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.     

Two-dimensional liquid chromatography/tandem mass spectrometry analysis of Gradiflow fractionated native human plasma

One of the major challenges facing protein analysis is the dynamic range of protein expression within massively complex samples (Corthals, G. L. et al.., Electrophoresis 2000, 21, 1104-1115). In plasma this difference is as great as ten orders of magnitude, and this is currently beyond the range of detection achievable by any of the analytical techniques. Plasma has the additional challenge of having a few highly abundant proteins, such as albumin, which mask the detection of lower abundance and biologically significant proteins. The use of the Gradiflow BF400 as a fractionation tool to deplete highly abundant albumin from human plasma is reported here. A sequential three-step protocol was performed on five plasma samples as part of the International Plasma Proteome Project organised by the HUPO; four containing different anticoagulants: EDTA, citrate, heparin and a control sample (NIBSC); and a serum sample. Plasma from an alternate source also underwent fractionation and served as an in-house control. Time modulation between 1 and 7 h was observed for the depletion of albumin from these samples. Following albumin depletion, each fraction was trypsin-digested and the peptides were fractionated further using a 2-D LC-MS/MS. Differences in the total number of proteins identified for each sample were also noted.     

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.     

Immunodepletion of albumin and immunoglobulin G from bovine plasma

Current MS-based proteomics has facilitated the identification of large numbers of proteins from complex mixtures. The bovine plasma proteome has the potential to provide a wealth of information concerning the biological state of an animal. However, during MS-based experiments, higher abundance proteins such as albumin and immunoglobulin G (IgG) can hinder the identification of potentially important proteins that are present in much lower abundance. While a variety of readily available technologies exist for the depletion of multiple high-abundance proteins from human, mouse and rat samples, there are few available for bovine. In this study, we report the depletion of >97% of albumin and >92% of IgG from bovine plasma.     

Depletion of highly abundant proteins in blood plasma by ammonium sulfate precipitation for 2D-PAGE analysis

Ammonium sulfate precipitation (ASP) was explored as a method for depleting some highly abundant proteins from blood plasma, in order to reduce the dynamic range of protein concentration and to improve the detection of low abundance proteins by 2D-PAGE. 40% ammonium sulfate saturation was chosen since it allowed depleting 39% albumin and 82% α-1-antitrypsin. ASP-depletion showed high reproducibility in 2D-PAGE analysis (4.2% variation in relative abundance of albumin), similar to that offered by commercial affinity-depletion columns. Besides, it allowed detecting 59 spots per gel, very close to the number of spots detected in immuno-affinity-depleted plasma. Thus, ASP at 40% saturation is a reliable depletion method that may help in proteomic analysis of blood plasma. Finally, ASP-depletion seems to be complementary to hydrophobic interaction chromatography (HIC)-depletion, and therefore an ASP-step followed by a HIC-step could probably deplete the most highly abundant plasma proteins, thus improving the detection of low abundance proteins by 2D-PAGE.     

Developing a strong anion exchange/RP (SAX/RP) 2D LC system for high‐abundance proteins depletion in human plasma

Human plasma is dominated by high‐abundance proteins which severely impede the detection of low‐abundance proteins. Unfortunately, now there is no efficient method for large‐scale depletion of high‐abundance proteins in human plasma. In this study, we developed a new strategy, strong anion exchange (SAX)/RP 2D LC system, which has potential for large‐scale depletion of high‐abundance proteins in human plasma. Separation gradients of the system were optimized to ensure an extensive separation of plasma proteins. Plasma was fractionated into 67 fractions by SAX. All these fractions were subjected a thorough separation by the 2D RPLC and 66 peaks with high UV absorption (>20 mAU) at 215 nm were collected. Proteins in these peaks were identified by LC‐MS/MS analysis. Results showed that 83 proteins could be identified in these peaks, 68 among them were reported to be high‐ or middle‐abundance proteins in plasma. All these proteins had definite retention times and were mapped in the 2D SAX‐RP system, which resulted in accurate depletion of high‐abundance proteins with ease. Our studies provide a convenient and effective method for large‐scale depletion of high‐abundance proteins and in‐depth research in human plasma proteomics.     

Comparison of different depletion strategies for improved resolution in proteomic analysis of human serum samples

Serum proteins may often serve as indicators of disease and is a rich source for biomarker discovery. However, the large dynamic range of proteins in serum makes the analysis very challenging because high-abundant proteins tend to mask those of lower abundance. A prefractionation step, such as depletion of a few high-abundant proteins before protein profiling, can assist in the discovery and detection of less abundant proteins that may prove to be informative biomarkers. In the present study, five different depletion columns were investigated considering efficiency, specificity, and reproducibility. Our research included quantitative determination of total protein, albumin, and immunoglobulin G (IgG) concentrations, one- and two-dimensional gels and mass spectrometric analysis of the serum samples before and after the depletion step. Our results showed that all five depletion columns tested removed albumin and IgG with high efficiency. We found that based on reproducibility and binding specificity, the Multiple Affinity Removal Column that removed a total of six high-abundant proteins (albumin, IgG, antitrypsin, IgA, transferring, and haptoglobin) offered the most promising depletion approach. Among the disposable (single-use) products, the ProteoExtract Albumin/IgG Removal kit displayed the best results. Depleted serum from the Multiple Affinity Removal column was further evaluated by 2-D gel electrophoresis (2-DE) analysis, and the results indicated increased resolution and improved intensity of low-abundant proteins in a reproducible fashion. Our study provides a comprehensive investigation of commercially available depletion columns and will be of high importance for future proteomic studies on serum samples.     

Serum/Plasma depletion with chicken immunoglobulin Y antibodies for proteomic analysis from multiple Mammalian species.

Plasma from different species is the most accessible and valuable source for biomarker discovery in clinical and animal samples. However, due to the high abundance of some proteins such as albumin and immunoglobulins, low-abundant proteins are often undetectable in proteomic analysis of plasma. We have established a plasma depletion scheme using chicken antibodies against various abundant proteins. This immunoaffinity purification procedure is able to deplete albumin across multiple species. The high binding capacity and specificity of the chicken antibody enables the efficient capture of its ligand from microliter volumes of plasma sample. The resulting two-dimensional gel analyses of the depleted and captured samples show significant enhancement of the low-abundant proteins and specific capture of the abundant ligand. By utilizing this sample preparation scheme, it is now possible to analyze the plasma proteome from multiple species in a potentially rapid and large-scale capacity for biomarker discovery, drug target discovery, and toxicology studies.     

Comparison of protein enrichment strategies for proteome analysis of plasma

Efforts to discover protein biomarkers in plasma are hampered by the high abundance of few proteins, which interfere with the detection of low‐abundant proteins. Different commercially available protein‐partitioning products were tested for their ability to lower the detection limit of proteins in 2‐D gels. Immuno‐depletion using polyclonal antibodies raised against the proteins of highest abundance (Seppro IgY14 System) was compared with a two‐step immuno‐depletion strategy, where depletion with the Seppro IgY14 column was followed by depletion with the Seppro IgY‐SuperMix system. The third strategy tested was protein pre‐fractionation using the ProteoMiner kit, where proteins compete for binding sites on bead‐bound peptide hexamers with different binding properties. The pre‐fractionated protein samples were analyzed using 2‐DE, which revealed stunning differences in protein patterns. However, detectable protein spots in the different plasma fractions contained exclusively high‐abundant proteins normally present in plasma at concentrations between 1 μg and 40 mg/mL.     

Depletion of the highly abundant protein albumin from human plasma using the Gradiflow

Analysis of complex protein samples by two-dimensional electrophoresis (2-DE) is often more difficult in the presence of a few predominant proteins. In plasma, proteins such as albumin mask proteins of lower abundance, as well as significantly limiting the amount of protein that can be loaded onto the immobilized pH gradient strip. In this paper the Gradiflow, a preparative electrophoresis system, has been used to deplete human plasma of the highly abundant protein albumin under native and denatured conditions. A three step protocol incorporating a charge separation to collect proteins with an isoelectric point greater than albumin and two size separations to isolate proteins larger and smaller than albumin, was used. When the albumin depleted fractions were analysed on pH 3-10 2-DE gels, proteins that were masked by albumin were revealed and proteins not seen in the unfractionated plasma sample were visualised. Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry analysis confirmed the identification of the protein that lies beneath albumin to be C4B-binding protein alpha chain. The liquid fractions from the Gradiflow separations were also analysed by liquid chromatography-tandem mass spectrometry to confirm the proteins were separated according to their size and charge mobility in an electric field.     

Depletion of highly abundant proteins in blood plasma by hydrophobic interaction chromatography for proteomic analysis

The proteomic analysis of plasma is extremely complex due to the presence of few highly abundant proteins. These proteins have to be depleted in order to detect low abundance proteins, which are likely to be of biomedical interest. In this work it was investigated the applicability of hydrophobic interaction chromatography (HIC) as a plasma fractionation method prior to two-dimensional gel electrophoresis (2DGE). The average hydrophobicity of the 56 main plasma proteins was calculated. Plasma proteins were classified as low, medium and highly hydrophobic through a cluster analysis. The highly abundant proteins showed a medium hydrophobicity, and therefore a HIC step was designed to deplete them from plasma. HIC performance was assessed by 2DGE, and it was compared to that obtained by a commercial immuno-affinity (IA) column for albumin depletion. Both methods showed similar reproducibility. HIC allowed partially depleting α-1-antitrypsin and albumin, and permitted to detect twice the number of spots than IA. Since albumin depletion by HIC was incomplete, it should be further optimized for its use as a complementary or alternative method to IA.     

Depletion of abundant human serum proteins by per se imprinted cryogels based on sample heterogeneity

Macroporous cryogels were prepared and used to deplete abundant proteins. It was accomplished based on the sample heterogeneity rather than any exogenous assistance. Human serum was added in monomer solutions to synthesize molecularly imprinted polymers; therein some abundant proteins were imprinted in the polyacrylamide cryogels. Meanwhile the rare components remained aqueous. Chromatography and electrophoresis showed that albumin, serotransferrin, and most globulins were depleted by columns packed with the molecularly imprinted polymers. After the depletion, lower abundance proteins were revealed by SDS‐PAGE, peptide fingerprint analysis, and identified by MALDI‐TOF‐MS. This is an example that a “per se imprint” protocol enables to gradually dimidiate proteomes, simplify sample complexities, and facilitate further proteome profiling or biomarker discovery.     

Depletion of multiple high-abundance proteins improves protein profiling capacities of human serum and plasma

Systematic detection of low-abundance proteins in human blood that may be putative disease biomarkers is complicated by an extremely wide range of protein abundances. Hence, depletion of major proteins is one potential strategy for enhancing detection sensitivity in serum or plasma. This study compared a recently commercialized HPLC column containing antibodies to six of the most abundant blood proteins ("Top-6 depletion") with either older Cibacron blue/Protein A or G depletion methods or no depletion. In addition, a prototype spin column version of the HPLC column and an alternative prototype two antibody spin column were evaluated. The HPLC polyclonal antibody column and its spin column version are very promising methods for substantially simplifying human serum or plasma samples. These columns show the lowest nonspecific binding of the depletion methods tested. In contrast other affinity methods, particularly dye-based resins, yielded many proteins in the bound fractions in addition to the targeted proteins. Depletion of six abundant proteins removed about 85% of the total protein from human serum or plasma, and this enabled 10- to 20-fold higher amounts of depleted serum or plasma samples to be applied to 2-D gels or alternative protein profiling methods such as protein array pixelation. However, the number of new spots detected on 2-D gels was modest, and most newly visualized spots were minor forms of relatively abundant proteins. The inability to detect low-abundance proteins near expected 2-D staining limits was probably due to both the highly heterogeneous nature of most plasma or serum proteins and masking of many low-abundance proteins by the next series of most abundant proteins. Hence, non2-D methods such as protein array pixelation are more promising strategies for detecting lower abundance proteins after depleting the six abundant proteins.     

Cross species applicability of abundant protein depletion columns for ribulose-1,5-bisphosphate carboxylase/oxygenase

In plants, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is an important enzyme in the Calvin cycle, catalyzing the first step of carbon fixation. Because of its critical role in photosynthesis, RuBisCO comprises 30-60% of the total protein content in green leaf tissue and represents a major protein which can interfere with determination of lower abundance proteins in plant proteomics. A potential solution to aid in the determination of low level proteins in plant proteomics are RuBisCO immunodepletion columns. Two formats, spin and LC, of Seppro IgY RuBisCO depletion columns were evaluated for cross species applicability. The spin and LC columns were found to deplete arabidopsis RuBisCO by greater than 90 and 98%, respectively, and automation could be achieved with the LC format. Canola RuBisCO was depleted to a similar extent, and there was evidence suggesting that corn and tobacco RuBisCO were also highly depleted in flow through fractions. Model proteins were spiked into samples to provide insight into the degree of non-specific binding. Finally, improved detection and identification of lower abundance proteins was demonstrated after depletion.     

Low plasma albumin levels are associated with increased plasma protein glycation and HbA1c in diabetes

Albumin is one of the most abundant plasma proteins and is heavily glycated in diabetes. In this study, we have addressed whether variation in the albumin levels influence glycation of plasma proteins and HbA1c. The study was performed in three systems: (1) streptozotocin (STZ)-induced diabetic mice plasma, (2) diabetic clinical plasma, and (3) in vitro glycated plasma. Diabetic mice and clinical plasma samples were categorized as diabetic high albumin plasma (DHAP) and diabetic low albumin plasma (DLAP) on the basis of their albumin levels. For the in vitro experiment, two albumin levels, high albumin plasma (HAP) and low albumin plasma (LAP), were created by differential depletion of plasma albumin. Protein glycation was studied by using a combination of two-dimensional electrophoresis (2DE), Western blotting, and LC-MS(E). In both mice and clinical experiments, an increased plasma protein glycation was observed in DLAP than in DHAP. Additionally, plasma albumin levels were negatively correlated with HbA1c. The in vitro experiment with differential depletion of albumin mechanistically showed that the low albumin levels are associated with increased plasma protein glycation and that albumin competes for glycation with other plasma proteins.     

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.     

Rat plasma proteomics: effects of abundant protein depletion on proteomic analysis

The proteomic analysis of plasma and serum samples represents a formidable challenge due to the presence of a few highly abundant proteins such as albumin and immunoglobulins. Detection of low abundance protein biomarkers requires therefore either the specific depletion of high abundance proteins with immunoaffinity columns and/or optimized protein fractionation methods based on charge, size or hydrophobicity. Here we describe the depletion of seven abundant rat plasma proteins with an immunoaffinity column with coupled antibodies directed against albumin, IgG, transferrin, IgM, haptoglobin, fibrinogen and alpha1-anti-trypsin. The IgY-R7-LC2 (Beckman Coulter) column showed high specificity for the targeted proteins and was able to efficiently remove most of the albumin, IgG and transferrin from rat plasma samples as judged by Western blot analysis. Depleted rat plasma protein samples were analyzed by SELDI-TOF MS, 2D SDS-PAGE and 2D-LC and compared to non-depleted plasma samples as well as to the abundant protein fraction that was eluted from the immunoaffinity column. Analysis of the depleted plasma protein fraction revealed improved signal to noise ratios, regardless of which proteomic method was applied. However, only a small number of new proteins were observed in the depleted protein fraction. Immunoaffinity depletion of abundant plasma proteins results in the significant dilution of the original sample which complicates subsequent analysis. Most proteomic approaches require specialized sample preparation procedures during which significant losses of less abundant proteins and potential biomarkers can occur. Even though abundant protein depletion reduces the dynamic range of the plasma proteome by about 2-3 orders of magnitude, the difference between medium-abundant and low abundant plasma proteins is still in the range of 7-8 orders of magnitude and beyond the dynamic range of current proteomic technologies. Thus, exploring the plasma proteome in greater detail remains a daunting task.     

Concentration-dependent internalization of a cytokine/cytokine receptor complex in human hematopoietic cells

Soluble steel factor (SF) is a potent stimulator of hematopoietic progenitor cell proliferation in vitro, and cytokine combinations that include SF can support extensive expansions of hematopoietic cells. Recently, we showed that very primitive progenitor cells from normal human bone marrow require exposure to very high concentrations of cytokines to maintain their primitive status while proliferating. These cells also display higher cell-specific cytokine uptake rates than more differentiated types of hematopoietic cells. As a first step toward identifying the mechanisms involved in mediating such cytokine dose-dependent effects, we have now investigated the kinetics of SF receptor (c-kit) internalization by human Mo7e cells exposed to different extracellular concentrations of soluble SF. Transfer of Mo7e cells to a higher concentration of SF caused an initially rapid downregulation of cell surface c-kit which was accompanied by a rapid depletion of extracellular SF. Confocal microscopy showed a concomitant increase in the number and intensity of intracellular c-kit aggregates. After the first 30 min, the cells continued to deplete SF from the medium but at a much slower rate. During this period, there was a gradual recovery of expression of c-kit on the cell surface. A mathematical analysis of bulk medium to cell-surface SF-mass transport indicated that the cytokine-depletion rates measured were not likely to have significantly depleted the SF concentration in the microenvironment of the cells. Taken together, these results underscore the importance of monitoring and appropriately regulating cytokine concentrations in hematopoietic cell expansion cultures. They may also help to explain the different biological responses exhibited by primitive hematopoietic cells exposed to different types and concentrations of cytokines for periods of days.     

Assessment of albumin removal from an immunoaffinity spin column: Critical implications for proteomic examination of the albuminome and albumin‐depleted samples

High abundance proteins in serum and plasma (e.g., albumin) are routinely removed during proteomic sample processing as they can mask lower abundance proteins and peptides of biological/clinical interest. A common method of albumin depletion is based on immunoaffinity capture, and many immunoaffinity devices are designed for multiple uses. In this case, it is critical that the albumin captured on the affinity matrix is stripped from the column prior to regeneration of the matrix and processing of subsequent samples, to ensure no carryover and that maximal binding sites are available for subsequent samples. The current study examines the ability of a manufacturer's protocol to remove the proteins and peptides captured by an immunoaffinity spin column. The data presented in the current work illustrate the difficulty in completely removing albumin from the immunoaffinity device, and consequently, may explain the variability and decreased efficiency shown for this device in previous studies. In summary, the current data present important considerations for the implementation of multiple‐use immunoaffinity devices for processing subsequent clinical samples in a proteomic workflow.     

High-abundance proteins depletion for serum proteomic analysis: concomitant removal of non-targeted proteins

In clinical and pharmaceutical proteomics, serum and plasma are frequently used for detection of early diagnostic biomarkers for therapeutic targets. Although obtaining these body fluid samples is non-invasive and easy, they contain some abundant proteins that mask other protein components present at low concentrations. The challenge in identifying serum biomarkers is to remove the abundant proteins, uncovering and enriching at the same time the low-abundance ones. The depletion strategies, however, could lead to the concomitant removal of some non-targeted proteins that may be of potential interest. In this study, we compared three different methods aimed to deplete high-abundance proteins from human serum, focusing on the identification of non-specifically bound proteins which might be eventually removed. A Cibacron blue-dye-based method for albumin removal, an albumin and IgG immunodepletion method and an immunoaffinity column (Multiple Affinity Removal System) that simultaneously removes a total of six high-abundance proteins, were investigated. The bound proteins were eluted, separated by two-dimensional gel electrophoresis and identified by Nano LC-CHIP-MS system. Flow-through fractions and bound fractions were also analysed with the ProteinChip technology SELDI-TOF-MS. Our results showed that the methods tested removed not only the targeted proteins with high efficiency, but also some non-targeted proteins. We found that the Multiple Affinity Removal Column improved the intensity of low-abundance proteins, displayed new protein spots and increased resolution. Notably, the column showed the lowest removal of untargeted proteins, proved to be the most promising depletion approach and a reliable method for serum preparation prior to proteomic studies.