Separation and identification of peptides by integrated multidimensional liquid chromatography-mass spectrometry (IMDLC-MS)

Abstract

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Integrated multidimensional liquid chromatography (IMDLC) is an innovative method used for separation of tryptic peptides. In this method a two-component column, containing strong cation exchange (SCX) and reverse-phase (RP) were used for two-dimensional liquid chromatography. To separate peptides on an SCX column two salts were used: ammonium formate and ammonium acetate. The salt was injected onto the column in the form of pulse in 13 different steps ranging from 0 to 100% through a quaternary pumping system. This novel method was tested on model peptides, small proteins and Arabidopsis thaliana proteins extracted from leaves. The proteins were digested with trypsin and the obtained tryptic peptides were used for the separation process. A total of 118 proteins were identified in Arabidopsis thaliana leaves. The two salts were compared and a larger number of peptides were identified when formate was used. This method is designed on a conventional HPLC with a quaternary pump yielding two dimensional separations.