Abstract
Liquid chromatography (LC) is essential for sample fractionation in shotgun proteomics applications. With suitable design, common LC separation chemistries, including reversed-phase (RP) and strong cation exchange (SCX) mode, can be combined in online multidimensional LC to greatly enhance the overall separation power and, thus, proteome coverage. This protocol describes the design and assembly of a flexible online multidimensional RP-SCX-RP LC system that is compatible with deep proteome profiling on common shotgun proteomics platforms.
The authors Maggie P.Y. Lam, Chun Hin Law, and Quan Quan have contributed equally to this work.
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Acknowledgment
This study was supported by the Hong Kong Research Grants Council (HKU7018/10P and HKU7016/11P). We thank AB SCIEX (Hong Kong) Limited for loan of the Eksigent NanoLC ultra 2D Plus system; Dr. Ricky P. W. Kong for performing some preliminary experiments and many helpful discussions; Dr. Tina Settineri, Eksigent (Dublin, CA, USA), Ms. Yuko P. Y. Lam, and Mr W.-Y. Yuen for their comments and suggestions; and the school of biological Science, HKU, for access to the 5600 mass spectrometer.
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S Figure 1 and detailed installation protocol. This material is available free of charge via the Internet at http://web.hku.hk/~ivankchu/ichu.html/RP_MDLC_shorgun_Proteomics. Other information is also available upon request.
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Lam, M.P.Y., Law, C.H., Quan, Q., Zhao, Y., Chu, I.K. (2014). Fully Automatable Multidimensional Reversed-Phase Liquid Chromatography with Online Tandem Mass Spectrometry. In: Martins-de-Souza, D. (eds) Shotgun Proteomics. Methods in Molecular Biology, vol 1156. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0685-7_3
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DOI: https://doi.org/10.1007/978-1-4939-0685-7_3
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-0684-0
Online ISBN: 978-1-4939-0685-7
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