Efficient and specific removal of albumin from human serum samples

Patient serum or plasma is frequently monitored for biochemical markers of disease or physiological status. Many of the rapidly evolving technologies of proteome analysis are being used to find additional clinically informative protein markers. The unusually high abundance of albumin in serum can interfere with the resolution and sensitivity of many proteome profiling techniques. We have used monoclonal antibodies against human serum albumin (HSA) to develop an immunoaffinity resin that is effective in the removal of both full-length HSA and many of the HSA fragments present in serum. This resin shows markedly better performance than dye-based resins in terms of both the efficiency and specificity of albumin removal. Immunoglobulins are another class of highly abundant serum protein. When protein G resin is used together with our immunoaffinity resin, Ig proteins and HSA can be removed in a single step. This strategy could be extended to the removal of any protein for which specific antibodies or affinity reagents are available.     

Cibacron Blue and proteomics: the mystery of the platoon missing in action

The use of Cibacron Blue columns (HiTrapBlue) in proteome analysis for removal of plasma albumin, for facilitating biomarker discovery, has not borne any fruit. In fact, the visibility of low-abundance proteins was obscured. It is here reported that, upon albumin sequestering from plasma, there is adsorption, via hydrophobic interaction, of a substantial number of plasma proteins, which are lost for subsequent analysis if the blue resin is eluted via an ion shock (2 M NaCl) or with a somewhat more robust eluant (5 M urea, 2 M thiourea, 2% CHAPS, 2% sulphobetain 3-10) as recommended by manufacturers. Such treatments, in fact, release at most 25 to 30 unique gene products, including albumin. If, however, the Affigel-Blue resin, after elution with either of the two above eluants, is further eluted with boiling 4% SDS in 25 mM DTT, all the missing proteins (amounting to at least 112 unique species) are desorbed and biomarker analysis can be conducted in a correct way. It is also suggested that such blue-resin treatment could be coupled to ProteoMiner adsorption, this coupled treatment further enhancing the chances of success for discovery of low-abundance proteins.     

Design of compounds having an enhanced tumour uptake,using serum albumin as a carrier. Part I

The search for a radioiodinated “cumulative” protein label, stored within cells following intracellular protein degradation, suggested that plasma protein turnover of tumours might be of use. While earlier investigators were primarily interested in metabolism and utilization of plasma proteins by tumours, we tried to utilize the tumour protein turnover to channel radioiodine labelled compounds, covalently bound to serum albumin, into neoplastic tissues. To identify those parameters which influence the tumour uptake and storage, we investigated a series of compounds having different chemical and physicochemical properties. Unbound, small molecular weight compounds were rapidly eliminated from the circulatory system. They had a prolonged biological half life if linked to serum albumin (SA), especially when derivatized with deoxysorbitol. Parallel with the prolongation of the biological half-life we noted a remarkable increase in tumour uptake, which was not accompanied by increased liver activity. Further-more, without thyroid blockade, we failed to detect significant radioiodine uptake in this organ after 24 or 72 h. This is due to the particular coupling mechanism, which may be relevant for other (radio)iodinated pharmaceuticals used in medicine. Glucose and aromatic amines, as well as aromatic aldehydes and glucamine react to form deoxysorbitol derivates, which then have similar biokinetics after linkage to serum albumin. This indicates that a new approach in tumour detection and possibly in tumour therapy may be possible when SA is used as a carrier molecule, using the described labelling procedure.     

A robust, streamlined, and reproducible method for proteomic analysis of serum by delipidation, albumin and IgG depletion, and two-dimensional gel electrophoresis

Serum is a readily available source for diagnostic assays, but the identification of disease-specific serum biomarkers has been impeded by the dominance of human serum albumin and immunoglobulins (Igs) in the serum proteome. There is a need to reduce the technical variation in serum processing and analysis to allow for a reproducible analysis of large cohorts. To this end, we have developed a rapid and reproducible procedure for sample preparation and high-resolution two-dimensional gel electrophoresis to analyze human serum. Serum is centrifuged at high speed to remove lipids and aggregated proteins, incubated with protein G resin to remove IgG, precipitated with NaCl/ethanol to deplete albumin, and slowly resolubilized in a sodium dodecyl sulfate (SDS)/N-(2-hydroxyethyl)piperazine-2'-(2-ethanesulfonic acid) (HEPES) buffer. The delipidated and IgG/albumin depleted serum proteins are focused on pH 4-7 linear large immobilized pH gradient strips, and then resolved by Bis-Tris SDS-polyacrylamide gel electrophoresis. The robustness and reproducibility of the optimized procedure was determined for three individual serum samples on three consecutive days. An image analysis of the nine silver-stained gels demonstrated that the intensity and localization of protein spots are highly reproducible. Our IgG and albumin depletion procedure will aid in screening the patient sera for normal biological variation and disease-specific biomarkers.     

Erythrocytes serve as a reservoir for cellular and extracellular sphingosine 1‐phosphate

Sphingosine 1‐phosphate (S1P) in blood is phosphorylated, stored, and transported by red blood cells (RBC). Release of S1P from RBC into plasma is a regulated process that does not occur in plasma‐ or serum‐free media. Plasma fractionation and incubations with isolated and recombinant proteins identified high density lipoprotein (HDL) and serum albumin (SA) as non‐redundant endogenous triggers for S1P release from RBC. S1P bound to SA and HDL was able to stimulate the S1P₁ receptor in calcium flux experiments. The binding capability of acceptor molecules triggers S1P release, as demonstrated with the anti‐S1P antibody Sphingomab?. More S1P was extracted from RBC membranes by HDL than by SA. Blood samples from anemic patients confirmed a reduced capacity for S1P release in plasma. In co‐cultures of RBC and endothelial cells (EC), we observed transcellular transportation of S1P as a second function of RBC‐associated S1P in the absence of SA and HDL and during tight RBC‐EC contact, mimicking conditions in tissue interstitium and capillaries. In contrast to S1P bound to SA and HDL, RBC‐associated S1P was significantly incorporated by EC after S1P lyase (SGPL1) inhibition. RBC‐associated S1P, therefore, has two functions: (1) It contributes to the cellular pool of SGPL1‐sensitive S1P in tissues after transcellular transportation and (2) it helps maintain extracellular S1P levels via SA and HDL independently from SGPL1 activity. J. Cell. Biochem. 109: 1232–1243, 2010. © 2010 Wiley‐Liss, Inc.     

Atlantic salmon (Salmo salar) serum albumin: cDNA sequence, evolution, and tissue expression

Atlantic salmon serum albumin is one of the most abundant proteins in salmon liver, representing 1.6% of all clones in a cDNA library made from salmon liver RNA. The DNA from a number of clones was sequenced to reveal an open reading frame of 1,827 bases encoding a 608-amino-acid protein. The sequenced 5' untranslated region is 69 bases long and the 3' untranslated region contains two putative polyadenylation signals and poly(A) tail. Sequence analysis of different clones indicates the presence of a second cDNA for salmon serum albumin. Multiple alignments of salmon serum albumin deduced amino acid sequence with Xenopus laevis, rat, bovine, and human serum albumins shows significant conservation of cysteine residues. The triple domain structure of serum albumin proteins is maintained. Unlike mammalian systems where serum albumin expression appears to be specific to liver only, salmon serum albumin is expressed in muscle also.     

Palmitate-binding, serum albumin-like proteins in salmonids

There has been considerable controversy over the existence of serum albumin in fish. One of the physiological functions of albumin is to bind free fatty acids. This characteristic was used to screen the plasma of seven species of salmonids. Each species contains a protein fraction that (i) binds palmitate, (ii) has a molecular mass similar to that of human serum albumin, and (iii) is one of the most rapidly migrating proteins when salmonid plasma is subjected to anodal polyacrylamide gel electrophoresis. We conclude therefore, that salmonids have serum albumins that are homologous to the serum albumin of higher vertebrates.     

The effect of low molecular weight ligands on relaxation of water protons in protein solutions

Interaction of low-molecular ligands (LML) isolated from blood serum albumin (SA) and serum proteins leads to higher T1 values for water protons compared with those observed in LML-free solutions, although the total amount of bound water increases. The latter was revealed by low-temperature NMR spectroscopy, as well as by the amount of water sorbed on SA + LML at a relative humidity P/Ps greater than 0.7. In the region 0.2 less than P/Ps less than 0.6 the amount of SA + LML-sorbed water decreased, as compared with that in SA indicating that the oppositely charged groups of LML screen some charged groups of the protein. A decrease of charge-to-charge interactions in solution, or with a high water content, leads to the hydration of those groups. The increase of the T1 value for water protons in solution is, probably, due to a hindered exchange between the sorbed water and bulk water. It is outlined that charge interactions between macromolecules may significantly affect water sorption by proteins.     

The dipolar origin of protein relaxation

1. A set of parameters is proposed to check the interpretation of the dielectric behaviour of protein solutions as a rigid-dipole relaxation of prolate ellipsoids of revolution in the frequency range between 20 kHz and 10 MHz. Besides the delta(b)-function of Scheraga, another analogous function (delta(a)) is presented to establish size and shape of globular proteins. A study of the influence of solvent viscosity on the dielectric dispersion also gives strong evidence in favour of rigid-dipole relaxation. 2. Measurements of the dielectric dispersion of monomer solutions of bovine serum albumin and transferrin are reported. Monomers of bovine serum albumin were obtained by fractionation on Sephadex G-150. Low-conductivity solutions of both proteins are obtained by passage through an ion-exchange resin. 3. Computer analysis of the experimental dispersion curves by use of a two-term Debye dispersion gives valuable information about transferrin and leads to an axial ratio 4.5 for a prolate ellipsoid of revolution. The dielectric increment of bovine serum albumin is very low and no conclusive results have yet been obtained.     

A fast and reproducible method for albumin isolation and depletion from serum and cerebrospinal fluid

Analysis of serum and plasma proteomes is a common approach for biomarker discovery, and the removal of high‐abundant proteins, such as albumin and immunoglobins, is usually the first step in the analysis. However, albumin binds peptides and proteins, which raises concerns as to how the removal of albumin could impact the outcome of the biomarker study while ignoring the possibility that this could be a biomarker subproteome itself. The first goal of this study was to test a new commercially available affinity capture reagent from Protea Biosciences and to compare the efficiency and reproducibility to four other commercially available albumin depletion methods. The second goal of this study was to determine if there is a highly efficient albumin depletion/isolation system that minimizes sample handling and would be suitable for large numbers of samples. Two of the methods tested (Sigma and ProteaPrep) showed an albumin depletion efficiency of 97% or greater for both serum and cerebrospinal fluid (CSF). Isolated serum and CSF albuminomes from ProteaPrep spin columns were analyzed directly by LC‐MS/MS, identifying 128 serum (45 not previously reported) and 94 CSF albuminome proteins (17 unique to the CSF albuminome). Serum albuminome was also isolated using Vivapure anti‐HSA columns for comparison, identifying 105 proteins, 81 of which overlapped with the ProteaPrep method.     

Depletion of Plasma Albumin for Proteomic Analysis of Bothrops jararaca Snake Plasma

The proteomic analysis of plasma samples represents a challenge as a result of the presence of highly abundant proteins such as albumin. To enable the detection of biomarkers, which are commonly low-abundance proteins, in complex blood fluids, it is necessary to remove high-abundance proteins efficiently. Moreover, there is a range of about 10 orders of magnitude for the abundance of different protein species in serum. Here, we describe for the first time a study of reptilian albumin depletion using resins usually used in mammalian plasma depletion procedures. We performed the depletion of albumin from Bothrops jaraca plasma using the HiTrap Blue high-performance column (GE Healthcare Life Sciences, Piscataway, NJ, USA) and the kit Albumin & IgG Depletion SpinTrap column (GE Healthcare Life Sciences). In addition, proteomic approaches were used to analyze reptilian plasma. Our results showed that B. jararaca albumin bound to both columns, but those interactions were not enough to remove a large amount of albumin to reach an enrichment of low-abundance proteins. Although the depletion techniques used in this work were not the best to remove B. jararaca plasma albumin, our present work highlights the similarity between B. jararaca and mammalian albumin, contributing to the knowledge of comparative hemostatic proteins.     

Isolation and characterization of denatured serum albumin from rats with endotoxicosis

Due to its rapid breakdown in the body, denatured serum albumin has not been identified in biological samples. In this study we attempted to determine whether denatured albumin could be identified in rats with endotoxicosis. Male Wistar rats were injected with lipopolysaccharide (LPS; 5 mg/kg body weight). Plasma albumin concentration decreased to one-third the normal level at 2 days after the injection. By using the purified IgG against the specific epitope of chemically denatured albumin, two immunoreactive plasma proteins (bands D2 and D3) were identified by native PAGE followed by Western blot analysis. The plasma concentration of these two proteins increased significantly at 1 and 1.5 days after LPS injection. Peptide mass fingerprinting using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI/TOF-MS) identified these two proteins as serum albumin. In order to characterize their conformational nature, ion-exchange chromatography was used to isolate D2 and D3 albumins from rats injected with LPS. Far- and near-UV circular dichroism (CD), tryptophan and 1-anilino-8-naphthalenesulfonate (ANS) fluorescence, and proteolytic susceptibility showed conformational alterations in the D2 and D3 albumins as compared with native albumin. These data indicate the presence of denatured albumin in circulating rat plasma, and this fact may contribute to a further understanding of the molecular mechanisms of albumin breakdown in physiological and pathophysiological conditions.     

Distribution of pyridoxal-5-phosphate between proteins and low molecular weight components of plasma: effect of acetaldehyde

It is found that approximately 65-70% of pyridoxal-P at physiological concentrations is bound to plasma proteins; 15% of its amount is bound to amino acids and peptides as a result of the Schiff base formation. Over 85% of pyridoxal-P associated with plasma proteins is bound to serum albumin. Inorganic phosphate and NaCl decrease the affinity of pyridoxal-P for albumin or other proteins. Acetaldehyde interacts with the alpha-amino group of the aspartic acid residue of the N-end of the polypeptide chain of the albumin molecule and with two epsilon-amino groups of the lysine residues having anomalously low value of pKa and deprotonated at physiological values of pH of the medium. Acetaldehyde competes with pyridoxal-P for the first (of the highest affinity) binding site of the coenzyme on serum albumin. Acetaldehyde is not bound at the second site of high affinity for pyridoxal-P on serum albumin.     

Immunodepletion of albumin for two-dimensional gel detection of new mouse acute-phase protein and other plasma proteins

Immunodepletion of albumin to improve the 2-D gel resolution of human plasma proteins has recently been described. With the importance of mouse models in many studies in which serum or plasma is often analyzed, we have adopted this approach to immunoprecipitate mouse albumin and evaluated its effectiveness for 2-D separation of mouse plasma proteins. Purified polyclonal antibodies against mouse albumin were effective depleting intact albumin as well as its numerous fragments from mouse plasma samples. Removal of albumin resulted in better resolution of mouse plasma proteins. Three proteins, alpha2-macroglobulin, coagulation factor XII, and hemopexin, that were previously either undetectable or poorly resolved, were identified from albumin-depleted 2-D gels by peptide mass fingerprinting. Albumin depletion also led to partial loss of several other proteins such as clusterin and gelsolin. This loss can be attributed to the interaction with albumin itself because the specificity of the antibody was demonstrated by Western blot. When applying this method to the 2-D separation of plasma from inflamed mouse induced by cutaneous burn injury with superimposed Pseudomonas aeruginosa infection, the upregulation of inter alpha-trypsin inhibitor heavy chain 4 (ITIH4) and hemopexin was unambiguously detected along with other mouse acute-phase proteins (APP), including haptoglobin and serum amyloid A. Based on the significant increase of ITIH4, we propose that this protein is a new member of mouse APP that are upregulated during the inflammatory response.     

A review on structure–affinity relationship of dietary flavonoids with serum albumins

Flavonoids are a class of plant secondary metabolites and among thousands of flavonoids few are considered as dietary flavonoids. Serum albumin (SA), the most abundant protein in plasma, functions as the most important carrier of vital drugs, including dietary flavonoids. The binding affinity of dietary flavonoids to SA is demonstrated to be governed by structure–affinity relationship (SAR) and its bioavailability. The present review summarizes the interactions of flavonoids categorized as flavanol, flavonol, flavone, isoflavone, flavanones, and anthocyanidins with SAs (bovine serum albumin and human serum albumin) in light of SAR. The key findings are: (1) the position and degree of hydroxylation highly influence the affinity of flavonoids to SAs, (2) glycosylation decreases and substitution of methoxy group increases the affinity of flavonoids for SAs, (3) catechin gallates have higher binding affinity to SAs than catechins and gallocatechins, (4) inorganic metal ions modulate the binding affinity of flavonoids to SAs, and (5) hydrophobic interaction plays a major role in the interactions of all flavonoids with SAs.     

The role of albumin in the hepatic transport of bilirubin: studies in mutant analbuminemic rats

Bilirubin and other cholephilic organic anions are bound to albumin in the circulation; their hepatic uptake involves a carrier-mediated process. To investigate the possible role of serum albumin in the transhepatic transport of a cholephilic ligand, plasma clearance of radioactive bilirubin and its biliary excretion as well as its interaction with plasma proteins were compared between normal and mutant analbuminemic rats (NAR). With a tracer amount of 3H-labeled bilirubin, its plasma clearance and biliary excretion were comparable in both animal groups. However, the plasma clearance of a loading dose of the ligand was significantly increased and its biliary recovery was low in NAR as compared with normal animals. In accord with these findings in vivo, gel permeation chromatographic analysis revealed that the bilirubin binding capacity of serum proteins was significantly lower in NAR than in control animals. When bilirubin was administered to NAR as a mixture with equimolar albumin, its plasma disappearance was considerably decreased and its biliary recovery was increased. Similar effects were observed when albumin was replaced by an equimolar amount of glutathione S-transferases (ligandins). These observations indicate that, although ligand-protein interaction in the circulation is important for directing bilirubin to the plasma membranes of the hepatocyte, this mechanism is not specific for albumin.     

Effective depletion of albumin using a new peptide-based affinity medium

Blood plasma and serum are very useful samples for the detection, identification and quantitation of proteins associated with both health and disease. However, analysis of plasma and serum is a challenge because traces of interesting polypeptides and proteins can be dominated by the very high concentration of albumin present. Albumin may be depleted by adsorption to immunoaffinity columns or to columns containing dyes such as Cibacron Blue, or by ultrafiltration, but these methods are far from ideal. We describe a new peptide-based affinity medium which is effective for removing albumin and is very specific. The albumin-binding capacity is at least 14 mg per mL of gel. The material may be reused hundreds of times after a simple regeneration step involving NaOH, with full retention of specificity and capacity. The material was tested with human and monkey plasma and serum and rat serum, and has been used to deplete litre volumes of human plasma. The development of other peptide-based affinity media to deplete abundant proteins is briefly discussed.     

Serum albumin prevents protein aggregation and amyloid formation and retains chaperone-like activity in the presence of physiological ligands

Although serum albumin has an established function as a transport protein, evidence is emerging that serum albumin may also have a role as a molecular chaperone. Using established techniques to characterize chaperone interactions, this study demonstrates that bovine serum albumin: 1) preferentially binds stressed over unstressed client proteins; 2) forms stable, soluble, high molecular weight complexes with stressed client proteins; 3) reduces the aggregation of client proteins when it is present at physiological levels; and 4) inhibits amyloid formation by both WT and L55P transthyretin. Although the antiaggregatory effect of serum albumin is maintained in the presence of physiological levels of Ca(2+) and Cu(2+), the presence of free fatty acids significantly alters this activity: stabilizing serum albumin at normal levels but diminishing chaperone-like activity at high concentrations. Moreover, here it is shown that depletion of albumin from human plasma leads to a significant increase in aggregation under physiologically relevant heat and shear stresses. This study demonstrates that serum albumin possesses chaperone-like properties and that this activity is maintained under a number of physiologically relevant conditions.