A robust, streamlined, and reproducible method for proteomic analysis of serum by delipidation, albumin and IgG depletion, and two-dimensional gel electrophoresis.
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 and immunoglobulins (Igs) in the serum proteome. There is a need to reduce the technical variation in serum processing and analysis to allow for a reproducible analysis of large cohorts. To this end, we have developed a rapid and reproducible procedure for sample preparation and high-resolution two-dimensional gel electrophoresis to analyze human serum. Serum is centrifuged at high speed to remove lipids and aggregated proteins, incubated with protein G resin to remove IgG, precipitated with NaCl/ethanol to deplete albumin, and slowly resolubilized in a sodium dodecyl sulfate (SDS)/N-(2-hydroxyethyl)piperazine-2'-(2-ethanesulfonic acid) (HEPES) buffer. The delipidated and IgG/albumin depleted serum proteins are focused on pH 4-7 linear large immobilized pH gradient strips, and then resolved by Bis-Tris SDS-polyacrylamide gel electrophoresis. The robustness and reproducibility of the optimized procedure was determined for three individual serum samples on three consecutive days. An image analysis of the nine silver-stained gels demonstrated that the intensity and localization of protein spots are highly reproducible. Our IgG and albumin depletion procedure will aid in screening the patient sera for normal biological variation and disease-specific biomarkers.
Fu
Qin
Q
Department of Medicine, Johns Hopkins University, Baltimore, MD, USA.
Garnham
Christopher P
CP
Elliott
Steven T
ST
Bovenkamp
Diane E
DE
Van Eyk
Jennifer E
JE
D001798:Blood Proteins / Q000737:chemistry / Q000302:isolation & purification*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007074:Immunoglobulin G / Q000097:blood* / Q000302:isolation & purification*; D008055:Lipids / Q000097:blood* / Q000302:isolation & purification; D020543:Proteome; D015203:Reproducibility of Results; D012709:Serum Albumin / Q000302:isolation & purification*
D001798:Blood Proteins; D007074:Immunoglobulin G; D008055:Lipids; D020543:Proteome; D012709:Serum Albumin