Multidimensional Liquid Chromatography Separations PDF Download

Sample complexity is one of the key challenges facing contemporary proteomic analysis. A variety of methods is commonly employed to reduce this complexity, both at an intact protein and digested peptide level. For complex lysates containing many thousands of proteins, orthogonal (mutually independent), multidimensional separation methods must be employed to provide sufficient resolution to characterize the appropriate number of different species. The most common of these methods are two dimensional gel electrophoresis (2DGE) of proteins and multidimensional liquid chromatographic separation (MuDPIT) of peptides, which rely on isoelectric focusing followed by mass separation in the former, and ion exchange followed by reversed phase separation in the latter. These methods have significant drawbacks in terms of sample bias, sample preparation and reproducibility, and therefore a new methodology that combines the positive aspects of both separation technologies in an automatable, reproducible form is highly desirable. New developments in column technology have allowed rapid improved-resolution separation of intact proteins in complex samples, coupled to improved methodology for peptide and protein identification. The separation of complex protein mixtures using both on- and off-line 2D liquid chromatography using derivitized polystyrene-divinylbenzene (PS-DVB) pellicular ion-exchange resins and PS-DVB monolithic reversed-phase columns is described. Proteolytic digestion of the fractions followed by rapid liquid chromatography-tandem mass spectrometry was used to complete the analysis. An alternative methodology, relying on direct analysis of the second dimension eluents by top-down (mass spectrometric analysis of intact proteins) methodology, using an Apex IV 12 T Fourier-transform ion cyclotron resonance mass spectrometer (Bruker Daltonics) and an HCT ion trap (Bruker Daltonics) equipped with electron transfer dissociation has allowed in-depth analysis of intact proteins. Sample types investigated to establish the utility of the methodology include bacterial lysates (Bordetella parapertusis, and Escherichia coli), a eukaryotic parasite (Leishmania donovani), and transformed human cell lines.