Mass spectrometry-based single-cell proteomics(MS-scP)is attracting tremendous attention because it is now technically feasible to quantify thousands of proteins in minute samples.Since protein amplification is still ...Mass spectrometry-based single-cell proteomics(MS-scP)is attracting tremendous attention because it is now technically feasible to quantify thousands of proteins in minute samples.Since protein amplification is still not possible,technological improvements in MS-scP focus on mini-mizing sample loss while increasing throughput,resolution,and sensitivity,as well as achieving measurement depth,accuracy,and stability comparable to bulk samples.Major advances in MS-sCP have facilitated its application in biological and even medical research.Here,we review the key advancements in MS-SCP technology and discuss the strategies of the typical proteomics workflow to improve MS-SCP analysis from single-cell isolation,sample preparation,and liquid chromatography separation to MS data acquisition and analysis.The review will provide an overall understanding of the development and applications of Ms-scP and inspire more novel ideas regarding the innovation of MS-SCPtechnology.展开更多
Chromatographic fingerprinting has been perceived as an essential tool for assessing quality and chemical equivalence of traditional Chinese medicine.However,this pattern-oriented approach still has some weak points i...Chromatographic fingerprinting has been perceived as an essential tool for assessing quality and chemical equivalence of traditional Chinese medicine.However,this pattern-oriented approach still has some weak points in terms of chemical coverage and robustness.In this work,we proposed a multiple reaction monitoring(MRM)-based fingerprinting method in which approximately 100 constituents were simultaneously detected for quality assessment.The derivative MRM approach was employed to rapidly design MRM transitions independent of chemical standards,based on which the large-scale fingerprinting method was efficiently established.This approach was exemplified on QiShenYiQi Pill(QSYQ),a traditional Chinese medicine-derived drug product,and its robustness was systematically evaluated by four indices:clustering analysis by principal component analysis,similarity analysis by the congruence coefficient,the number of separated peaks,and the peak area proportion of separated peaks.Compared with conventional ultraviolet-based fingerprints,the MRM fingerprints provided not only better discriminatory capacity for the tested normal/abnormal QSYQ samples,but also higher robustness under different chromatographic conditions(i.e.,flow rate,apparent pH,column temperature,and column).The result also showed for such large-scale fingerprints including a large number of peaks,the angle cosine measure after min-max normalization was more suitable for setting a decision criterion than the unnormalized algorithm.This proof-of-concept application gives evidence that combining MRM technique with proper similarity analysis metrices can provide a highly sensitive,robust and comprehensive analytical approach for quality assessment of traditional Chinese medicine.展开更多
Sclerotinia sclerotiorum(Lib.)de Bary is a necrotrophic plant pathogen that causes cottony rot,watery soft rot,stem rot,white mold,and other disease symptoms in over 700 plant hosts around the world.Destruction of eco...Sclerotinia sclerotiorum(Lib.)de Bary is a necrotrophic plant pathogen that causes cottony rot,watery soft rot,stem rot,white mold,and other disease symptoms in over 700 plant hosts around the world.Destruction of economically important crops,the lack of resistant cultivars,and the general challenge of controlling diseases caused by this broad-based pathogen call for continued research.However,in recent years,mass spectrometry-based proteomics analyses have been used to acquire a fundamental and in-depth molecular understanding of this fungal pathogen.In this review,we describe the characteristics of the Sclerotinia sclerotiorum pathogen and examine its virulence factors,secreted proteins,and host suppression mechanisms.Furthermore,we review recent proteomics studies and extrapolate their primary findings for the identification and functional characterization of Sclerotinia sclerotiorum proteins.Finally,we discuss key findings that shape the understanding of the virulent factors and pathogenesis of Sclerotinia sclerotiorum and outline directions for future proteomic investigations of plant pathogens.展开更多
基金supported by the National Natural Science Foundation of China(Grant No.32371500)the National Key R&D Program of China(Grant No.2022YFA1304500).
文摘Mass spectrometry-based single-cell proteomics(MS-scP)is attracting tremendous attention because it is now technically feasible to quantify thousands of proteins in minute samples.Since protein amplification is still not possible,technological improvements in MS-scP focus on mini-mizing sample loss while increasing throughput,resolution,and sensitivity,as well as achieving measurement depth,accuracy,and stability comparable to bulk samples.Major advances in MS-sCP have facilitated its application in biological and even medical research.Here,we review the key advancements in MS-SCP technology and discuss the strategies of the typical proteomics workflow to improve MS-SCP analysis from single-cell isolation,sample preparation,and liquid chromatography separation to MS data acquisition and analysis.The review will provide an overall understanding of the development and applications of Ms-scP and inspire more novel ideas regarding the innovation of MS-SCPtechnology.
基金financially supported by the National Natural Science Foundation of China(Grant No.81803714)the Fundamental Research Funds for the Central Universities(Grant No.2019QNA7041).
文摘Chromatographic fingerprinting has been perceived as an essential tool for assessing quality and chemical equivalence of traditional Chinese medicine.However,this pattern-oriented approach still has some weak points in terms of chemical coverage and robustness.In this work,we proposed a multiple reaction monitoring(MRM)-based fingerprinting method in which approximately 100 constituents were simultaneously detected for quality assessment.The derivative MRM approach was employed to rapidly design MRM transitions independent of chemical standards,based on which the large-scale fingerprinting method was efficiently established.This approach was exemplified on QiShenYiQi Pill(QSYQ),a traditional Chinese medicine-derived drug product,and its robustness was systematically evaluated by four indices:clustering analysis by principal component analysis,similarity analysis by the congruence coefficient,the number of separated peaks,and the peak area proportion of separated peaks.Compared with conventional ultraviolet-based fingerprints,the MRM fingerprints provided not only better discriminatory capacity for the tested normal/abnormal QSYQ samples,but also higher robustness under different chromatographic conditions(i.e.,flow rate,apparent pH,column temperature,and column).The result also showed for such large-scale fingerprints including a large number of peaks,the angle cosine measure after min-max normalization was more suitable for setting a decision criterion than the unnormalized algorithm.This proof-of-concept application gives evidence that combining MRM technique with proper similarity analysis metrices can provide a highly sensitive,robust and comprehensive analytical approach for quality assessment of traditional Chinese medicine.
基金This work was supported by the South African National Research Foundation(NRF,No.TTK170413227119)SARChI program of the Department of Science and Technology and the National Research Foundation for post-doctoral fellowship funding.
文摘Sclerotinia sclerotiorum(Lib.)de Bary is a necrotrophic plant pathogen that causes cottony rot,watery soft rot,stem rot,white mold,and other disease symptoms in over 700 plant hosts around the world.Destruction of economically important crops,the lack of resistant cultivars,and the general challenge of controlling diseases caused by this broad-based pathogen call for continued research.However,in recent years,mass spectrometry-based proteomics analyses have been used to acquire a fundamental and in-depth molecular understanding of this fungal pathogen.In this review,we describe the characteristics of the Sclerotinia sclerotiorum pathogen and examine its virulence factors,secreted proteins,and host suppression mechanisms.Furthermore,we review recent proteomics studies and extrapolate their primary findings for the identification and functional characterization of Sclerotinia sclerotiorum proteins.Finally,we discuss key findings that shape the understanding of the virulent factors and pathogenesis of Sclerotinia sclerotiorum and outline directions for future proteomic investigations of plant pathogens.
