Enrichment of low‐abundant serum proteins by albumin/immunoglobulin G immunoaffinity depletion under partly denaturing conditions

Abstract

We present a simple protocol for affinity depletion to remove the two most abundant serum proteins, albumin and immunoglobulin G (IgG). Under native conditions, albumin/IgG were efficiently removed and several proteins were enriched as shown by two-dimensional electrophoresis (2-DE). Besides that, partly denaturing conditions were established by adding 5 or 20% acetonitrile (ACN) in order to disrupt the binding of low-molecular-weight (LMW) proteins to the carrier proteins albumin/IgG. 2-DE results showed that the total number of detected LMW proteins increased under denaturing conditions when compared to native conditions. Interestingly, the presence of 5% ACN in serum revealed better enrichment of LMW proteins when compared to 20% ACN condition. Seven randomly distributed spots in albumin/IgG depleted serum samples under 5% ACN condition were picked from the 2-DE gels and identified by mass spectrometry (MS). The intensity of five LMW protein spots increased under denaturing conditions when compared to native conditions. Three of the seven identified spots (serum amyloid P, vitamin D-binding protein, and transthyretin) belong to a group of relatively low-abundant proteins, which make up only 1% of all serum proteins. The method presented here improves the resolution of the serum proteome by increasing the number of visualized spots on 2-D gels and allowing the detection and MS identification of LMW proteins and proteins of lower abundance.

Human body fluid proteome analysis

“…Immunodepletion can be effectively realized with IgY antibody microbeads or peptidebased affinity medium [23,24]. Protein precipitation with TCA/acetone or NaCl/ethanol also appears to be useful for depletion of albumins [25,26].…”

“…For instance, partly denaturing conditions by adding 5 or 20% ACN may disrupt the binding between LMW and albumin/IgG, resulting in increased number of proteins detected under denaturing conditions when compared to native conditions. The presence of 5% ACN in serum provided better enrichment of LMW proteins compared to 20% ACN condition [26]. In addition, rather than depletion of highly abundant proteins, it would be promising to develop methods such as functionalized nanoparticles [29] to enrich the low abundant proteins/peptides or reduce the protein concentration range [30].…”

Two‐dimensional gel electrophoresis as tool for proteomics studies in combination with protein identification by mass spectrometry

“…to isolate and map phosphorylation sites of proteins via immobilized metal affinity chromatography [41] or by antibody column chromatography. This technique is employed to deplete serum samples from their major protein constituents, such as albumin, immunoglobulins, transferrin, haptoglobin, or lipoproteins [42]. Removal of many of these major proteins from serum samples makes it possible to identify relatively low abundant proteins [43,44].…”

Current Insights in Mammalian Glycosylation: Implication of Glycoproteomics as Next-Generation Biomarkers in Diabetes Mellitus

“…Table 1 showed various enrichment techniques of glycoproteins. Since, various biological samples of serum, plasma constitute mixtures of glycoproteins, therefore, extensive chromatographic separations is required that uses ionexchange, affinity column separation, size exclusion and hydrophobic separation to minimize the complexity of proteins and enhance accuracy [27]. Recently develops a bottom-up approach has been developed for analysis of glycoprotein [28].…”