比较纳升反相色谱-串联质谱与毛细管区带电泳-串联质谱用于自顶向下蛋白质组学（英文）
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摘要
自顶向下蛋白质组学的一个重要难题是缺乏与质谱可以在线连用并且可以提供高效蛋白质分离的液相分离技术。毛细管区带电泳与纳升反相色谱都可以与质谱在线连用,并且在复杂蛋白质样品分析方面也都有了显著的提升。在这里,我们首次比较了先进的纳升反相色谱-串联质谱与毛细管区带电泳-串联质谱平台用于自顶向下蛋白质组学分析。相对于纳升反相色谱-质谱而言,毛细管区带电泳-质谱可以将标准蛋白质样品的消耗量降低10倍,而且保持与纳升反相色谱-质谱相当的蛋白质信号强度。有意思的是,与毛细管区带电泳-质谱相比,纳升反相色谱-质谱可以获得更高的蛋白质分子的气相价态。这个现象可能是由于反相流动相中的高浓度乙腈使得蛋白质变性的更加充分。从1微克的大肠杆菌蛋白质样品中,毛细管区带电泳-串联质谱可以鉴定到159个蛋白质和513个蛋白质变体,而纳升反相色谱-串联质谱仅鉴定到105个蛋白质和277个蛋白质变体。当将大肠杆菌蛋白质的上样量提高到8微克时,纳升反相色谱-串联质谱可以鉴定到245个蛋白质和1 004个蛋白质变体。由于纳升反相色谱-串联质谱具有比毛细管区带电泳-串联质谱更高的上样量与更宽的分离窗口,当蛋白质样品量不受限制时,纳升反相色谱-串联质谱具有明显的优势。但是,在痕量样品分析方面,毛细管区带电泳-串联质谱具有更大的潜力。
One of the major shortcomings in top-down proteomics is the lack of efficient separations for intact proteins that can be effectively coupled to mass spectrometry. Capillary zone electrophoresis(CZE)and nanoflow reversed-phase liquid chromatography(nanoRPLC)are two methods that can be coupled to mass spectrometry directly and have been recently advanced in terms of their ability to separate intact proteins in complex biological mixtures. In this work,for the first time,we compared the state-of-the-art nanoRPLC-MS/MS and CZE-MS/MS platforms for top-down characterization of a standard protein mixture and an Escherichia coli(E.coli)proteome sample. CZE-MS produced comparable signals of standard proteins to RPLC-MS with 10-times less sample consumption. Interestingly,the proteins in RPLC-MS tended to have higher charge states than in CZE-MS,most likely due to the high acetonitrile concentration in RPLC mobile phase,leading to the more extensive unfolding of proteins in RPLC compared to in CZE. CZE-MS/MS identified 159 proteins and 513 proteoforms using 1-μg E. coli proteins in a single run and outperformed RPLC-MS/MS using 1-μg E. coli proteins in terms of protein and proteoform identifications(159 vs. 105 proteins and 513 vs. 277 proteoforms). The RPLC-MS/MS using 8-μg E. coli proteins identified 245 proteins and 1 004 proteoforms in a single run,and the data was much better than that from CZE-MS/MS(1-μg E. coli proteins)regarding the number of identifications because of the 8-times higher sample loading amount and significantly wider separation window of RPLCMS/MS compared to CZE-MS/MS.
One of the major shortcomings in top-down proteomics is the lack of efficient separations for intact proteins that can be effectively coupled to mass spectrometry. Capillary zone electrophoresis(CZE)and nanoflow reversed-phase liquid chromatography(nanoRPLC)are two methods that can be coupled to mass spectrometry directly and have been recently advanced in terms of their ability to separate intact proteins in complex biological mixtures. In this work,for the first time,we compared the state-of-the-art nanoRPLC-MS/MS and CZE-MS/MS platforms for top-down characterization of a standard protein mixture and an Escherichia coli(E.coli)proteome sample. CZE-MS produced comparable signals of standard proteins to RPLC-MS with 10-times less sample consumption. Interestingly,the proteins in RPLC-MS tended to have higher charge states than in CZE-MS,most likely due to the high acetonitrile concentration in RPLC mobile phase,leading to the more extensive unfolding of proteins in RPLC compared to in CZE. CZE-MS/MS identified 159 proteins and 513 proteoforms using 1-μg E. coli proteins in a single run and outperformed RPLC-MS/MS using 1-μg E. coli proteins in terms of protein and proteoform identifications(159 vs. 105 proteins and 513 vs. 277 proteoforms). The RPLC-MS/MS using 8-μg E. coli proteins identified 245 proteins and 1 004 proteoforms in a single run,and the data was much better than that from CZE-MS/MS(1-μg E. coli proteins)regarding the number of identifications because of the 8-times higher sample loading amount and significantly wider separation window of RPLCMS/MS compared to CZE-MS/MS.
引文
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[8] Han X,Wang Y,Aslanian A,et al.J Proteome Res,2014,13:6078
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[12] Catherman A D,Durbin K R,Ahlf D R,et al.Mol Cell Proteomics,2013,12:3465
[13] Shishkova E,Hebert A S,Westphall M S,et al.Anal Chem,2018,90:11503
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[15] Wang L,MacDonald D,Huang X,et al.Electrophoresis,2016,37:1143
[16] Li C,Tan X F,Lim T K,et al.Sci Rep,2016,6:24329
[17] Zubarev R A.Proteomics,2013,13:723
[18] Aebersold R,Agar J N,Amster I J,et al.Nat Chem Biol,2018,14:206
[19] Li Y,Champion M M,Sun L,et al.Anal Chem,2012,84:1617
[20] Sun L,Knierman M D,Zhu G,et al.Anal Chem,2013,85:5989
[21] Zhao Y,Sun L,Zhu G,et al.J Proteome Res,2016,15:3679
[22] Haselberg R,de Jong G J,Somsen G W.Anal Chem,2013,85:2289
[23] Jorgenson J W,Lukacs K D.Science,1983,222:266
[24] Imami K,Monton M R,Ishihama Y,et al.J Chromatogr A,2007,1148:250
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[31] McCool E N,Lubeckyj R,Shen X,et al.J Vis Exp,2018,140:e58644
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[33] Kou Q,Xun L,Liu X.Bioinformatics,2016,32:3495
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[35] Keller A,Nesvizhskii A I,Kolker E,et al.Anal Chem,2002,74:5383
[36] Elias J E,Gygi S P.Nat Methods,2007,4:207
[37] Yang Z,Shen X,Chen D,et al.Anal Chem,2018,90:10479
[38] Lubeckyj R A,Basharat A R,Shen X,et al.J Am Soc Mass Spectrom,2019,doi:10.1007/s13361-019-02167-w