Improved Hydrophobic Peptide Recovery During Online 2D Nano LC/MS/MS for Proteomic Analysis

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Liu, Finch, Gebler
ASMS 2006 May 28-Jun 1 Seattle, Wa
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ASMS 2006 Proteomics: New Methods II A 2D (SCX/RP) nanoLC/MS/MS system has been developed with improved separation efficiency and peptide recovery. The 2D separation was improved with an band re-focusing effect, made possible with the use of an analytical column which was more hydrophobic than the trap column. The chromatographic peaks acquired with the developed 2D method were equivalent in shape to those obtained with a 1D direct injection method run with the same conditions. Many hydrophobic peptides were recovered with the use of a RP step gradient applied to the SCX column after a conventional salt gradient.  Due to stronger hydrophobic interaction with the SCX column, these peptides could have not been eluted from the SCX column, and therefore not characterized with 2D without the application of the RP step gradient. The developed 2D method can be employed as an effective tool to characterize peptides with improved chromatographic resolution, MS sensitivity and sequence coverage.

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