Evaluation of multidimensional (ion-exchange/reversed-phase) protein separations using linear and step gradients in the first dimension

Detection and expression analysis of recombinant proteins in plant‐derived complex mixtures using nanoUPLC‐MSE

“…These peptides showed no trace of post-translational modification; however, that possibility cannot be discarded because an additional 6 CTAG peptides were not detected. The orthogonal separations [48] obtained using SCX columns [49,50] or recent technologies at the first-dimension linear gradient with fractions at different pH levels and with high-resolution separations in both the first and the second dimensions [51] were permitted because of the complexity of the chromatogram in this particular sample ( Fig. 2A).…”

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

“…Methods that use IPG gels to fractionate samples between liquid-filled chambers are of particular interest due to claimed ease of protein recovery and minimal fraction overlap [34][35][36][37][38]. Other chromatographic methods applied to protein fractionation prior to RPLC-MS include strong anion exchange chromatography [39,40] and size-exclusion chromatography (SEC) [41], but RPLC itself is an excellent fractionation method. Separations compatible with direct MS interfacing that are orthogonal to RPLC include CIEF and CZE.…”

The development of an evaluation method for capture columns used in two-dimensional liquid chromatography

“…Importantly, interface techniques could also be valuable to the development of on-line comprehensive 2D-LC. Common interfaces include capture columns [9,16,17], sample loops [2,18], parallel columns [1,19] and vacuum solvent evaporation [20]. In this study, we present a new method for evaluating and selecting appropriate capture columns for 2D-LC systems.…”

“…At the same time, switching the mobile phase on the capture column can avoid incompatibility between two-dimensional mobile phases. Berger and co-workers [16] developed a 2D-LC system using a strong anion-exchange (SAX) column as the first separation dimension and a Symmetry 300 C4 column as the reversed-phase second dimension; trapping of the analytes from the SAX column was accomplished using two Symmetry 300 C4 columns as capture columns for separating proteins. Sweeney and Shalliker [9] reported a 2D-LC system based on a Sephasil C4 column and an Activon Valupak ODS column that uses C18 as the capture column to isolate and concentrate polystyrene oligomers.…”