Fractionation of the human plasma proteome for monoclonal antibody proteomics-based biomarker discovery

András Kovács, Edit Sperling, József Lázár, Attila Balogh, János Kádas, Ákos Szekrényes, László Takács, István Kurucz, András Guttman

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12 Citations (Scopus)

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 500mL normal pooled human plasma, this process resulted in 783 fractions with the average protein concentration of 1mg/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 114226918 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.

Original languageEnglish
Pages (from-to)1916-1925
Number of pages10
JournalELECTROPHORESIS
Volume32
Issue number15
DOIs
Publication statusPublished - Aug 1 2011

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Keywords

  • Chromatography
  • Dot blot screening
  • Fractionation
  • Human plasma
  • MAb proteomics

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry

Cite this

Kovács, A., Sperling, E., Lázár, J., Balogh, A., Kádas, J., Szekrényes, Á., Takács, L., Kurucz, I., & Guttman, A. (2011). Fractionation of the human plasma proteome for monoclonal antibody proteomics-based biomarker discovery. ELECTROPHORESIS, 32(15), 1916-1925. https://doi.org/10.1002/elps.201100018