Using size exclusion chromatography-RPLC and RPLC-CIEF as two-dimensional separation strategies for protein profiling

Bottom-up proteomics (analyzing peptides that result from protein digestion) has demonstrated capability for broad proteome coverage and good throughput. However, due to incomplete sequence coverage, this approach is not ideally suited to the study of modified proteins. The modification complement of a protein can best be elucidated by analyzing the intact protein. 2-DE, typically coupled with the analysis of peptides that result from in-gel digestion, is the most frequently applied protein separation technique in MS-based proteomics. As an alternative, numerous column-based liquid phase techniques, which are generally more amenable to automation, are being investigated. In this work, the combination of size-exclusion chromatography (SEC) fractionation with RPLC-Fourier-transform ion cyclotron resonance (FTICR)-MS is compared with the combination of RPLC fractionation with CIEF-FTICR-MS for the analysis of the Shewanella oneidensis proteome. SEC-RPLC-FTICR-MS allowed the detection of 297 proteins, as opposed to 166 using RPLC-CIEF-FTICR-MS, indicating that approaches based on LC-MS provide better coverage. However, there were significant differences in the sets of proteins detected and both approaches provide a basis for accurately quantifying changes in protein and modified protein abundances.     

Ground state and excited state properties of ethylene isomers studied by a combined Feynman path integral-ab initio approach

Ground state and excited state properties of ethylene, C₂H₄, and several ethylene isomers have been studied by Feynman path integral Monte Carlo (PIMC) simulations. The PIMC treatment of the atomic nuclei has been combined with different electronic Hamiltonians in order to analyse the influence of the nuclear degrees of freedom on electronic quantities. Electronic expectation values at the minimum of the potential energy surface (PES) have been compared with PIMC based ensemble averaged values. Ensemble averaged quantities have been derived by Hamiltonians of the ab initio type and a tight-binding (TB) one-electron model. The combined influence of anharmonicities in the interatomic potential and the quantum fluctuations of the atomic nuclei lead to ensemble averaged bondlengths r _g which are significantly larger than the parameters r _e, at the minimum of the PES. The implications of this bond length elongation for the electronic properties of ethylene are discussed. The occupied canonical molecular orbitals (CMOs) of ethylene are destabilized under the influence of the nuclear degrees of freedom while virtual CMOs are stabilized. These shifts of one-electron energies suggest a comparison of electronic excitation energies at the minimum of the PES with PIMC based ensemble averages. The quantum fluctuations of the nuclei cause a strong redistribution in the intensities of electronic transitions. Transitions, which are dipole allowed in the planar D_2h geometry of ethylene, lose intensity under the influence of nuclear quantum effects, and vice versa for electronic excitations that are dipole forbidden under D_2h symmetry. This ‘vibrational borrowing’ is enhanced with decreasing atomic masses. The Feynman centroid density has been used to calculate the anharmonic vibrational wavenumbers of C₂H₄ and C₂D₄. The results of the present PIMC simulations have been employed to emphasize general problems of electronic structure calculations based on a single nuclear configuration (i.e. the configuration at the minimum of the PES).