Department of Biochemistry and Molecular Pharmacology, Thomas Jefferson University, Jefferson Center for Biomedical Research, Doylestown, Pennsylvania 18901
¶ Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111
|| Isis Innovation, Ewert House, Ewert Place, Summertown, Oxford OX2 7SG, United Kingdom
** Daniel Baugh Institute for Functional Genomics/Computational Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
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ABSTRACT |
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Serum presents some unique challenges to efforts to characterize its protein content by any single method (for review, see Ref. 1). Notably, different protein species are present in serum over a very wide range of abundance. A small number of highly abundant proteins such as albumin, immunoglobulins, 1-antitrypsin, transferrin, and haptoglobins are present in concentrations in the milligram to tens of milligrams per milliliter range and together account for as much as 80% of the total serum protein. A larger number of proteins, including many that are, or could be, diagnostically significant, are present at far lower concentrations. Removal of abundant serum proteins will help in the discovery and detection of less abundant proteins that may prove to be informative disease markers.
Over the last several decades, attempts have been made to remove albumin, the single most abundant serum protein, from serum (or plasma) and other body fluids, often by methods based on the high affinity of albumin for certain blue textile dyes (24). These methods can be effective in removing albumin, although they suffer from a lack of specificity as many serum proteins in addition to albumin will bind to the dye-based resins. Other methods reported include a proprietary polypeptide affinity matrix that removes albumin together with IgG, but is now apparently unavailable (5), and a method based on size separation in a centrifugal filtration device that was, perhaps predictably, unsuccessful (6).
In the work reported here, we have investigated the efficacy of an immunoaffinity resin for improving the specificity and efficiency of removing albumin from human serum samples. The use of immunoaffinity resins for the selective removal or purification of proteins from solution is well established. However, their application to the problem of albumin removal is not entirely straightforward, primarily due to the enormous load of this single protein species in serum and plasma. We show that by using high affinity antibodies heavily loaded on a beaded resin support, albumin and a large number of albumin fragments can be essentially quantitatively removed from human serum samples. Furthermore, when a protein G affinity resin is used together with the immunoaffinity resin, IgG and human serum albumin (HSA)1 are simultaneously removed from the samples. Similar methods could be applied to the removal of additional abundant proteins.
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EXPERIMENTAL PROCEDURES |
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Preparation of Antibody Affinity Resin
Antibodies were coupled to N-hydroxysuccinimide-activated Sepharose 4 Fast Flow resin (Amersham Biosciences) at a concentration of 15 mg antibody per milliliter resin, following procedures recommended by the manufacturer. After antibody coupling, the resin was washed first with 0.1 M NaCl, 0.1 M glycine, pH 2.8, then with 0.1 M NaCl, 0.1 M glycine, pH 9.0, and finally with 0.01 M Tris, pH 8.0. The resin was equilibrated with Tris-buffered saline/Tween-20 (TBS/T; 0.02 M Tris, 0.15 M NaCl, pH 7.6 with 0.1% Tween-20) prior to use or storage at 4 °C.
Removal of HSA from Serum Samples
Antibody Affinity ResinHuman serum was diluted into one column volume or less of TBS/T and applied to immunoaffinity resin that had been equilibrated in the same buffer. Proteins were allowed to bind while mixing gently for 3060 min at room temperature. Unbound proteins were recovered from the resin by gravity flow in a standard column format (PolyPrep column; Bio-Rad) and then immediately reapplied to the column. This step was repeated so that unbound proteins were collected and immediately reapplied a total of four times. The column was then washed five times, each time with a single column volume of TBS/T, and all washes were combined as the unbound fraction of proteins. Bound proteins were eluted from the column with a total of five washes, each with a single column volume of 0.1 M NaCl, 0.1 M glycine, pH 2.8. Eluted fractions were neutralized by the addition of 0.05 volume of 1 M Tris, pH 8.0, and combined. Proteins from both the unbound and eluted fractions were concentrated by precipitation with 5 volumes of acetone.
SwellGel BlueAlbumin was removed from human serum samples using a SwellGel Blue Albumin Removal Kit (Pierce) according to the manufacturers instructions. Samples containing 650 µg of total protein (260 µg albumin) were loaded onto a single removal disc, where each disc is reported to have a binding capacity of >2 mg of albumin.
Addition of Protein G Resin to Immunoaffinity ResinFor the removal of both HSA and IgG in a single column, 0.1 column volume of protein G immobilized on Sepharose 4B Fast Flow (Sigma) was added to the immunoaffinity resin. Columns were washed, run, and regenerated exactly as for the immunoaffinity resin alone.
Two-dimensional Gel Electrophoresis (2DE)
2DE was carried out as described previously (8) with minor modifications. Acetone precipitated proteins were redissolved in sample buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 65 mM dithiothreitol, 5 mM tributylphosphine, and 0.4% ampholytes (Servalyt 3-10). Proteins were focused in an 18-cm pH 3-10NL immobilized pH gradient strip (Amersham Biosciences) after rehydration at 50 V for 14 h. Gel strips were equilibrated in 6 M urea, 2% SDS, 1.5% dithiothreitol, 30% glycerol, 50 mM Tris, pH 8.8, for 10 min followed by incubation in the same solution, but replacing dithiothreitol with 3% iodoacetamide, for an additional 10 min. The gel strips were placed on an 818% polyacrylamide gel for resolution in the second dimension. Gels were stained with silver (9) or colloidal Coomassie blue (10) and imaged with a FluorChem 16-bit charge-coupled device camera (Alpha Innotech, San Leandro, CA).
For immunoblotting, proteins were transferred to a polyvinylidene difluoride membrane and the blots were stained with SyproRuby protein blot stain (Bio-Rad). Blots were probed with goat anti-HSA (Fitzgerald Industries, Concord, MA) and developed using horseradish peroxidase-conjugated anti-goat antibody (Sigma) and SuperSignal West Pico detection reagents (Pierce).
Peptide Mass Fingerprint Analysis of Proteins
Protein spots excised from colloidal Coomassie blue-stained two-dimensional (2D) gels were destained, reduced, and alkylated and then digested with trypsin (11). Peptides were further treated by elution from ZipTip-C18 reversed phase pipette tips (Millipore). Recovered peptides were prepared for MALDI-TOF mass spectrometry by mixing with alpha-cyano-4-hydroxy cinnamic acid, 1% formic acid in 50% acetonitrile, and droplets were allowed to dry on the MALDI sample plate. Peptide mass maps were obtained using a Voyager DE (Applied Biosystems, Foster City, CA) MALDI-TOF mass spectrometer operated in positive ion reflectron mode. Proteins were identified from the peptide mass maps using MASCOT (Matrix Science, London, UK) to search the nonredundant protein data base (12).
Antibody Array
The following mouse monoclonal antibodies were used for capture: anti-HSA (Research Diagnostics, Flanders, NJ), anti-hepatitis B surface antigen (HBsAg; Fitzgerald Industries), anti-alphafetoprotein (AFP; U.S. Biological), and anti-hepatitis B e antigen (HBeAg; Fitzgerald Industries). Antibodies were diluted to a concentration of 0.5 mg/ml in PBS plus 0.02% Tween-20 and spotted onto Hydrogel slides (PerkinElmer Life Sciences) with a MicroGrid TAS (BioRobotics, Cambridge, UK) robotic spotter using MicroSpot 2500 pins. Slides were incubated in a humid chamber overnight and then washed briefly in TBS/T before blocking for 2 h in TBS/T plus 1% gelatin (Bio-Rad). Slides were rinsed briefly in TBS/T and then incubated with antigen solution (see below) for 2 h at room temperature. Slides were washed three times for 20 min in TBS/T and then incubated for 2 h at room temperature with antibodies that had been labeled with Cy3 fluorescent dye (Amersham Biosciences) according to manufacturers instructions. Antibodies used for detection were goat polyclonal anti-human AFP, anti-HBsAg, and anti-HSA (Fitzgerald Industries). Slides were again washed with TBS/T and then imaged using a ScanArray 5000XL scanner (PerkinElmer Life Sciences).
Antigen solution was prepared by adding purified AFP (Fitzgerald Industries) and HBsAg (Chemicon International, Temecula, CA) to human serum at a concentration of 1000 ng/ml each. The spiked serum samples were diluted into TBS/T and fractionated on immunoaffinity resin as described above. Proteins recovered in flow-through and eluted fractions were concentrated by acetone precipitation and resuspended in TBS/T. Unfractionated, spiked samples were similarly diluted, acetone precipitated, and redissolved in TBS/T. Antibody microarrays were incubated with the antigen solution at a concentration equivalent to a 1:10 dilution of the starting serum sample, so that AFP and HBsAg would each be present at a maximum of 100 ng/ml in any fraction.
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RESULTS |
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Hybridomas were prepared from the spleens of mice immunized with full-length HSA, as described in "Experimental Procedures." Supernatants from individual hybridoma clones were first tested for reactivity against full-length HSA by ELISA. In order to identify hybridomas producing antibodies reactive against different HSA epitopes, clones showing strong reactivity to the full-length molecule were further tested against recombinant His-tagged proteins representing each of the three major domains of HSA (see Fig. 1). Antibodies against epitopes in two different domains (I and II) were chosen for large scale preparation and purification. These two antibodies were covalently attached to Sepharose resin to prepare an immunoaffinity reagent, as described in "Experimental Procedures."
Efficiency and Specificity of HSA Removal from Serum
To test the efficiency of HSA removal from human serum, 10 µl of serum, containing 700 µg of total protein and an estimated 400 µg of HSA, was incubated with 0.25 ml of the antibody affinity resin in a column format (see "Experimental Procedures"). Proteins that did not bind to the column (the flow-through fraction) and proteins specifically bound by the resin and then eluted (the eluted fraction) were prepared and examined by 2DE. The abundant HSA protein is evident as a large, poorly resolved and clearly overloaded spot when total serum proteins are displayed in these 2D gels (Fig. 2a). After separation of serum proteins on the affinity column, the flow-through fraction shows essentially no remaining HSA (Fig. 2b) while the HSA is recovered in the fraction eluted from the column (Fig. 2c).
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While HSA is by far the most abundant protein seen by 2DE of proteins that bind to and are eluted from the column, many other "spots" are present in the gel (Fig. 2c). A large number of these have been shown to be albumin or albumin-related fragments by two methods. First, eluted proteins separated by 2DE were transferred to polyvinylidene difluoride membranes for detection by immunoblotting using a polyclonal antibody against HSA. Almost all spots visualized by staining of the blot were also detected by reaction with the anti-HSA antibody, as shown in Fig. 3. This suggests that proteins (or protein fragments) that bind to the column are doing so by specific interactions with the antibody, rather than by nonspecific binding to the resin substrate or to HSA itself.
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Improvements in the Resolution and Detection of Serum Proteins in 2D Gels after HSA Removal
Small regions of the 2D gels shown in Fig. 2, a and b, are enlarged in Fig. 5 to demonstrate the improvement in the data available from 2D gels, even in the absence of significant overloading of the proteins that remain after albumin depletion. Many of the abundant proteins that were obscured by albumin are now visible (compare resolution in upper left of each image). Less abundant proteins that were lost in the confusion of albumin fragments become distinct and are now available for analysis (see spots indicated by arrows). Minor proteins that were previously hidden by comigration with albumin fragments or smears can now be resolved (see boxed region and three-dimensional depictions of that region below each image). It is evident that higher protein loads are now possible so that proteins of this and even somewhat lower abundance classes can be detected and analyzed without overwhelming the gel with albumin and its fragments. We expect that similar improvements will be realized using alternative fractionation technologies, such as liquid chromatography linked to mass spectrometry, for the analysis of serum proteins.
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Ten microliters of serum, containing 400 µg of HSA, was loaded onto SwellGel Blue resin with a capacity of >2 mg of HSA, as stated by the manufacturer. The column was processed according to the manufacturers instructions, and proteins in the flow-through and eluted fractions were analyzed by 2DE, as before. Results, shown in Fig. 6, indicate that a large portion of the HSA was not bound by the resin. Proteins that bound to and were eluted from the resin include albumin, many albumin fragments, and a large number of nonalbumin proteins. In particular, significant amounts of relatively abundant serum proteins such as Ig heavy and light chains, apolipoprotein A1, serotransferrin, haptoglobin, and
1-antitrypsin were bound by the column. One-dimensional gel analysis of several sequential wash and elution fractions suggested that this was not the result of incomplete washing during the initial binding steps, but instead represents proteins, in addition to HSA, that bind to the column under the recommended conditions (data not shown). We conclude that immunoaffinity reagents are far superior to the dye-based resin in terms of both the efficiency and specificity of albumin removal.
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Human serum was spiked with purified AFP and HBsAg, each at a concentration of 1000 ng/ml serum. The sample was then treated to remove HSA using the immunoaffinity resin as described above. The total spiked sample (prior to HSA removal), the immunoaffinity column flow-through fraction, and proteins eluted after binding to the column were all assayed by an antibody microarray for the presence of HSA, AFP, and HBsAg. The array also contained antibody to HBeAg (anti-HBeAg) as a negative control. As shown in Fig. 7a, this method easily detected HSA, AFP, and HBsAg in the unfractionated, total sample. Both AFP and HBsAg are present in the flow-through fraction (Fig. 7b) but are undetectable above background in the eluted fraction (Fig. 7c). As expected, HSA is found in the eluted fraction, but is undetectable above background in the flow-through fraction (Fig. 7, b and c). These results provide further evidence of the high degree of specificity that is possible when using an immunoaffinity reagent for the removal of albumin from serum samples.
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Simultaneous Removal of HSA and Immunoglobulin Chains
Removal of additional abundant proteins can be beneficial in the analysis of serum proteins, and therefore we attempted to remove albumin together with the IgG class of immunoglobulins. These proteins can account for 20% of total serum proteins and are present at a concentration of
816 mg/ml (17). To remove IgG, we took advantage of its well-known affinity for protein G. Protein G resin was added to the HSA immunoaffinity resin, and serum samples were processed as above. Fig. 8 shows a 2D gel of serum where both HSA and IgG have been removed. Very small amounts of IgG heavy chains remain in the flow-through fraction, and a more specific affinity reagent (e.g. anti-IgG antibody) might be required for their complete removal. Because protein G binds IgG, but has only a low affinity for other classes of human immunoglobulins, we expect that the light chain proteins that remain in the sample are derived primarily from these other immunoglobulin classes. Again, an affinity reagent with specificity for light chains (e.g. protein L or anti-light chain antibodies) could be used to achieve a more complete removal, if necessary. It was possible that protein G on the resin would interact with the densely loaded anti-HSA immunoaffinity resin when the two were used together in the same column, thereby reducing binding capacity. However, we saw no difference in binding capacity when the two resins were used together in the same column, or sequentially in separate columns (data not shown). Therefore, HSA and most IgG proteins are readily removed from serum samples in a single column.
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DISCUSSION |
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Removal of albumin from serum samples is problematic because of its extremely high concentration. A high specificity and high capacity resin is required. While Cibacron Blue and related dyes have been shown to bind albumin with high affinity (2), they also bind many other abundant serum proteins (3). Dye-based resins that have been optimized for albumin binding are commercially available, but our results suggest that they still lack sufficient specificity. The need to compromise between specificity and capacity may account for the failure of the product we tested to perform well in either area.
Antibodies are an obvious choice as affinity reagents with the specificity required for selective removal of any single protein species. In the case of albumin removal, however, the quantity of antibody needed for essentially complete depletion of albumin, even from microliter quantities of plasma or serum, makes the use of antibodies problematic. To maximize the value of the antibodies used in our resin, we selected individual monoclonal antibodies that each react with high affinity to a unique epitope on the albumin molecule. This not only provides a renewable antibody source, but also is a reagent that can benefit from cooperative binding to different regions of the protein. By using antibodies reactive against different regions of the molecule, we are able to capture a large number of the albumin fragments that are present in serum samples. While our resin was effective when made with only one (not shown) or two of our antibodies, it is possible that further efficiencies could be achieved with additional antibodies.
Several modifications to our procedure that could improve its applicability in different circumstances are possible. For instance, the use of formats such as multi-well plates that are suitable for high throughput will be explored. The use of antibody fragments or single-chain antibodies derived from the anti-HSA monoclonals might be beneficial if they can be produced less expensively or loaded onto substrate at higher concentrations.
In the ideal, it would be best to use a nonproteinaceous surface or resin for the specific removal of proteins from any sample. This would eliminate the potential for contaminating the sample with protein leached from the resin and would likely be a more stable and less expensive product. To date, however, no affinity resin that can match the specificity and binding capacity of monoclonal antibodies has been discovered. Covalent binding of antibody to the resin has minimized the leaching problem, and the ability to reuse the resin helps to keep the cost reasonable for research purposes.
Our research involves the examination of proteins in human serum samples by 2DE and antibody microarrays, and both of these techniques benefit from the removal of the bulk of the albumin and IgG. Removal of highly abundant serum proteins improves the detection of less abundant proteins by 2DE, as well as the display of proteins that comigrate and are otherwise obscured by HSA or IgG. The removal of the large and poorly resolved spots created by these highly abundant proteins, and the numerous smaller spots that represent albumin fragments, also helps in computer-assisted gel analysis, particularly with spot delineation, quantitation, and gel-to-gel matching. We expect that many of the new technologies being developed for protein profiling will benefit in similar ways by the improvements in sensitivity and reduction of background that result from the removal of a small number of very abundant proteins from samples prior to analysis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Published, MCP Papers in Press, May 16, 2003, DOI 10.1074/mcp.M300026-MCP200
1 The abbreviations used are: HSA, human serum albumin; 2DE, two-dimensional gel electrophoresis; AFP, alpha fetoprotein; HBsAg, hepatitis B surface antigen; HBeAg, hepatitis B e antigen; ELISA, enzyme-linked immunosorbent assay; 2D, two dimensional; TBS/T, Tris-buffered saline/Tween-20; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight mass spectrometry.
* This work was supported by the National Cancer Institute, Early Detection Research Network, and by an appropriation from the Commonwealth of Pennsylvania. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Department of Biochemistry and Molecular Pharmacology, Thomas Jefferson University, Jefferson Center for Biomedical Research, Doylestown, PA 18901. Tel.: 215-489-4946; Fax: 215-489-4920; E-mail: laura.steel{at}jefferson.edu
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REFERENCES |
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