title: Rat plasma proteomics: effects of abundant protein depletion on proteomic analysis.
abstract: The proteomic analysis of plasma and serum samples represents a formidable challenge due to the presence of a few highly abundant proteins such as albumin and immunoglobulins. Detection of low abundance protein biomarkers requires therefore either the specific depletion of high abundance proteins with immunoaffinity columns and/or optimized protein fractionation methods based on charge, size or hydrophobicity. Here we describe the depletion of seven abundant rat plasma proteins with an immunoaffinity column with coupled antibodies directed against albumin, IgG, transferrin, IgM, haptoglobin, fibrinogen and alpha1-anti-trypsin. The IgY-R7-LC2 (Beckman Coulter) column showed high specificity for the targeted proteins and was able to efficiently remove most of the albumin, IgG and transferrin from rat plasma samples as judged by Western blot analysis. Depleted rat plasma protein samples were analyzed by SELDI-TOF MS, 2D SDS-PAGE and 2D-LC and compared to non-depleted plasma samples as well as to the abundant protein fraction that was eluted from the immunoaffinity column. Analysis of the depleted plasma protein fraction revealed improved signal to noise ratios, regardless of which proteomic method was applied. However, only a small number of new proteins were observed in the depleted protein fraction. Immunoaffinity depletion of abundant plasma proteins results in the significant dilution of the original sample which complicates subsequent analysis. Most proteomic approaches require specialized sample preparation procedures during which significant losses of less abundant proteins and potential biomarkers can occur. Even though abundant protein depletion reduces the dynamic range of the plasma proteome by about 2-3 orders of magnitude, the difference between medium-abundant and low abundant plasma proteins is still in the range of 7-8 orders of magnitude and beyond the dynamic range of current proteomic technologies. Thus, exploring the plasma proteome in greater detail remains a daunting task.
authors.affiliation: Phytonutrients Laboratory, Beltsville Human Nutrition Research Center, Beltsville, MD 20705, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Linke
authors.forename: Sundari
authors.initials: S
authors.lastname: Doraiswamy
authors.forename: Earl H
authors.initials: EH
authors.lastname: Harrison
mesh_terms: D000818:Animals; D001798:Blood Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D015153:Blotting, Western; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D006242:Haptoglobins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D007074:Immunoglobulin G / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D007075:Immunoglobulin M / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods*; D051381:Rats; D015203:Reproducibility of Results; D012709:Serum Albumin / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014168:Transferrin / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D000515:alpha 1-Antitrypsin / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D001798:Blood Proteins; D006242:Haptoglobins; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D012709:Serum Albumin; D014168:Transferrin; D000515:alpha 1-Antitrypsin; D005340:Fibrinogen