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Multidimensional liquid chromatography (MDLC) coupled to mass spectrometry is a valuable strategy for bottom-up or top-down workflows in proteomics. A large number of different MDLC approaches have been described for the separation of intact proteins and peptides.
Off-line MDLC techniques have several advantages over on-line approaches, including: (1) higher flexibility with respect to column dimensions and mobile phase selection, (2) easier method development and, (3) the ability to re-analyze the fractionated effluent.
Here we present a fully automated method for off-line MDLC of peptides and proteins. The method allows for a combination of different column dimensions and chemistries to optimize the MDLC separation. Effluent from the first dimension column is directed to an eight-port injection valve and fractionated through the injection needle in a well plate or sample vials.
The second dimension separation utilizes a column-switching configuration for on-line desalting, sample concentration, and direct interface to mass spectrometer. Alternatively, for protein separations, a second fractionation step can be implemented after the reversed-phase separation to allow for proteolytic digestion.
Methods have been developed for 2D-LC of peptides and proteins using ion-exchange and reversed-phase chromatography. Fractionation of the ion-exchange separation and subsequent injection of the fractions is performed automatically onto capillary PS-DVB monolithic columns. The fast mass transfer kinetics of the monolithic columns allows for fast gradient separations and a reduction of the total analysis time of the 2D-LC method. Interfacing the 2D-LC separation with tandem MS sequencing of peptides shows that peptides elute typically in one or two strong cation-exchange fractions. In addition, the system shows good precision performance for injection and fractionation of samples.