title: Fractionation of the human plasma proteome for monoclonal antibody proteomics-based biomarker discovery.
abstract: mAb proteomics, a reversed biomarker discovery approach, is a novel methodology to recognize the proteins of biomarker potential, but requires subsequent antigen identification steps. While in case of high-abundant proteins, it generally does not represent a problem, for medium or lower abundant proteins, the identification step requires a large amount of sample to assure the proper amount of antigen for the ID process. In this article, we report on the use of combined chromatographic and precipitation techniques to generate a large set of fractions representing the human plasma proteome, referred to as the Analyte Library, with the goal to use the relevant library fractions for antigen identification in conjunction with mAb proteomics. Starting from 500 mL normal pooled human plasma, this process resulted in 783 fractions with the average protein concentration of 1 mg/mL. First, the serum albumin and immunoglobulins were depleted followed by prefractionation by ammonium sulfate precipitation steps. Each precipitate was then separated by size exclusion chromatography, followed by cation and anion exchange chromatography. The 20 most concentrated ion exchange chromatography fractions were further separated by hydrophobic interaction chromatography. All chromatography and precipitation steps were carefully designed aiming to maintain the native forms of the intact proteins throughout the fractionation process. The separation route of vitamin D-binding protein (an antibody proteomics lead) was followed in all major fractionation levels by dot blot assay in order to identify the library fraction it accumulated in and the identity of the antigen was verified by Western blot.
authors: Horváth Laboratory of Bioseparation Sciences, Research Centre for Molecular Medicine, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary.
authors: András
authors: A
authors: Kovács
authors: Edit
authors: E
authors: Sperling
authors: József
authors: J
authors: Lázár
authors: Attila
authors: A
authors: Balogh
authors: János
authors: J
authors: Kádas
authors: Akos
authors: A
authors: Szekrényes
authors: László
authors: L
authors: Takács
authors: István
authors: I
authors: Kurucz
authors: András
authors: A
authors: Guttman
mesh_terms: D000645:Ammonium Sulfate / Q000737:chemistry; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000737:chemistry*; D015415:Biomarkers / Q000032:analysis* / Q000378:metabolism; D001798:Blood Proteins / Q000032:analysis* / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D015151:Immunoblotting; D020543:Proteome / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*
chemical_list: D000911:Antibodies, Monoclonal; D015415:Biomarkers; D001798:Blood Proteins; D020543:Proteome; D000645:Ammonium Sulfate