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Automated ultra-high-pressure multidimensional protein identification technology (UHP-MudPIT) for improved peptide identification of proteomic samples
- Source :
- Analytical Chemistry. July 15, 2006, Vol. 78 Issue 14, p5109, 10 p.
- Publication Year :
- 2006
-
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 ~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.
Details
- Language :
- English
- ISSN :
- 00032700
- Volume :
- 78
- Issue :
- 14
- Database :
- Gale General OneFile
- Journal :
- Analytical Chemistry
- Publication Type :
- Academic Journal
- Accession number :
- edsgcl.148867042