Tailoring orthogonal proteomic routines to understand protein separation during ion exchange chromatography

Abstract

Original PaperTailoring orthogonal proteomic routines to understand protein separation during ion exchange chromatography Surface charge, molecular weight, and folding state are known to influence protein chromatographic behaviour onto ion exchangers. Experimentally, information related to such factors can be gathered via 2-DE methods. The application of 2-D PAGE under denaturing/reducing conditions was already shown to reveal separation trends within a large protein population from cell extracts. However, ion-exchange chromatography normally runs under native conditions. A tailored protocol consisting in a first separation based on IEF on Immobiline TM strips under native conditions followed by a second dimension SDS-PAGE run was adopted. The chromatographic versus electrophoretic separation behaviours of two model proteins, thaumatin (TAU) and BSA, were compared to better understand which proteomic routine would be better suited to anticipate IEX chromatographic separations. It was observed that the information contained in the pI value obtained with the adapted 2-DE protocol showed better correlation with the IEX chromatographic behaviour. On the other hand, chromatographic separations performed in the presence of urea as a denaturant have demonstrated the potential influence of hydrodynamic radius/ conformation on protein separation. Moreover, the information provided by such 2-D system correlated well with the chromatographic behaviour of an additional set of pure proteins. An initial prediction of protein ion-exchange chromatographic behaviour could be possible utilizing an experimental approach based on 2-DE running under milder chemical conditions. This technique provides information that more closely resembles the separation behaviour observed with a complex biotechnological feedstock.

On-chip technologies for multidimensional separations

“…A myriad of chromatographic and electrophoretic approaches, including high pressure liquid chromatography (HPLC), isoelectric focusing (IEF), and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), have been combined in various ways to improve and optimize the information available from a single assay. [3][4][5][6][7][8][9] The number of useful assay combinations increases when mass spectrometry (MS), which aids in identifying the chemical composition of a sample based upon the mass-to-charge ratio of its constituent species, is added as an analysis dimension. [10][11][12] Further, developers of 2D separations seek to surmount daunting challenges in control of the physicochemical interface between the dimensions.…”