on 05-01-201210:38 AM - edited on 10-15-202112:02 PM by AnalyteGuru
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.