Depletion of High-Abundance Proteins in Plasma by Immunoaffinity Subtraction for Two-Dimensional Difference Gel Electrophoresis Analysis

Abstract

Blood plasma is believed the most complex human-derived proteome, containing other tissue proteome subsets. Almost all body cells communicate with the plasma, either directly or through tissues or biological fluids, and many of these cells release at least a part of their content into the plasma upon damage or death. A comprehensive, systematic characterization of the plasma proteome in the healthy and diseased states will greatly facilitate the development of biomarkers for early disease detection, clinical diagnosis, and therapy. However, the characterization of human plasma proteome is a very complicated task, owing to the wide dynamic range of concentration that separates the most abundant proteins and the less common ones (10-12 orders of magnitude). The removal of its predominant proteins by affinity chromatography using an FPLC system improves the presence of low-abundance proteins in two-dimensional gel electrophoresis (2DE). The "Multiple Affinity Removal System" (Agilent Technologies) retains albumin, IgG, IgA, haptoglobin, transferrin, and antitrypsin with high specificity and reproducibility. After depletion, we have independently analyzed the flow-through (low-abundance proteins), and the retained fractions, by 2DE (4.0-7.0 pH range). Image analysis of the stained gels revealed that more than 300 spots appeared in the retained fraction and about 1800 spots appeared in the nonretained fraction. This methodology is a valuable tool for clinical proteomics, because its reproducibility allows comparative studies and quantitative analysis by 2DE or two-dimensional differential gel electrophoresis of plasma or sera samples from subjects with different pathological or physiological conditions. In addition, the method allows the comparison of experimental results from different laboratories.

Proteomic Analysis of Human Plasma Proteins by Two-Dimensional Gel Electrophoresis and by Antibody Arrays Following Depletion of High-Abundance Proteins

“…Since, human plasma can be easily obtained, several laboratories have developed various proteomic strategies for the purpose of using plasma proteins as a source for possible disease-related biomarkers [7][8][9][10][11][12][13][14][15]22]. However, the great variety of protein types present, as well as the extended dynamic range of protein concentrations greatly hinders the identification of plasma biomarkers.…”

“…Although these depletion methods have shown good efficiency for albumin and IgG depletion, they often do not completely extract the targeted proteins or alternatively they nonspecifically bind some other plasma proteins. Recently, immunoaffinity methods have been developed to improve both the efficiency and the specificity for depletion of several abundant proteins in addition to albumin and IgGs in plasma or serum [7][8][9][10][11][12][13][14]. These chromatographic affinity methods enable specific depletion of 6-9 of the most abundant plasma proteins.…”

Combinatorial peptide ligand libraries: The conquest of the ‘hidden proteome’ advances at great strides

“…SDS-PAGE, 2-D electrophoresis, SEC, HPLC and the like) in turn followed by MS analysis (although one can go directly to MS if the first steps have been already successful as such in digging into the deep proteome). Immuno-affinity separation of proteins using immunoglobulin G (IgG) or immunoglobulin yolk (IgY) as first reported by Pieper et al [17] had become quite popular and had generated great hopes in biomarker discovery [18,19]. It had been increasingly accepted as the most effective sample preparation process in plasma proteomics studies.…”

Vascular proteomics

“…These data should be evaluated with caution since SELDI-TOF is a controversial technique from which methodological and bioinformatic shortcomings have been reported, supporting the view that SELDI profiling cannot be considered a reliable proteomic approach [114]. We have removed six of the most abundant plasma proteins using the Multiple Affinity Removal System (Agilent Technologies) coupled to FPLC system (AKTA Purifier, Amersham Biosciences) [115], and both, the unretained flow-through fractions (low-abundance proteins) and the retained proteins (highly abundant) of plasma from ACS patients and healthy controls were analyzed by 2-DE. More than 1800 spots were detected by silver staining of the 2-DE of flow-through fractions whereas about 320 could be visualized in the 2-DE containing the retained peak.…”