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

Anal Chem. 2016 Jan 5;88(1):583-91. doi: 10.1021/acs.analchem.5b04000. Epub 2015 Dec 14.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cations / chemistry
  • Chromatography, High Pressure Liquid
  • Histidine / analysis*
  • Hydrogen-Ion Concentration
  • Internet
  • Nanotechnology*
  • Peptides / analysis*
  • Peptides / chemistry*
  • Proteomics*
  • Spectrometry, Mass, Electrospray Ionization*
  • Tandem Mass Spectrometry*


  • Cations
  • Peptides
  • Histidine