In this study, we selected adult normal pituitary gland tissues from six patients during operations for pituitary microadenomas via the transsphenoidal approach for extended normal pituitary tissue resection around th...In this study, we selected adult normal pituitary gland tissues from six patients during operations for pituitary microadenomas via the transsphenoidal approach for extended normal pituitary tissue resection around the tumor, and analyzed the protein expression of human normal pituitary using two-dimensional high-performance liquid chromatography combined with LTQ-Orbitrap mass spectrometry proteomics technology. The ten most highly expressed proteins in normal human pituitary were： alpha 3 type VI collagen isoform 5 precursor （abundance among tall pituitary proteins 1.30%）, fibrinogen beta chain preproprotein （0.99%）, vimentin （0.73%）, prolactin （0.69%）, ATP synthase, H~ transporting and mitochondrial F1 complex beta subunit precursor （0.52%）, keratin I （0.49%）, growth hormone （0.45%）, carbonic anhydrase I （0.40%）, heat shock protein 90 kDa I （0.31%）, and annexin V （0.30%）. Based on the biological function classifications of these proteins, the top three categories by content were neuroendocrine proteins （abundance among all pituitary proteins, 40.1%）, catalytic and metabolic proteins （28.3%）, and cell signal transduction proteins （9.8%）. Based on cell positioning classification, the top three categories were cell organelle （24.5%） membrane （20.8%）, and cytoplasm （13.0%）. Based on biological process classification, the top three categories of proteins are involved in physiological processes （42.9%）, cellular processes （40.4%）, and regulation of biological processes （9.1%）. Our experimental findings indicate that a protein expression profile database of normal human pituitary can be precisely and efficiently established by proteomics technology.展开更多
An offline two-dimensional system combining a rat cardiac mascle cell membrane chromatography time-of-flight mass spectrometry （CMC-TOF/MS） with a high performance liquid chromatography time-of-flight mass spectrome...An offline two-dimensional system combining a rat cardiac mascle cell membrane chromatography time-of-flight mass spectrometry （CMC-TOF/MS） with a high performance liquid chromatography time-of-flight mass spectrometry （HPLC-TOF/MS） was established for investigating the parent components and metabolites in rat urine samples after administration of the roots of Aconitum carmichaeli. On the basis of the analysis of the first dimension, retention components of the urine sample were collected into 30 fractions （one fraction per minute）. Then offline analysis of the second dimension was carried out. 34 compounds including 24 parent alkaloids and 10 potential metabolites were identified from the dosed rat urine, and then binding affinities of different compounds on cell membranes were compared and influences of some functional groups on activity were estimated with the semi-quantification and curve fitting method. As a result, binding affinities decreased along with the process of deacylation, debenzoylation and demethylation, which may be related to the alleviation of toxicity in the procedure of herb processing or metabolism. Moreover, some minor components in rat urine （Songorine, 14-benzoylneoline, Deoxyaconitine, etc. ） exerted relatively strong affinity on cell membranes are worth exploring. The results delivered by the system suggest that the CMC can be applied to in vivo study.展开更多
A comprehensive two-dimensional liquid chromatographic system (2D SCX/RP) is con- structed with a 10-port-2-way valve using strong cation exchange chromatography (Hypersil SCX, 100 mm×4.6 mm I.D.) followed by rev...A comprehensive two-dimensional liquid chromatographic system (2D SCX/RP) is con- structed with a 10-port-2-way valve using strong cation exchange chromatography (Hypersil SCX, 100 mm×4.6 mm I.D.) followed by reversed phase chromatography (Hypersil BDS C18, 15 mm×4.6 mm I.D.) to separate the complex peptides from globin peptic hydrolysate. After the sample was loaded on the SCX column, the phosphate buffer (pH 4.0) was used to elute the peptides. Then, elutes flowed through the interface and the peptides focused on the head of the trapping columns (Hypersil BDS C18, 15 mm×4.6 mm I.D.) but salt passed into the waste. After the valve was switched, the samples were flushed with a backward flow into the RP analytical column. The peptides on the SCX were eluted with 12 discontinuous steps linearly increasing salt concentrations. The peptides enriched on the trapping column were desalted and separated by the RP columns. The resolution and the resolved peaks of the 2D SCX/RP system were greatly increased and the total peak capacity reached as high as 2280.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
基金supported by the National NaturalScience Foundation of China, No. 81200890
文摘In this study, we selected adult normal pituitary gland tissues from six patients during operations for pituitary microadenomas via the transsphenoidal approach for extended normal pituitary tissue resection around the tumor, and analyzed the protein expression of human normal pituitary using two-dimensional high-performance liquid chromatography combined with LTQ-Orbitrap mass spectrometry proteomics technology. The ten most highly expressed proteins in normal human pituitary were： alpha 3 type VI collagen isoform 5 precursor （abundance among tall pituitary proteins 1.30%）, fibrinogen beta chain preproprotein （0.99%）, vimentin （0.73%）, prolactin （0.69%）, ATP synthase, H~ transporting and mitochondrial F1 complex beta subunit precursor （0.52%）, keratin I （0.49%）, growth hormone （0.45%）, carbonic anhydrase I （0.40%）, heat shock protein 90 kDa I （0.31%）, and annexin V （0.30%）. Based on the biological function classifications of these proteins, the top three categories by content were neuroendocrine proteins （abundance among all pituitary proteins, 40.1%）, catalytic and metabolic proteins （28.3%）, and cell signal transduction proteins （9.8%）. Based on cell positioning classification, the top three categories were cell organelle （24.5%） membrane （20.8%）, and cytoplasm （13.0%）. Based on biological process classification, the top three categories of proteins are involved in physiological processes （42.9%）, cellular processes （40.4%）, and regulation of biological processes （9.1%）. Our experimental findings indicate that a protein expression profile database of normal human pituitary can be precisely and efficiently established by proteomics technology.
基金supported by the National Natural Science Foundation of China(No.30873196)Project of Modernization of Traditional Chinese Medicine of Shanghai(No.09dZ1975100)
文摘An offline two-dimensional system combining a rat cardiac mascle cell membrane chromatography time-of-flight mass spectrometry （CMC-TOF/MS） with a high performance liquid chromatography time-of-flight mass spectrometry （HPLC-TOF/MS） was established for investigating the parent components and metabolites in rat urine samples after administration of the roots of Aconitum carmichaeli. On the basis of the analysis of the first dimension, retention components of the urine sample were collected into 30 fractions （one fraction per minute）. Then offline analysis of the second dimension was carried out. 34 compounds including 24 parent alkaloids and 10 potential metabolites were identified from the dosed rat urine, and then binding affinities of different compounds on cell membranes were compared and influences of some functional groups on activity were estimated with the semi-quantification and curve fitting method. As a result, binding affinities decreased along with the process of deacylation, debenzoylation and demethylation, which may be related to the alleviation of toxicity in the procedure of herb processing or metabolism. Moreover, some minor components in rat urine （Songorine, 14-benzoylneoline, Deoxyaconitine, etc. ） exerted relatively strong affinity on cell membranes are worth exploring. The results delivered by the system suggest that the CMC can be applied to in vivo study.
基金Financial support from the National Natural Science Foundation of China (Grant Nos. 20175029 and 20375040) is gratefully acknowledged.
文摘A comprehensive two-dimensional liquid chromatographic system (2D SCX/RP) is con- structed with a 10-port-2-way valve using strong cation exchange chromatography (Hypersil SCX, 100 mm×4.6 mm I.D.) followed by reversed phase chromatography (Hypersil BDS C18, 15 mm×4.6 mm I.D.) to separate the complex peptides from globin peptic hydrolysate. After the sample was loaded on the SCX column, the phosphate buffer (pH 4.0) was used to elute the peptides. Then, elutes flowed through the interface and the peptides focused on the head of the trapping columns (Hypersil BDS C18, 15 mm×4.6 mm I.D.) but salt passed into the waste. After the valve was switched, the samples were flushed with a backward flow into the RP analytical column. The peptides on the SCX were eluted with 12 discontinuous steps linearly increasing salt concentrations. The peptides enriched on the trapping column were desalted and separated by the RP columns. The resolution and the resolved peaks of the 2D SCX/RP system were greatly increased and the total peak capacity reached as high as 2280.
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.
