title: Development of Online pH Gradient-Eluted Strong Cation Exchange Nanoelectrospray-Tandem Mass Spectrometry for Proteomic Analysis Facilitating Basic and Histidine-Containing Peptides Identification.
abstract: 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.
authors.affiliation: 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.
authors.forename: Jingjing
authors.initials: J
authors.lastname: Xu
authors.affiliation: 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.|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.
authors.forename: Jing
authors.initials: J
authors.lastname: Gao
authors.affiliation: 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.
authors.forename: Chengli
authors.initials: C
authors.lastname: Yu
authors.affiliation: 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.
authors.forename: Han
authors.initials: H
authors.lastname: He
authors.affiliation: Department of Analytical Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences , Shanghai, 201203, China.
authors.forename: Yiming
authors.initials: Y
authors.lastname: Yang
authors.affiliation: 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.
authors.forename: Daniel
authors.initials: D
authors.lastname: Figeys
authors.affiliation: 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.|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.
authors.forename: Hu
authors.initials: H
authors.lastname: Zhou
mesh_terms: D002412:Cations / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D006639:Histidine / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D020407:Internet; D036103:Nanotechnology; D010455:Peptides / Q000032:analysis* / Q000737:chemistry*; D040901:Proteomics; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D002412:Cations; D010455:Peptides; D006639:Histidine