detection of multi-phosphopeptide was developed via optimizing the sample loading amount on per mg of matrix. Mass spectra were obtained on a Nano liquid chromatography-electrospray ionization-quadrupole time of flight-mass spectrometry （LC-ESI-qTOF-MS）. The enrichment selectivity of double-phosphopeptide could be enhanced with the increase of loading amount on per mg of matrix when taking the mixture of mono-, double-, and non-phosphopeptides as probe. Furthermore, the multi-phosphopeptide enrichment selectivity was enhanced under the condition of optimized loading amount when taking the product of typtic digestion a-casein as test sample. When the loading amount was 573 pmol/mg matrix, up to 20 a-casein phosphopeptide signals（including 15 multi-phosphopeptides） were detected. The result is much better than that of our previous report. The reduction of ion-suppression effect arised from the existence of high-abundance non- or mono-phosphopeptides and the stronger interactions between multiphosphopepides and the matrix were contributed to the result. The study could be helpful to the better utilization of Click OEG-CD matrix in the enrichment of multi-phosphopeptide from complex biosamples in the subsequent investigation.
基金supported by the Creative Research Group Project of National Natural Science Foundation of China(Grant No.21021004)the National Basic Research Program(973 Program)(Nos.2012CB910601,2012CB910101)the Analytical Method Innovation Program of MOST(No.2010IM030500)(H.Z.)。
文摘The human serum proteome is closely associated with the state of the body.Endogenous peptides derived from proteolytic enzymes cleaving on serum proteins are widely studied due to their potential application in disease-specific marker discovery.However,the reproducibility of peptidome analysis of endogenous peptides is significantly influenced by the proteolytic enzymes within body fluids,thereby limiting the clinical use of the endogenous peptides.We comprehensively investigated the N and C terminus of endogenous peptides using peptidomics.The cleavage site patterns of the N and C terminus and adjacent sites from all the identified endogenous peptides were highly conserved under different sample preparation conditions,including long-term incubation at 37℃ and pretreatment with repeated freeze-thaw cycles.Furthermore,a distinguishable cleavage site pattern was obtained when a different disease serum was analyzed.The conserved cleavage site pattern derived from proteolytic enzymes holds potential in highly specific disease diagnosis.
