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.