Multiplexed Two-Dimensional Liquid Chromatography for MALDI and Nanoelectrospray Ionization Mass Spectrometry in Proteomics

We developed a multiplexed two-dimensional separation system based on reversed phase (RP) − strong cation exchange (SCX) chromatography as a front-end device for matrix-assisted laser desorption ionization (MALDI) or nanoelectrospray ionization (nanoESI) mass spectrometry. Tryptic peptide mixtures were fractionated on a reversed-phase HPLC column, and each fraction was loaded onto multiplexed SCX microcolumns. Because this second chromatography was carried out in parallel, the analysis time is independent of the fraction number in the first RP−HPLC separation. The resultant samples were desalted/concentrated and eluted onto a MALDI plate with matrix-containing elution solutions in parallel, or eluted with optimized solutions for nanoESI and loaded onto nanoESI sprayers by an automated instrument. The soluble portion of HCT116 lysate was digested and fractionated using a 48-plexed chromatography system. Approximately 1000 unique peaks were detected in MALDI−MS with 3000 MS/MS spectra, while 724 peptides with ultrahigh peptide mass accuracy (sub-ppm error) were identified in nanoESI−FTICR mass spectrometry with five integrated selected ion monitoring scans. Since MS measurement with this off-line LC−LC approach is not restricted by continuous LC elution, it is expected to be useful especially in cases where repeated analysis with different scan modes or long-term data acquisition is required.

Keywords: multiplexed chromatography • two-dimensional chromatography • StageTip • C18−SCX fractionation • proteomics • MALDI−MS • nanoelectrospray−MS • HCT116