In this study we systematically analyzed the elution condition of tryptic peptides and the characteristics of identi?ed peptides in reverse phase liquid chromatogra- phy and electrospray tandem mass spectrometry (RPLC...In this study we systematically analyzed the elution condition of tryptic peptides and the characteristics of identi?ed peptides in reverse phase liquid chromatogra- phy and electrospray tandem mass spectrometry (RPLC-MS/MS) analysis. Fol- lowing protein digestion with trypsin, the peptide mixture was analyzed by on-line RPLC-MS/MS. Bovine serum albumin (BSA) was used to optimize acetonitrile (ACN) elution gradient for tryptic peptides, and Cytochrome C was used to retest the gradient and the sensitivity of LC-MS/MS. The characteristics of identi?ed peptides were also analyzed. In our experiments, the suitable ACN gradient is 5% to 30% for tryptic peptide elution and the sensitivity of LC-MS/MS is 50 fmol. Analysis of the tryptic peptides demonstrated that longer (more than 10 amino acids) and multi-charge state (+2, +3) peptides are likely to be identi?ed, and the hydropathicity of the peptides might not be related to whether it is more likely to be identi?ed or not. The number of identi?ed peptides for a protein might be used to estimate its loading amount under the same sample background. Moreover, in this study the identi?ed peptides present three types of redundancy, namely iden- ti?cation, charge, and sequence redundancy, which may repress low abundance protein identi?cation.展开更多
High-resolution liquid chromatography separation is essential to in-depth proteomic profiling of complex biological samples.Herein,we established an ion-pair reversed-phase×reversed-phase two-dimensional liquid c...High-resolution liquid chromatography separation is essential to in-depth proteomic profiling of complex biological samples.Herein,we established an ion-pair reversed-phase×reversed-phase two-dimensional liquid chromatography(IPRP×RP 2DLC)strategy for comprehensive proteomic analysis.Both RPLC separation dimensions were performed at low pH,with trifluoroacetic acid(TFA)and formic acid(FA)as mobile phase addictive,respectively.As the good separation resolution offered by ion-pairing effect of TFA,the fractionation efficiency was greatly improved with 74.0%peptides identified in just one fraction.Comparing with conventional high pH RP fractionation,the overall separation rate of IPRP was about 1.6 times that of high-pH RP,which increased the number of identified peptides by 21%.Further,2169 proteins and 8540 peptides were confidently identified from crude serum sample by our IPRP×RP 2DLC strategy,exhibiting great potential in clinical proteomics in the future.展开更多
Background The pathogenesis of autism spectrum disorders remains elusive and currently there are no diagnostic or pre-dictive biomarkers in autism available. Proteomic profiling has been used in a wide range of neurod...Background The pathogenesis of autism spectrum disorders remains elusive and currently there are no diagnostic or pre-dictive biomarkers in autism available. Proteomic profiling has been used in a wide range of neurodevelopmental disorder studies, which could produce deeper perceptions of the molecular bases behind certain disease and potentially becomes useful in discovering biomarkers in autism spectrum disorders. Methods Serum samples were collected from autistic children about 3 years old in age (n = 32) and healthy controls (n = 20) in similar age and gender. The samples were identified specific proteins that are diff erentially expressed by magnetic bead-based pre-fractionation and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF-MS). Results Eight protein peaks were significantly different in autistic children from the healthy controls (P < 0.0001). The two peaks with the most significant diff erences were 6428 and 7758 Da in size. Conclusion According to diff erences in serum protein profiles between the autistic children and healthy controls, this study identified a set of diff erentially expressed proteins those are significant for further evaluation and might function as biomark-ers in autism.展开更多
目的基于超高效液相色谱-串联质谱法(ultra performance liquid chromatography-tandem mass spectrometry,UPLC-MS/MS)建立乳制品中乳铁蛋白、乳桥蛋白、奶牛A1β-酪蛋白和A2β-酪蛋白、水牛A2β-酪蛋白的检测方法,开展市售乳制品中功...目的基于超高效液相色谱-串联质谱法(ultra performance liquid chromatography-tandem mass spectrometry,UPLC-MS/MS)建立乳制品中乳铁蛋白、乳桥蛋白、奶牛A1β-酪蛋白和A2β-酪蛋白、水牛A2β-酪蛋白的检测方法,开展市售乳制品中功能性蛋白质检测和乳制品真实性鉴评分析。方法乳蛋白经酶解后,应用超高效液相色谱-四极杆-静电场轨道阱高分辨质谱法(ultra performance liquid chromatography-triple quadrupole-Orbitrap high resolution mass spectrometry,UPLC-Q/Orbitrap HRMS)和UPLC-MS/MS筛选获得可用于定性定量分析的特征肽段,基于UPLC-MS/MS建立同时检测LF、LPN、奶牛A1β-CN、奶牛A2β-CN和水牛A2β-CN的方法并应用于乳制品检测。结果5种待测蛋白在相应的浓度范围内线性关系良好(r^(2)>0.99),加标回收率在82.1%~105.5%之间,相对标准偏差均小于5%,78份乳制品的检测结果表明同类样品LF含量差异较大,不同类液体乳中LPN均值差异不大。结论本方法性能良好,适用于乳制品中乳桥蛋白等功能性蛋白的同时检测,也可以实现乳制品中乳蛋白物种来源的鉴别。展开更多
AIM: To identify differentially expressed hydrophobic proteins in colorectal cancer. METHODS: Eighteen pairs of colorectal cancerous tissues in addition to tissues from normal mucosa were analysed. Hydrophobic protein...AIM: To identify differentially expressed hydrophobic proteins in colorectal cancer. METHODS: Eighteen pairs of colorectal cancerous tissues in addition to tissues from normal mucosa were analysed. Hydrophobic proteins were extracted from the tissues, separated using 2-D gel electrophoresis and analysed using Liquid Chromatography Tandem Mass Spectrometry (LC/MS/MS). Statistical analysis of the proteins was carried out in order to determine the significance of each protein to colorectal cancer (CRC) and also their relation to CRC stages, grades and patients’ gender. RESULTS: Thirteen differentially expressed proteins which were expressed abundantly in either cancerous or normal tissues were identified. A number of these proteins were found to relate strongly with a particular stage or grade of CRC. In addition, the association of these proteins with patient gender also appeared to be significant.CONCLUSION: Stomatin-like protein 2 was found to be a promising biomarker for CRC, especially in female patients. The differentially expressed proteins identified were associated with CRC and may act as drug target candidates.展开更多
基金This work was partially supported by grants fromthe National Basic Research Program (973) of China(2004CB520804), and National Natural Science Foun-dation of China (No. 30270657, 30230150, 3037030).
文摘In this study we systematically analyzed the elution condition of tryptic peptides and the characteristics of identi?ed peptides in reverse phase liquid chromatogra- phy and electrospray tandem mass spectrometry (RPLC-MS/MS) analysis. Fol- lowing protein digestion with trypsin, the peptide mixture was analyzed by on-line RPLC-MS/MS. Bovine serum albumin (BSA) was used to optimize acetonitrile (ACN) elution gradient for tryptic peptides, and Cytochrome C was used to retest the gradient and the sensitivity of LC-MS/MS. The characteristics of identi?ed peptides were also analyzed. In our experiments, the suitable ACN gradient is 5% to 30% for tryptic peptide elution and the sensitivity of LC-MS/MS is 50 fmol. Analysis of the tryptic peptides demonstrated that longer (more than 10 amino acids) and multi-charge state (+2, +3) peptides are likely to be identi?ed, and the hydropathicity of the peptides might not be related to whether it is more likely to be identi?ed or not. The number of identi?ed peptides for a protein might be used to estimate its loading amount under the same sample background. Moreover, in this study the identi?ed peptides present three types of redundancy, namely iden- ti?cation, charge, and sequence redundancy, which may repress low abundance protein identi?cation.
基金National Key R&D Program of China(No.2019YFE0119300)National Natural Science Foundation of China(No.32088101)+1 种基金Original Innovation Project of CAS(No.ZDBS-LY-SLH032)Project of Dalian Institute of Chemical Physics,CAS,China(No.DICPI202007).
文摘High-resolution liquid chromatography separation is essential to in-depth proteomic profiling of complex biological samples.Herein,we established an ion-pair reversed-phase×reversed-phase two-dimensional liquid chromatography(IPRP×RP 2DLC)strategy for comprehensive proteomic analysis.Both RPLC separation dimensions were performed at low pH,with trifluoroacetic acid(TFA)and formic acid(FA)as mobile phase addictive,respectively.As the good separation resolution offered by ion-pairing effect of TFA,the fractionation efficiency was greatly improved with 74.0%peptides identified in just one fraction.Comparing with conventional high pH RP fractionation,the overall separation rate of IPRP was about 1.6 times that of high-pH RP,which increased the number of identified peptides by 21%.Further,2169 proteins and 8540 peptides were confidently identified from crude serum sample by our IPRP×RP 2DLC strategy,exhibiting great potential in clinical proteomics in the future.
文摘Background The pathogenesis of autism spectrum disorders remains elusive and currently there are no diagnostic or pre-dictive biomarkers in autism available. Proteomic profiling has been used in a wide range of neurodevelopmental disorder studies, which could produce deeper perceptions of the molecular bases behind certain disease and potentially becomes useful in discovering biomarkers in autism spectrum disorders. Methods Serum samples were collected from autistic children about 3 years old in age (n = 32) and healthy controls (n = 20) in similar age and gender. The samples were identified specific proteins that are diff erentially expressed by magnetic bead-based pre-fractionation and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF-MS). Results Eight protein peaks were significantly different in autistic children from the healthy controls (P < 0.0001). The two peaks with the most significant diff erences were 6428 and 7758 Da in size. Conclusion According to diff erences in serum protein profiles between the autistic children and healthy controls, this study identified a set of diff erentially expressed proteins those are significant for further evaluation and might function as biomark-ers in autism.
文摘目的基于超高效液相色谱-串联质谱法(ultra performance liquid chromatography-tandem mass spectrometry,UPLC-MS/MS)建立乳制品中乳铁蛋白、乳桥蛋白、奶牛A1β-酪蛋白和A2β-酪蛋白、水牛A2β-酪蛋白的检测方法,开展市售乳制品中功能性蛋白质检测和乳制品真实性鉴评分析。方法乳蛋白经酶解后,应用超高效液相色谱-四极杆-静电场轨道阱高分辨质谱法(ultra performance liquid chromatography-triple quadrupole-Orbitrap high resolution mass spectrometry,UPLC-Q/Orbitrap HRMS)和UPLC-MS/MS筛选获得可用于定性定量分析的特征肽段,基于UPLC-MS/MS建立同时检测LF、LPN、奶牛A1β-CN、奶牛A2β-CN和水牛A2β-CN的方法并应用于乳制品检测。结果5种待测蛋白在相应的浓度范围内线性关系良好(r^(2)>0.99),加标回收率在82.1%~105.5%之间,相对标准偏差均小于5%,78份乳制品的检测结果表明同类样品LF含量差异较大,不同类液体乳中LPN均值差异不大。结论本方法性能良好,适用于乳制品中乳桥蛋白等功能性蛋白的同时检测,也可以实现乳制品中乳蛋白物种来源的鉴别。
基金Supported by RU Grant, No.1001/PFARMASI/815007 and Universiti Sains Malaysia
文摘AIM: To identify differentially expressed hydrophobic proteins in colorectal cancer. METHODS: Eighteen pairs of colorectal cancerous tissues in addition to tissues from normal mucosa were analysed. Hydrophobic proteins were extracted from the tissues, separated using 2-D gel electrophoresis and analysed using Liquid Chromatography Tandem Mass Spectrometry (LC/MS/MS). Statistical analysis of the proteins was carried out in order to determine the significance of each protein to colorectal cancer (CRC) and also their relation to CRC stages, grades and patients’ gender. RESULTS: Thirteen differentially expressed proteins which were expressed abundantly in either cancerous or normal tissues were identified. A number of these proteins were found to relate strongly with a particular stage or grade of CRC. In addition, the association of these proteins with patient gender also appeared to be significant.CONCLUSION: Stomatin-like protein 2 was found to be a promising biomarker for CRC, especially in female patients. The differentially expressed proteins identified were associated with CRC and may act as drug target candidates.
