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Automated ultra-high-pressure multidimensional protein identification technology (UHP-MudPIT) for improved peptide identification of proteomic samples

Reputable Mentor II
Reputable Mentor II
Motoyama A, Venable JD, Ruse CI, Yates JR 3rd.
Anal Chem. 2006 Jul 15;78(14):5109-18.
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.
Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
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