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Anion and cation mixed-bed ion exchange for enhanced multidimensional separations of peptides and phosphopeptides

Academic Article
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Overview

authors

  • Motoyama, A.
  • Xu, T.
  • Ruse, C. I.
  • Wohlschlegel, J. A.
  • Yates III, John

publication date

  • May 2007

journal

  • Analytical Chemistry  Journal

abstract

  • Shotgun proteomics typically uses multidimensional LC/MS/MS analysis of enzymatically digested proteins, where strong cation-exchange (SCX) and reversed-phase (RP) separations are coupled to increase the separation power and dynamic range of analysis. Here we report an on-line multidimensional LC method using an anion- and cation-exchange mixed bed for the first separation dimension. The mixed-bed ion-exchange resin improved peptide recovery over SCX resins alone and showed better orthogonality to RP separations in two-dimensional separations. The Donnan effect, which was enhanced by the introduction of fixed opposite charges in one column, is proposed as the mechanism responsible for improved peptide recovery by producing higher fluxes of salt cations and lower populations of salt anions proximal to the SCX phase. An increase in orthogonality was achieved by a combination of increased retention for acidic peptides and moderately reduced retention of neutral to basic peptides by the added anion-exchange resin. The combination of these effects led to approximately 100% increase in the number of identified peptides from an analysis of a tryptic digest of a yeast whole cell lysate. The application of the method to phosphopeptide-enriched samples increased by 94% phosphopeptide identifications over SCX alone. The lower pKa of phosphopeptides led to specific enrichment in a single salt step resolving acidic phosphopeptides from other phospho- and non-phosphopeptides. Unlike previous methods that use anion exchange to alter selectivity or enrich phosphopeptides, the proposed format is unique in that it works with typical acidic buffer systems used in electrospray ionization, making it feasible for online multidimensional LC/MS/MS applications.

subject areas

  • Chromatography, Liquid
  • Ion Exchange Resins
  • Peptides
  • Phosphopeptides
  • Proteomics
  • Trypsin
  • Yeasts
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Identity

PubMed Central ID

  • PMC2538959

International Standard Serial Number (ISSN)

  • 0003-2700

Digital Object Identifier (DOI)

  • 10.1021/ac062292d

PubMed ID

  • 17411013
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Additional Document Info

start page

  • 3623

end page

  • 3634

volume

  • 79

issue

  • 10

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