Three dimensional liquid chromatography coupling ion exchange chromatography/hydrophobic interaction chromatography/reverse phase chromatography for effective protein separation in top-down proteomics

Analytical Chemistry
Santosh G ValejaYing Ge

Abstract

To address the complexity of the proteome in mass spectrometry (MS)-based top-down proteomics, multidimensional liquid chromatography (MDLC) strategies that can effectively separate proteins with high resolution and automation are highly desirable. Although various MDLC methods that can effectively separate peptides from protein digests exist, very few MDLC strategies, primarily consisting of 2DLC, are available for intact protein separation, which is insufficient to address the complexity of the proteome. We recently demonstrated that hydrophobic interaction chromatography (HIC) utilizing a MS-compatible salt can provide high resolution separation of intact proteins for top-down proteomics. Herein, we have developed a novel 3DLC strategy by coupling HIC with ion exchange chromatography (IEC) and reverse phase chromatography (RPC) for intact protein separation. We demonstrated that a 3D (IEC-HIC-RPC) approach greatly outperformed the conventional 2D IEC-RPC approach. For the same IEC fraction (out of 35 fractions) from a crude HEK 293 cell lysate, a total of 640 proteins were identified in the 3D approach (corresponding to 201 nonredundant proteins) as compared to 47 in the 2D approach, whereas simply prolonging the gradients i...Continue Reading

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Citations

Feb 19, 2016·Analytical Chemistry·Erin GemperlineLingjun Li
Jun 17, 2016·Annual Review of Analytical Chemistry·Timothy K TobyNeil L Kelleher
Aug 16, 2016·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Xia YanYing-Kun Qiu
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May 3, 2019·Proteomics·Leah V SchafferLloyd M Smith
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Apr 1, 2017·Expert Review of Proteomics·Ping JinEdouard C Nice
Jul 28, 2016·Expert Review of Proteomics·Wenxuan CaiYing Ge
Jun 29, 2018·Nature Communications·Michiel van de WaterbeemdAlbert J R Heck
May 14, 2021·Journal of the American Society for Mass Spectrometry·Jake A MelbyYing Ge
May 29, 2021·Mass Spectrometry Reviews·Jessica L NickersonAlan A Doucette
Aug 9, 2019·Journal of the American Society for Mass Spectrometry·Scott A UgrinDonald F Hunt
May 4, 2018·Analytical Chemistry·Andrea F G GarganoGovert W Somsen
Apr 18, 2017·Journal of Proteome Research·Timothy P ClelandJennifer S Brodbelt
Dec 5, 2020·Journal of the American Society for Mass Spectrometry·John R CorbettSteven M Patrie
Feb 6, 2017·Journal of Proteome Research·Luca FornelliNeil L Kelleher
Jul 18, 2017·Biochimica Et Biophysica Acta. Molecular Cell Research·Andreas Tholey, Alexander Becker

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