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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• 作者：Jingjing Xu ; Jing Gao ; Chengli Yu ; Han He ; Yiming Yang ; Daniel Figeys ; Hu Zhou
• 刊名：Analytical Chemistry
• 出版年：2016
• 出版时间：January 5, 2016
• 年：2016
• 卷：88
• 期：1
• 页码：583-591
• 全文大小：611K
• ISSN：1520-6882

文摘

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.