Increasing the Separation Capacity of Intact Histone Proteoforms Chromatography Coupling Online Weak Cation Exchange-HILIC to Reversed Phase LC UVPD-HRMS.
Increasing the Separation Capacity of Intact Histone Proteoforms Chromatography Coupling Online Weak Cation Exchange-HILIC to Reversed Phase LC UVPD-HRMS.
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DOI：
10.1021/acs.jproteome.8b00458
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发表时间：
2018-11-02
影响因子：
4.4
通讯作者：
Paša-Tolić L
中科院分区：
文献类型：
--
作者：
Gargano AFG;Shaw JB;Zhou M;Wilkins CS;Fillmore TL;Moore RJ;Somsen GW;Paša-Tolić L
自上而下的蛋白质组学是一种新兴的分析策略，以表征组合蛋白质翻译后修饰（PTM）。然而，样品的复杂性和化学上密切相关的proteoforms之间的小质量差异往往限制了分离采用单一的液相色谱（LC）原理可达到的分辨率。特别是，对于超修饰的蛋白质，如组蛋白，需要广泛和耗时的分级分离，以实现深蛋白质型覆盖。在此，我们提出了第一个在线纳米流综合二维液相色谱（nLC×LC）平台自顶向下质谱分析组蛋白蛋白质型。所描述的二维LC系统结合了在亲水相互作用LC条件下的弱阳离子交换色谱（即，基于电荷和结晶度的分离）与反相液相色谱（即，基于疏水性的分离）。两个独立的化学选择性在纳米流（300 nL/min）下运行，并与采用紫外光解离的高分辨率质谱（UVPD-HRMS）在线耦合。相对于一维方法，nLC×LC工作流程增加了可观察到的完整蛋白质质量的数量，并允许从有限的样品量（约1.5 μg）开始表征数百种蛋白质型。
Top-down proteomics is an emerging analytical strategy to characterize combinatorial protein post-translational modifications (PTMs). However, sample complexity and small mass differences between chemically closely related proteoforms often limit the resolution attainable by separations employing a single liquid chromatographic (LC) principle. In particular, for ultramodified proteins like histones, extensive and time-consuming fractionation is needed to achieve deep proteoform coverage. Herein, we present the first online nanoflow comprehensive two-dimensional liquid chromatography (nLC×LC) platform top-down mass spectrometry analysis of histone proteoforms. The described two-dimensional LC system combines weak cation exchange chromatography under hydrophilic interaction LC conditions (i.e., charge- and hydrophilicity-based separation) with reversed phase liquid chromatography (i.e., hydrophobicity-based separation). The two independent chemical selectivities were run at nanoflows (300 nL/min) and coupled online with high-resolution mass spectrometry employing ultraviolet photodissociation (UVPD-HRMS). The nLC×LC workflow increased the number of intact protein masses observable relative to one-dimensional approaches and allowed characterization of hundreds of proteoforms starting from limited sample quantities (∼1.5 μg).
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影响因子：
4.4
作者：
Liu X;Hengel S;Wu S;Tolić N;Pasa-Tolić L;Pevzner PA
通讯作者：
Pevzner PA
影响因子：
4.4
作者：
LeDuc, Richard D.;Fellers, Ryan T.;Early, Bryan P.;Greer, Joseph B.;Thomas, Paul M.;Kelleher, Neil L.
通讯作者：
Kelleher, Neil L.
DOI：
10.1074/mcp.o114.046573
发表时间：
2015-04
期刊：
Molecular & cellular proteomics : MCP
影响因子：
--
作者：
Maile TM;Izrael-Tomasevic A;Cheung T;Guler GD;Tindell C;Masselot A;Liang J;Zhao F;Trojer P;Classon M;Arnott D
通讯作者：
Arnott D
影响因子：
14.8
作者：
Garcia, Benjamin A.;Mollah, Sahana;Hunt, Donald F.
通讯作者：
Hunt, Donald F.
影响因子：
3.4
作者：
Britton LM;Gonzales-Cope M;Zee BM;Garcia BA
通讯作者：
Garcia BA