Intact Protein Separation by One- and Two-Dimensional Liquid Chromatography for the Comparative Proteomic Separation of Partitioned Serum or Plasma

  • Simon Sheng
  • Helena Skalnikova
  • Andrew Meng
  • John Tra
  • Qin Fu
  • Allen Everett
  • Jennifer Van Eyk
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 728)

Abstract

Serum- and plasma-based biomarker discovery requires technologies with specific capabilities: sufficient proteome coverage and depth, technical reproducibly, and the scalability to enable analysis on a large number of samples at reasonable cost. We have shown that plasma samples processed using IgY LC10 Proteome Partitioning kits to remove the most highly abundant proteins selectively, followed by intact protein separation by two-dimensional liquid chromatography (2DLC, chromatofocusing, and reversed phase) can uniquely enrich for middle to lower-abundant proteins. Equally, 1DLC (reversed phase) separation of intact proteins is complementary to 2DLC. The serial use of a single piece of equipment can be prohibitively time consuming and thus, this chapter also describes the harmonization of multiple LC instruments in order to minimize technical variation and ensure reproducibility. These technical improvements allow large numbers of individual clinical samples to be analyzed with multiple instruments in a timely manner, while retaining optimal reproducibility and allowing precise differential analysis at the proteome scale.

Key words

Liquid chromatography Chromatofocusing Reversed phase Reproducibility Protein load Plasma Serum Proteomics Biomarkers 

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Simon Sheng
  • Helena Skalnikova
  • Andrew Meng
  • John Tra
  • Qin Fu
  • Allen Everett
  • Jennifer Van Eyk
    • 1
  1. 1.Department of MedicineJohns Hopkins UniversityBaltimoreUSA

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