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.
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Acknowledgments
The authors would like to acknowledge Beckman Coulter for their support. H.S. fellowship in the J.E. Van Eyk’s laboratory was partially supported by Czech Ministry of Education project No. LC07017. The study was funded by the NHLBI Proteomics Innovation Contract N01-HV-28180 (JEV) and the Institute for Clinical and Translational Science Award (Grant NO 1U54RR023561-01A1).
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Sheng, S. et al. (2011). Intact Protein Separation by One- and Two-Dimensional Liquid Chromatography for the Comparative Proteomic Separation of Partitioned Serum or Plasma. In: Simpson, R., Greening, D. (eds) Serum/Plasma Proteomics. Methods in Molecular Biology, vol 728. Humana Press. https://doi.org/10.1007/978-1-61779-068-3_2
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DOI: https://doi.org/10.1007/978-1-61779-068-3_2
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