Intact Protein Separation by One- and Two-Dimensional Liquid Chromatography for the Comparative Proteomic Separation of Partitioned Serum or Plasma
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 wordsLiquid chromatography Chromatofocusing Reversed phase Reproducibility Protein load Plasma Serum Proteomics Biomarkers
- 1.Fu Q, Sheng S, Van Eyk JE (2007) Development of biomarker development pipeline: search for myocardial ischemia biomarkers. Book Chapter, Clinical Proteomics: From Diagnosis to Therapy, J. Van Eyk and M.J. Dunn (Eds), Wiley-VCH.Google Scholar
- 9.Gundry RL, White MY, Murray CI, Kane LA, Fu Q, Stanley BA, Van Eyk JE (2009) Preparation of proteins and peptides for mass spectrometry analysis in a bottom-up proteomics workflow. Curr. Protoc. Mol. Biol. Chapter 10:Unit10.25Google Scholar