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Quantitative assessment of human serum high‐abundance protein depletion
Author(s) -
Stempfer Rene,
Kubicek Markus,
Lang Irene M.,
Christa Noehammer,
Gerner Christopher
Publication year - 2008
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200800211
Subject(s) - polyclonal antibodies , shotgun , immunogenicity , chemistry , proteome , blood proteins , protein purification , chromatography , antibody , proteomics , biology , microbiology and biotechnology , biochemistry , immunology , gene
The aim of this study is to quantify the effectivity of the depletion of human high‐abundance serum and plasma proteins for improved protein identification and disease marker candidate discovery and to assess the risk of concomitant removal of relevant marker proteins. 2‐DE and bottom‐up shotgun MS combining 2‐D capillary chromatography with MS/MS were applied in parallel for the analysis of fractions resulting from the depletion procedure. For many proteins the factors of enrichment by the depletion were obvious allowing their enhanced detection and identification upon high‐abundance protein depletion. Nano‐liquid chromatography linked MS allowed the efficient identification of several low‐abundant proteins that were not identified on the 2‐DE gels. Resolving the fractions that were eluted from the matrix upon depletion indicated unspecific binding of disease relevant proteins in plasma samples from acute myocardial infarction patients. The unspecific binding to the depletion matrix of inflammatory markers spiked into the serum was found to depend on the type of capturing agent used. Polyclonal avian antibodies (IgY) displayed the least unspecific binding due to the high immunogenicity of mammalian proteins in avian hosts.