Development of an Online 2D Ultrahigh-Pressure Nano-LC System for High-pH and Low-pH Reversed Phase Separation in Top-Down Proteomics.
Development of an Online 2D Ultrahigh-Pressure Nano-LC System for High-pH and Low-pH Reversed Phase Separation in Top-Down Proteomics.
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DOI：
10.1021/acs.analchem.0c03395
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发表时间：
2020-10-06
影响因子：
7.4
通讯作者：
Wu S
中科院分区：
文献类型：
--
作者：
Wang Z;Yu D;Cupp-Sutton KA;Liu X;Smith K;Wu S
新型高分辨率分离技术的发展对于推进高通量自上而下蛋白质组学所需的复杂样品分析至关重要。最近，我们的团队开发了一种离线的2D高pH RPLC/低pH RPLC分离方法，并证明了这两种RPLC格式之间具有良好的正交性。具体地，超高压长毛细管柱RPLC分离已被应用作为第二维低pH RPLC分离以提高分离分辨率。为了进一步提高离线方法的通量和灵敏度，我们开发了一种在线2D超高压纳米LC系统，用于自上而下蛋白质组学中的高pH和低pH RPLC分离。使用具有稀释设置的在线微阱柱来收集从第一维高pH分离洗脱的蛋白质，并将级分注射用于第二维中的超高压长毛细管柱低pH RPLC分离。该自动化平台能够从5 μg完整的E.大肠杆菌细胞裂解物在10个在线收集的馏分。在这里，我们已经证明，我们的在线2D pH RP/RPLC系统与自上而下的蛋白质组学相结合，具有对质量有限的样品进行深度蛋白质组表征的潜力，因为它允许在低微克样品量下从复杂的生物样品中鉴定数百种完整的蛋白质型。
The development of novel high-resolution separation techniques is crucial for advancing the complex sample analysis necessary for high-throughput top-down proteomics. Recently, our group developed an offline 2D high-pH RPLC/low-pH RPLC separation method and demonstrated good orthogonality between these two RPLC formats. Specifically, ultrahigh-pressure long capillary column RPLC separation has been applied as the second dimensional low-pH RPLC separation for the improvement of separation resolution. To further improve the throughput and sensitivity of the offline approach, we developed an online 2D ultrahigh-pressure nano-LC system for high-pH and low-pH RPLC separations in top-down proteomics. An online microtrap column with a dilution setup was used to collect eluted proteins from the first dimension high-pH separation and inject the fractions for ultrahigh-pressure long capillary column low-pH RPLC separation in the second dimension. This automatic platform enables the characterization of 1000+ intact proteoforms from 5 μg of intact E. coli cell lysate in 10 online-collected fractions. Here, we have demonstrated that our online 2D pH RP/RPLC system coupled with top-down proteomics holds the potential for deep proteome characterization of mass-limited samples because it allows the identification of hundreds of intact proteoforms from complex biological samples at low microgram sample amounts.
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影响因子：
7.4
作者：
Chen B;Brown KA;Lin Z;Ge Y
通讯作者：
Ge Y
三维液相色谱耦合离子交换色谱/疏水相互作用色谱/反相色谱法，用于自上而下的蛋白质组学中有效蛋白质分离。
影响因子：
7.4
作者：
Valeja SG;Xiu L;Gregorich ZR;Guner H;Jin S;Ge Y
通讯作者：
Ge Y
影响因子：
3.4
作者：
Wang, Yuexi;Yang, Feng;Gritsenko, Marina A.;Wang, Yingchun;Clauss, Therese;Liu, Tao;Shen, Yufeng;Monroe, Matthew E.;Lopez-Ferrer, Daniel;Reno, Theresa;Moore, Ronald J.;Klemke, Richard L.;Camp, David G., II;Smith, Richard D.
通讯作者：
Smith, Richard D.
影响因子：
4.4
作者：
Gargano AFG;Shaw JB;Zhou M;Wilkins CS;Fillmore TL;Moore RJ;Somsen GW;Paša-Tolić L
通讯作者：
Paša-Tolić L
DOI：
10.1007/s13361-016-1532-8
发表时间：
2017-04-01
影响因子：
3.2
作者：
Choi, Sam B.;Zamarbide, Marta;Nemes, Peter
通讯作者：
Nemes, Peter