Depletion of high-abundance proteins from human plasma using a combination of an affinity and pseudo-affinity column (original) (raw)
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Pseudospecific magnetic affinity beads for immunoglobulin-G depletion from human serum
Journal of Applied Polymer Science, 2007
Magnetic poly(ethylene glycol dimethacrylate-N-methacryloyl-(L)-histidine methyl ester) [m-poly(EGDMA-MAH)] beads were prepared by suspension polymerization for the affinity depletion of immunoglobulin-G (IgG) from human serum in a batch system. Elemental analysis of the magnetic beads for nitrogen was estimated as 70 μmol MAH/g polymer. IgG adsorption onto the m-poly(EGDMA) was negligible. Higher adsorption value (up to 46.8 mg/g) was obtained in which the m-poly(EGDMA-MAH) beads were used. IgG adsorption capacity of the magnetic beads increased with an increase in the concentration of IgG. The maximum IgG adsorption was observed at pH 6.5 for MOPS buffer. IgG molecules could be repeatedly adsorbed and eluted with these adsorbents, without noticeable loss in their IgG adsorption capacity. Adsorption capacity decreased for both increasing salt concentration and temperature. In this study, we show that m-poly(EGDMA-MAH) beads (wherein IgG molecules bind directly with the matrix) can be used directly for affinity depletion without further modification. Higher adsorption value was obtained from human serum (up to 85.7 mg/g). The elution results demonstrated that the adsorption of IgG to the adsorbent was reversible. The depletion efficiencies for IgG were above 85% for all studied concentrations. Eluted portion was analyzed for testing the IgG removal efficiency by two dimensional gel electrophoresis. Eluted proteins include mainly IgG, and a small number of nonalbumin proteins such as apolipoprotein A1, serotransferrin, haptoglobulin, and α1-antitrypsin. IgA was not identified in eluted fraction. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2007
Biotechnology and Applied Biochemistry, 2010
High-abundant plasma proteins pose a challenge in a large number of proteomics-based technologies. Depletion of these high-abundant proteins has proven to be a fruitful strategy to circumvent masking of lowerabundant proteins that could serve as valuable biomarker candidates. However, current strategies often do not meet the throughput requirements of largescale proteomic studies. In the present paper, a flexible and parallelized method for the depletion of highabundant proteins is described, allowing the removal of the two most abundant proteins from 48 blood-derived samples in less than 15 min using Affibody molecules as affinity ligands. A sample-processing platform like this should be suitable for a number of proteomics technologies; its flexibility in buffer composition allows for different types of downstream applications.
Analytical Letters, 2007
An in-tandem study comprising two affinity chromatographic columns, Blue Avidgel P (for the selective removal of albumin) and an aza-arenophilic gel (Gel A) containing a nitrogen capping nucleophile (for the selective removal of IgG), was conducted to obtain high purity IgG from serum. The results demonstrated that highly pure IgG (.97%) can be obtained from the phenethylamine capped aza-arenophilic gel column after the serum was passed through the Blue Avidgel P column in contrast to the 38% IgG purity obtained when the serum was passed through a Gel A column alone under similar conditions. Further studies show that IgG selectivity is not dependent on the nature of the support material used in the synthesis of these aza-arenophilic gels since the synthesis of aza-arenophilic gels using either Sepharose or Fractogel yield similar results.
A method for the rapid depletion of albumin and immunoglobulin from human plasma
PROTEOMICS, 2004
In studies of the plasma proteome, the high abundance of proteins such as albumin and immunoglobulin impedes the investigation of lower abundance proteins that may be more suitable as biomarkers of disease. We report the specific removal of 98% of albumin and 80% of immunoglobulin heavy chain from human plasma by affinity chromatography, and the subsequent improvement in the number of spots detected and their resolution following two-dimensional gel electrophoresis.
Journal of Applied Polymer Science, 2010
Fractionation of human plasma on ion exchanger resin was performed on Amberlite IRC-718 saturated with metal ions. Depletion of human immunoglobulin G was carried out by column chromatography using Tris-HCl, pH 7 at different concentrations. Results showed that, when Cu þ2 and Ni þ2 were adsorbed on the resin, one or two fractions of purified IgG were obtained, respectively. Whereas Fe þ2 and Zn þ2 , both retain IgG and serum albumin or serum albumin alone. Furthermore, the Ni þ2resin retention of serum proteins is too strong that the use of 700 mMTris-HCl cannot liberate any other proteins than nonadsorbed serum albumin. In conclusion, this investigation demonstrates that immobilized metal ion affinity chromatography with Cu 2þ , Ni 2þ , and Fe 2þ immobilized on Amberlite IRC-718 has the potential to be developed as part of a process to purify IgG out of untreated human plasma as acceptable adsorption and elution levels of IgG could be achieved.
2009
High-abundant plasma proteins pose a challenge in a large number of proteomics-based technologies. Depletion of these high-abundant proteins has proven to be a fruitful strategy to circumvent masking of lowerabundant proteins that could serve as valuable biomarker candidates. However, current strategies often do not meet the throughput requirements of largescale proteomic studies. In the present paper, a flexible and parallelized method for the depletion of highabundant proteins is described, allowing the removal of the two most abundant proteins from 48 blood-derived samples in less than 15 min using Affibody molecules as affinity ligands. A sample-processing platform like this should be suitable for a number of proteomics technologies; its flexibility in buffer composition allows for different types of downstream applications.
Biomedical Chromatography, 2009
In plasma proteomics, before a proteome analysis, it is essential to prepare protein samples without high-abundance proteins, including albumin, via specific preparation techniques, such as immunoaffinity capture. However, our preliminary experiments suggested that functional changes with use alter the ability of the immunoaffinity column. Thus, in this study, to evaluate the changes of the removal ability of abundant proteins from plasma by the immunoaffinity column, plasma proteome analysis was performed for the long-term test for the reproducibility of the affinity column using the fluorogenic derivatizationliquid chromatography-tandem mass spectrometry method combined with an IgY column. The specific adsorption for albumin decreased with an increase in the number of the column usage before its expiration date. Moreover, it was demonstrated that hydrophobic high molecular weight compounds in plasma adsorbed onto the column materials surface contributed to the functional changes from specific immunoaffinity adsorption into hydrophobic interaction. These results suggested that, in quantitative plasma proteomics studies, it is important to keep in mind the risk of not only the nonselective loss but also the changes in the adsorption ability of the immunoafinity column.
Journal of Chromatography A, 2007
A butyl methacrylate-co-ethylene dimethacrylate (BuMA-co-EDMA) monolith was synthesized by UV initiated polymerization at the inlet end of a 75 m I.D. fused silica capillary that had been previously coated with a protein compatible polymer, poly(vinyl)alcohol. The monolith was used for on-line preconcentration of proteins followed by capillary electrophoresis (CE) separation. For the analysis of standard proteins (cytochrome c, lysozyme and trypsinogen A) this system proved reproducible. The run-to-run %RSD values for migration time and corrected peak area were less than 5%, which is typical of CE. As measured by frontal analysis using lysozyme as solute, saturation of a 1 cm monolith was reached after loading 48 ng of protein. Finally, the BuMA-co-EDMA monolithic preconcentrator was coupled to a protein G monolithic column via a zero dead volume union. The coupled system was used for on-line removal of IgG, preconcentration of standard proteins and CE separation. This system could be a valuable sample preparation tool for the analysis of low abundance proteins in complex samples such as human serum, in which high abundance proteins, e.g., human serum albumin (HSA) and immunoglobulin G (IgG), hinder identification and quantification of low abundance proteins. Published by Elsevier B.V.