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Development of Online pH Gradient-Eluted Strong Cation Exchange Nanoelectrospray-Tandem Mass Spectrometry for Proteomic Analysis Facilitating Basic and Histidine-Containing Peptides Identification

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Department of Analytical Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China
CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai, 201203, China
§ University of Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing 100049, China
Department of Biochemistry, Microbiology and Immunology, and Department of Chemistry and Biomolecular Sciences, University of Ottawa, 451 Smyth Road, Ottawa, Ontario K1H 8M5, Canada
SIMMUOMICS Laboratory, Joint Research Laboratory of Translational “OMICS” between Shanghai Institute of Materia Medica, Chinese Academy of Sciences, China and University of Ottawa, Canada, 555 Zuchongzhi Road, Shanghai, 201203, China
*E-mail: [email protected]. Phone: 613-562-5800 ext. 8202.
*E-mail: [email protected]. Phone: +86-021-50806706.
Cite this: Anal. Chem. 2016, 88, 1, 583–591
Publication Date (Web):December 8, 2015
Copyright © 2015 American Chemical Society

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    A novel one-dimensional online pH gradient-eluted strong cation exchange-nanoelectrospray ionization-tandem mass spectrometry (SCX-nano-ESI-MS/MS) method was developed for protein identification and tested with a mixture of six standard proteins, total lysate of HuH7 and N2a cells, as well as membrane fraction of N2a cells. This method utilized an online nanoflow SCX column in a nano-LC system coupled with a nanoelectrospray high-resolution mass spectrometer. Protein digests were separated on a nanoflow SCX column with a pH gradient and directly introduced into a mass spectrometer through nanoelectrospray ionization. More than five thousand unique peptides were identified in each 90 min LC–MS/MS run using 500 nanogram of protein digest either from total cell lysate or from membrane fraction. The unique peptide overlap between online strong cation exchange nano-ESI-MS/MS (SCXLC–MS/MS) and reverse phase nano-ESI-MS/MS (RPLC–MS/MS) is only ≤30%, which indicated these two methods were complementary to each other. The correlation coefficient of retention time and theoretical isoelectric point (pI) of identified peptides in SCXLC–MS/MS was higher than 0.4, which showed that peptides elution in SCXLC–MS/MS was dependent on their charge states. Furthermore, SCXLC–MS/MS showed identification capability for a higher proportion of basic peptides compared to the RPLC–MS/MS method, especially for histidine-containing peptides. Our SCXLC–MS/MS method is an excellent alternative method to the RPLC–MS/MS method for analysis of standard proteins, total cell and membrane proteomes.

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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b04000.

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    Cited By

    This article is cited by 11 publications.

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    5. Xiaoyun Gong, Xingchuang Xiong, Yingchen Zhao, Sijian Ye, and Xiang Fang . Boosting the Signal Intensity of Nanoelectrospray Ionization by Using a Polarity-Reversing High-Voltage Strategy. Analytical Chemistry 2017, 89 (13) , 7009-7016.
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