Automated Ultra-High-Pressure Multidimensional Protein Identification Technology (UHP-MudPIT) for Improved Peptide Identification of Proteomic Samples

Multidimensional separation is one of the most successful approaches for proteomics studies that deal with complex samples. We have developed an automated ultra-high-pressure multidimensional liquid chromatography system that operates up to ∼20 kpsi to improve separations and increase protein coverage from limited amount of samples. The reversed-phase gradient is operated in the constant-flow mode opposed to the constant-pressure mode, which is typical of previous ultra-high-pressure systems. In contrast to constant-pressure systems, the gradient shape is fully controllable and can be optimized for the type of samples to be run. The system also features fast sample loading/desalting using a vented column approach to improve sample throughput. This approach was validated on a soluble fraction from yeast lysate where we achieved ∼30% more protein identifications using a 60-cm-long triphasic capillary column than with our traditional approach. Advantages of the use of a relatively long reversed-phase column (∼50 cm) for MudPIT-type experiments are also discussed.