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 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.
Motoyama
Akira
A
Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Venable
John D
JD
Ruse
Cristian I
CI
Yates
John R
JR
D000595:Amino Acid Sequence; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D011312:Pressure; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics / Q000295:instrumentation* / Q000379:methods*
D010455:Peptides; D011506:Proteins