title: Intact protein separation by one- and two-dimensional liquid chromatography for the comparative proteomic separation of partitioned serum or plasma.
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.
authors.affiliation: Department of Medicine, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: Simon
authors.initials: S
authors.lastname: Sheng
authors.forename: Helena
authors.initials: H
authors.lastname: Skalnikova
authors.forename: Andrew
authors.initials: A
authors.lastname: Meng
authors.forename: John
authors.initials: J
authors.lastname: Tra
authors.forename: Qin
authors.initials: Q
authors.lastname: Fu
authors.forename: Allen
authors.initials: A
authors.lastname: Everett
authors.forename: Jennifer
authors.initials: J
authors.lastname: Van Eyk
mesh_terms: D001798:Blood Proteins / Q000737:chemistry / Q000302:isolation & purification*; D005591:Chemical Fractionation; D002853:Chromatography, Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase; D005374:Filtration; D006801:Humans; D007074:Immunoglobulin G / Q000302:isolation & purification; D007136:Immunoglobulins / Q000302:isolation & purification; D008055:Lipids / Q000302:isolation & purification; D013058:Mass Spectrometry; D010949:Plasma / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D044967:Serum / Q000737:chemistry*; D012709:Serum Albumin / Q000302:isolation & purification; D014461:Ultracentrifugation
chemical_list: D001798:Blood Proteins; C016512:IgY; D007074:Immunoglobulin G; D007136:Immunoglobulins; D008055:Lipids; D012709:Serum Albumin