Multi‐component immunoaffinity subtraction chromatography: An innovative step towards a comprehensive survey of the human plasma proteome

Abstract

In order to discover novel protein markers indicative of disease processes or drug effects, the proteomics technology platform most commonly used consists of high resolution protein separation by two-dimensional electrophoresis (2-DE), mass spectrometric identification of proteins from stained gel spots and a bioinformatic data analysis process supported by statistics. This approach has been more successful in profiling proteins and their disease- or treatment-related quantitative changes in tissue homogenates than in plasma samples. Plasma protein display and quantitation suffer from several disadvantages: very high abundance of a few proteins; high heterogeneity of many proteins resulting in long charge trains; crowding of 2-DE separated protein spots in the molecular mass range between 45-80 kD and in the isoelectric point range between 4.5 and 6. Therefore, proteomic technologies are needed that address these problems and particularly allow accurate quantitation of a larger number of less abundant proteins in plasma and other body fluids. The immunoaffinity-based protein subtraction chromatography (IASC) described here removes multiple proteins present in plasma and serum in high concentrations effectively and reproducibly. Applying IASC as an upfront plasma sample preparation process for 2-DE, the protein spot pattern observed in gels changes dramatically and at least 350 additional lower abundance proteins are visualized. Affinity-purified polyclonal antibodies (pAbs) are the immunoaffinity reagents used to specifically remove the abundant proteins such as albumin, immunoglobulin G, immunoglobulin A, transferrin, haptoglobin, alpha-1-antitrypsin, hemopexin, transthyretin, alpha-2-HS glycoprotein, alpha-1-acid glycoprotein, alpha-2-macroglobulin and fibrinogen from human plasma samples. To render the immunoaffinity subtraction procedure recyclable, the pAbs are immobilized and cross-linked on chromatographic matrices. Antibody-coupled matrices specific for one protein each can be pooled to form mixed-bed IASC columns. We show that up to ten affinity-bound plasma proteins with similar solubility characteristics are eluted from a mixed-bed column in one step. This facilitates automated chromatographic processing of plasma samples in high throughput, which is desirable in proteomic disease marker discovery projects.

Quantitative analysis of the low molecular weight serum proteome using 18O stable isotope labeling in a lung tumor xenograft mouse model

“…Logically, depletion of one or more of the high abundance, high molecular weight proteins would assist the detection and identification of low abundance species. In an effort to decrease this dynamic range of protein concentration, numerous affinity purification and extraction methods have been developed to enrich the smaller, less-abundant proteins and peptides in complex biofluids [9,10]. Recently, a simple methodology was developed to deplete serum of high molecular weight proteins by size partitioning through the use of centrifugal ultrafilters [2].…”

Nanotechnologies in proteomics

“…An alternative procedure to 2-DE separation of proteins is high-resolution chromatography [12]. Compared to 2-DE, high-resolution chromatography is more convenient for analysis: it is automatable and is capable of removing highabundance proteins from a mixture for the subsequent revelation of low-abundance proteins -which are then subjected to 2-DE procedure [25]. For direct identification of proteins in complex mixtures, a chromatographic column is coupled to mass spectrometer.…”

Setup for human sera MALDI profiling: The case of rhEPO treatment

“…Moreover, the high cost, the scarce selectivity, or long operation times, impose the development of new depletion systems based on multiple affinity columns [30,31]. The latter are characterized by the presence of specific antibodies derivatized columns which specifically and selectively deplete a number of high-abundant proteins in serum plus their proteolytic products and molecular forms [32][33][34][35][36][37]. The selection of a methodological approach providing optimal reduction of the serum dynamic range with high reproducibility represents a critical point for the translation of MALDI serum profiling to clinical laboratories.…”