Intact protein separations are required for the reduction of complexity of samples analyzed in proteomics. The analysis of intact proteins is typically performed by gel electrophoresis (GE), however optimization of LC based protein separations offers unique opportunities for the detection and identification of proteins. Biomarker discovery research is an example that could benefit from such separation workflows.

With the combination of the unique Off-line 2-D LC workflow and dedicated protein separation columns, such as the ProSwift™ Monolithic Ion-Exchange and Reversed-Phase and PepSwift™ Monolithic Reversed-Phase , the UltiMate^®3000 Proteomics MDLC provides a turnkey solution for proteomics research.

The ProSwift WAX-1S monolithic column, 1.0-mm x 50-mm, allows for the efficient ion-exchange separation of protein samples. The separated sample is then fractionated using the UltiMate 3000 Proteomics MDLC well-plate sampler . Automated reinjection onto the PepSwift RP monolithic column (200-µm x 50-mm or 500-µm x 50-mm) assures high-resolution separation of these WAX-fractions. With flow rates of 3.0 µL/min (200-µm) or 20 µL/min (500-µm) convenient and efficient coupling to mass spectrometry is ensured.

Key features of the application are:

• Reduction of sample complexity
• Separation of PTMs, splice variants, and isoforms
• Perfectly suited for use in fully automated off-line 2-D LC/MS workflows
• Targeted analysis of fractions of interest

Reconstructed 2-D gel plot (2-D retention map) of an E. coli separation. The upper trace shows the ion-exchange separation in the first dimension and the lower trace the 2-D reversed-phase separation of the corresponding fraction selected from the reconstructed 2-D gel plot.