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

Academic Article
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Overview

authors

  • Motoyama, A.
  • Venable, J. D.
  • Ruse, C. I.
  • Yates III, John

publication date

  • July 2006

journal

  • Analytical Chemistry  Journal

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.

subject areas

  • Amino Acid Sequence
  • Molecular Sequence Data
  • Peptides
  • Pressure
  • Proteins
  • Proteomics
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Identity

International Standard Serial Number (ISSN)

  • 0003-2700

Digital Object Identifier (DOI)

  • 10.1021/ac060354u

PubMed ID

  • 16841936
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Additional Document Info

start page

  • 5109

end page

  • 5118

volume

  • 78

issue

  • 14

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