A rapid, economical, and reproducible method for human serum delipidation and albumin and IgG removal for proteomic analysis.
Serum is a readily available source for diagnostic assays, but the identification of disease-specific serum biomarkers has been impeded by the dominance of human serum albumin (HSA) and immunoglobulin G (IgG) in the serum proteome. Therefore, in order to observe lower-abundance serum proteins, removal or depletion of at least these two proteins is required. However, the depletion method needs to be inexpensive and reproducible. We describe such a protocol that combines delipidation by centrifugation, IgG removal with Protein G Sepharose, and HSA depletion with sodium chloride/ethanol precipitation. The protocol is streamlined to increase reproducibility and is compatible with many proteomic platforms, including two-dimensional gel electrophoresis, and high-performance liquid chromatography either offline or coupled online with a mass spectrometer. The reproducible depletion of lipids, IgG, and HSA permits a higher load of the remaining serum proteins, facilitating the identification of disease biomarkers.
Fu
Qin
Q
Department of Medicine, Johns Hopkins University, Baltimore, MD, USA.
Bovenkamp
Diane E
DE
Van Eyk
Jennifer E
JE
D015415:Biomarkers / Q000097:blood; D001797:Blood Protein Electrophoresis; D001798:Blood Proteins / Q000032:analysis* / Q000302:isolation & purification; D002846:Chromatography, Affinity; D005260:Female; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood / Q000302:isolation & purification*; D008055:Lipids / Q000097:blood / Q000302:isolation & purification; D008297:Male; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D012709:Serum Albumin / Q000302:isolation & purification*
D015415:Biomarkers; D001798:Blood Proteins; D007074:Immunoglobulin G; D008055:Lipids; D012709:Serum Albumin