Sodium dodecyl sulfate(SDS) is a powerful solubilizing detergent which is often used during the separation of highly complex protein mixtures by one- or two-dimensional(2D) gel electrophoresis. Matrix-assisted laser d...Sodium dodecyl sulfate(SDS) is a powerful solubilizing detergent which is often used during the separation of highly complex protein mixtures by one- or two-dimensional(2D) gel electrophoresis. Matrix-assisted laser desorption/ionization mass spectrometry(MALDI-MS) is a widely used technique for mass spectrometric analysis of some protein molecules compared to other techniques. But the presence of SDS or some salts usually leads to signal deterioration when using MALDI-MS. A method for using nitrocellulose membrane as the solid-phase carrier combined with n-octyl-β-D-glucopyranoside in the matrix highly enhances the sensitivity of the molecular mass determination of lysozyme. This technique has the advantage that the signal-to-noise of the molecular weight profile is improved compared with the mass spectrum and the profile is relatively easy to interpret.展开更多
A liquid-phase isoelectric focusing electrophoresis system(Rotofor) was used as the prefractionation tool for the sample preparation in the MALDI-MS analysis of a protein mixture. Each fraction collected was then dire...A liquid-phase isoelectric focusing electrophoresis system(Rotofor) was used as the prefractionation tool for the sample preparation in the MALDI-MS analysis of a protein mixture. Each fraction collected was then directly subjected to MALDI-TOF-MS analysis. By this approach, we are able to resolve two types of hemoglobins, A and C, which cannot be successfully separated by means of the traditional SDS-PAGE method.展开更多
A novel sensing chip was designed for MALDI-MS quantitation of acid phosphatase(ACP).The ACP sensing chip was constructed through non-covalent interaction of streptavidin and biotin for the assembly of biotinylated pe...A novel sensing chip was designed for MALDI-MS quantitation of acid phosphatase(ACP).The ACP sensing chip was constructed through non-covalent interaction of streptavidin and biotin for the assembly of biotinylated peptide substrate on biotinylated polyethylene-glycol(PEG)modified indium-tin oxide(ITO)slide.In the presence of ACP,the peptide substrate was dephosphorylated under acidic condition to generate a new mass signal.The quantitative assay of ACP was achieved with the mass signal ratio of product to the sum of product and left peptide substrate.Under optimal detection conditions,the ratio was linearly correlated with the concentration of ACP in the range of 0.05–12 g/L with a detection limit(LOD)of 0.04 g/L.The designed ACP sensing chip has been used to analyze ACP in complex clinical samples,which exhibited high selectivity,good repeatability,and admirably anti-interference ability.This work further demonstrates the concept of MS sensing and the application of MALDI-MS in quantitative analysis,and provides a convenient method for the quantitation of proteases in clinical diagnosis.展开更多
Halogens are elements in soil and water that occur as organic and inorganic species.Studies have shown that inorganic halogens are closely related to animal and plant life.Matrix-assisted laser desorption/ionization m...Halogens are elements in soil and water that occur as organic and inorganic species.Studies have shown that inorganic halogens are closely related to animal and plant life.Matrix-assisted laser desorption/ionization mass spectrometry(MALDI-MS)can achieve high-throughput and rapid analysis,which is also an essential characteristic in some environmental studies.However,there is currently no report on MALDI-MS analysis for water-soluble inorganic halogen compounds.In this study,we aimed to develop a reliable and reproductive method to detect inorganic halogens in soil and water by MALDI-MS.Based on sinapic acid(SA)and silver trifluoromethanesulfonate(SA/AgOTf)as the matrix in negative-ion mode,MALDI-MS showed excellent MS signals related to halogens.Under the optimum conditions,the method achieved satisfactory linearity(R^(2)>0.99)and repeatability(RSD<18%).The LODs for Cl^(−),Br^(−)and I^(−)were 0.76,0.11 and 0.10μg/mL,respectively.The concentration of inorganic halogens from 24 samples was successfully determined by MALDI-MS.In summary,an innovative method was developed for the analysis of inorganic halogens by MALDI-MS using a novel SA/AgOTf matrix,extending the application of MALDI-MS in inorganic compounds.展开更多
【目的】芽孢杆菌的热抗性及潜在安全风险可能威胁蜂蜜产品品质,因此需要优化蜂蜜中芽孢杆菌检测前处理工艺,揭示芽孢杆菌污染特征,并评估加工环节风险,为建立科学有效的质量安全管控体系提供理论依据。【方法】为提升蜂蜜中芽孢杆菌检...【目的】芽孢杆菌的热抗性及潜在安全风险可能威胁蜂蜜产品品质,因此需要优化蜂蜜中芽孢杆菌检测前处理工艺,揭示芽孢杆菌污染特征,并评估加工环节风险,为建立科学有效的质量安全管控体系提供理论依据。【方法】为提升蜂蜜中芽孢杆菌检测结果的准确性,本研究选取60—80℃温度区间开展热处理条件优化试验,系统探究可同步实现高效灭活杂菌与保留芽孢杆菌芽孢浓度的最优工艺条件。对150份市售蜂蜜样本与150份原料蜜样本中的芽孢杆菌进行芽孢计数,并进一步对菌株分离纯化,同时结合基质辅助激光解吸电离飞行时间质谱(Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectometry,MALDI-TOF MS)和基于二代测序对细菌基因组测序的方法鉴定蜂蜜中芽孢杆菌类型,并统计各蜂蜜样品中芽孢杆菌的种类及检出率,同时开展食品安全风险评估。模拟蜂蜜的加工环节,探究40—60℃热处理对蜂蜜中蜡样芽孢杆菌芽孢生长的影响,并明确其变化趋势。【结果】80℃/10 min热处理可完全灭活蜂蜜中的干扰菌(大肠杆菌和酿酒酵母),并且维持芽孢存活率在同一数量级;对150份市售蜂蜜样本与150份原料蜜样本的芽孢杆菌计数结果及鉴定结果进行统计分析,结果显示:市售蜂蜜芽孢杆菌总检出率为38.7%,以蜡样芽孢杆菌(20.7%)、枯草芽孢杆菌(12.7%)及短小芽孢杆菌(11.3%)为主;原料蜜中芽孢杆菌总体检出率为23.3%,以蜡样芽孢杆菌(16.7%)、短小芽孢杆菌(8.0%)和地衣芽孢杆菌(3.3%)为主,对比市售蜂蜜和原料蜜中检出芽孢杆菌浓度最高的样品,结果显示均为蜡样芽孢杆菌的单一污染。此外,模拟蜂蜜加工热处理条件(40—60℃),结果显示蜡样芽孢杆菌芽孢数量与温度-时间呈正相关,随处理温度升高与处理时间延长,其芽孢数量呈现上升趋势。芽孢杆菌种类相关性分析显示,市售蜂蜜中的芽孢杆菌污染主要源自原料蜜携带,环境菌的检出间接表明蜂蜜在蜜蜂采集、原料蜜采收及后续加工等环节中存在被微生物污染的可能性。【结论】80℃热处理10 min为蜂蜜中芽孢杆菌检测的最优热处理条件;蜡样芽孢杆菌为蜂蜜中的主要风险菌株,建议蜂蜜生产企业可将芽孢杆菌列为原料蜜质量管控的核心监测指标之一。展开更多
文摘Sodium dodecyl sulfate(SDS) is a powerful solubilizing detergent which is often used during the separation of highly complex protein mixtures by one- or two-dimensional(2D) gel electrophoresis. Matrix-assisted laser desorption/ionization mass spectrometry(MALDI-MS) is a widely used technique for mass spectrometric analysis of some protein molecules compared to other techniques. But the presence of SDS or some salts usually leads to signal deterioration when using MALDI-MS. A method for using nitrocellulose membrane as the solid-phase carrier combined with n-octyl-β-D-glucopyranoside in the matrix highly enhances the sensitivity of the molecular mass determination of lysozyme. This technique has the advantage that the signal-to-noise of the molecular weight profile is improved compared with the mass spectrum and the profile is relatively easy to interpret.
文摘A liquid-phase isoelectric focusing electrophoresis system(Rotofor) was used as the prefractionation tool for the sample preparation in the MALDI-MS analysis of a protein mixture. Each fraction collected was then directly subjected to MALDI-TOF-MS analysis. By this approach, we are able to resolve two types of hemoglobins, A and C, which cannot be successfully separated by means of the traditional SDS-PAGE method.
基金the National Natural Science Foundation of China(21635005,21827812,21890741,21974063)the National Key Research and Development Program of China(2018YFC1004704)the Fundamental Research Funds for the Central Universities(14380200)。
文摘A novel sensing chip was designed for MALDI-MS quantitation of acid phosphatase(ACP).The ACP sensing chip was constructed through non-covalent interaction of streptavidin and biotin for the assembly of biotinylated peptide substrate on biotinylated polyethylene-glycol(PEG)modified indium-tin oxide(ITO)slide.In the presence of ACP,the peptide substrate was dephosphorylated under acidic condition to generate a new mass signal.The quantitative assay of ACP was achieved with the mass signal ratio of product to the sum of product and left peptide substrate.Under optimal detection conditions,the ratio was linearly correlated with the concentration of ACP in the range of 0.05–12 g/L with a detection limit(LOD)of 0.04 g/L.The designed ACP sensing chip has been used to analyze ACP in complex clinical samples,which exhibited high selectivity,good repeatability,and admirably anti-interference ability.This work further demonstrates the concept of MS sensing and the application of MALDI-MS in quantitative analysis,and provides a convenient method for the quantitation of proteases in clinical diagnosis.
基金This study was jointly supported by the National Natural Science Foundation of China(21906137)the Science and Technology Program of Zhejiang Province(LGC21B050008).
文摘Halogens are elements in soil and water that occur as organic and inorganic species.Studies have shown that inorganic halogens are closely related to animal and plant life.Matrix-assisted laser desorption/ionization mass spectrometry(MALDI-MS)can achieve high-throughput and rapid analysis,which is also an essential characteristic in some environmental studies.However,there is currently no report on MALDI-MS analysis for water-soluble inorganic halogen compounds.In this study,we aimed to develop a reliable and reproductive method to detect inorganic halogens in soil and water by MALDI-MS.Based on sinapic acid(SA)and silver trifluoromethanesulfonate(SA/AgOTf)as the matrix in negative-ion mode,MALDI-MS showed excellent MS signals related to halogens.Under the optimum conditions,the method achieved satisfactory linearity(R^(2)>0.99)and repeatability(RSD<18%).The LODs for Cl^(−),Br^(−)and I^(−)were 0.76,0.11 and 0.10μg/mL,respectively.The concentration of inorganic halogens from 24 samples was successfully determined by MALDI-MS.In summary,an innovative method was developed for the analysis of inorganic halogens by MALDI-MS using a novel SA/AgOTf matrix,extending the application of MALDI-MS in inorganic compounds.
文摘【目的】芽孢杆菌的热抗性及潜在安全风险可能威胁蜂蜜产品品质,因此需要优化蜂蜜中芽孢杆菌检测前处理工艺,揭示芽孢杆菌污染特征,并评估加工环节风险,为建立科学有效的质量安全管控体系提供理论依据。【方法】为提升蜂蜜中芽孢杆菌检测结果的准确性,本研究选取60—80℃温度区间开展热处理条件优化试验,系统探究可同步实现高效灭活杂菌与保留芽孢杆菌芽孢浓度的最优工艺条件。对150份市售蜂蜜样本与150份原料蜜样本中的芽孢杆菌进行芽孢计数,并进一步对菌株分离纯化,同时结合基质辅助激光解吸电离飞行时间质谱(Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectometry,MALDI-TOF MS)和基于二代测序对细菌基因组测序的方法鉴定蜂蜜中芽孢杆菌类型,并统计各蜂蜜样品中芽孢杆菌的种类及检出率,同时开展食品安全风险评估。模拟蜂蜜的加工环节,探究40—60℃热处理对蜂蜜中蜡样芽孢杆菌芽孢生长的影响,并明确其变化趋势。【结果】80℃/10 min热处理可完全灭活蜂蜜中的干扰菌(大肠杆菌和酿酒酵母),并且维持芽孢存活率在同一数量级;对150份市售蜂蜜样本与150份原料蜜样本的芽孢杆菌计数结果及鉴定结果进行统计分析,结果显示:市售蜂蜜芽孢杆菌总检出率为38.7%,以蜡样芽孢杆菌(20.7%)、枯草芽孢杆菌(12.7%)及短小芽孢杆菌(11.3%)为主;原料蜜中芽孢杆菌总体检出率为23.3%,以蜡样芽孢杆菌(16.7%)、短小芽孢杆菌(8.0%)和地衣芽孢杆菌(3.3%)为主,对比市售蜂蜜和原料蜜中检出芽孢杆菌浓度最高的样品,结果显示均为蜡样芽孢杆菌的单一污染。此外,模拟蜂蜜加工热处理条件(40—60℃),结果显示蜡样芽孢杆菌芽孢数量与温度-时间呈正相关,随处理温度升高与处理时间延长,其芽孢数量呈现上升趋势。芽孢杆菌种类相关性分析显示,市售蜂蜜中的芽孢杆菌污染主要源自原料蜜携带,环境菌的检出间接表明蜂蜜在蜜蜂采集、原料蜜采收及后续加工等环节中存在被微生物污染的可能性。【结论】80℃热处理10 min为蜂蜜中芽孢杆菌检测的最优热处理条件;蜡样芽孢杆菌为蜂蜜中的主要风险菌株,建议蜂蜜生产企业可将芽孢杆菌列为原料蜜质量管控的核心监测指标之一。
