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

Molecular Biology

Journal of Cytology & Histology

Author(s): Motoyama A, Venable JD, Ruse CI, Yates JR rd

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Abstract 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 approximately 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 approximately 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 (approximately 50 cm) for MudPIT-type experiments are also discussed. This article was published in Anal Chem and referenced in Journal of Cytology & Histology

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