Automated LC-LC-MS-MS platform using binary ion-exchange and gradient reversed-phase chromatography for improved proteomic analyses

A simple multidimensional liquid chromatography system utilizing an isocrat ic pump and a HPLC system is described for the comprehensive proteomic anal ysis of complex peptide digest mixtures by coupled LC-LC-MS-MS techniques. A binary ion-exchange separation was achieved through the use of a strong c ation-exchange column followed by a reversed-phase column for data-dependen t LC-MS-MS analysis of the unbound analytes, and following salt elution (an d concomitant column reequilibration), the bound analytes. Off-line validat ion of the platform showed near quantitative recovery of fractionated pepti des and essentially complete ion-exchange partitioning. In comparative anal yses of a highly complex peptide digest mixture a >40% increase in the numb er of peptide and protein identifications was achieved using this multidime nsional platform compared to an unfractionated control. (C) 2001 Elsevier S cience B.V. All rights reserved.