The human red blood cell proteome and interactome

The red blood cell or erythrocyte is easily purified, readily available, and has a relatively simple structure. Therefore, it has become a very well studied cell in terms of protein composition and function. RBC proteomic studies performed over the last five years, by several laboratories, have identified 751 proteins within the human erythrocyte. As RBCs contain few internal structures, the proteome will contain far fewer proteins than nucleated cells. In this minireview, we summarize the current knowledge of the RBC proteome, discuss alterations in this partial proteome in varied human disease states, and demonstrate how in silico studies of the RBC interactome can lead to considerable insight into disease diagnosis, severity, and drug or gene therapy response. To make these latter points we focus on what is known concerning changes in the RBC proteome in Sickle Cell Disease.     

Challenges for red blood cell biomarker discovery through proteomics

Red blood cells are rather unique body cells, since they have lost all organelles when mature, which results in lack of potential to replace proteins that have lost their function. They maintain only a few pathways for obtaining energy and reducing power for the key functions they need to fulfill. This makes RBCs highly sensitive to any aberration. If so, these RBCs are quickly removed from circulation, but if the RBC levels reduce extremely fast, this results in hemolytic anemia. Several causes of HA exist, and proteome analysis is the most straightforward way to obtain deeper insight into RBC functioning under the stress of disease. This should result in discovery of biomarkers, typical for each source of anemia. In this review, several challenges to generate in-depth RBC proteomes are described, like to obtain pure RBCs, to overcome the wide dynamic range in protein expression, and to establish which of the identified/quantified proteins are active in RBCs. The final challenge is to acquire and validate suited biomarkers unique for the changes that occur for each of the clinical questions; in red blood cell aging (also important for transfusion medicine), for thalassemias or sickle cell disease. Biomarkers for other hemolytic anemias that are caused by dysfunction of RBC membrane proteins (the RBC membrane defects) or RBC cytosolic proteins (the enzymopathies) are sometimes even harder to discover, in particular for the patients with RBC rare diseases with unknown cause. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.     

Proteome analysis of red deer antlers

Deer antlers are the only mammalian organs capable of repeated regeneration. Although antlers are known to develop from pedicles, which arise from antlerogenic cells of cranial periosteum, their developmental process is not fully elucidated. For example, while endocrine and environmental factors influence the antler development, it is still unclear which signaling pathways are involved in the transduction of such stimuli. To study the developmental process of antlers and identify proteins functioning in their growth, we have established proteome maps of red deer (Cervus elaphus) antlers. With two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry, we analyzed more than 800 protein spots and identified approximately 130 individual proteins derived from the growing tip of antlers. The overall profile of the antler proteome was dissimilar to those of other types of tissue. Also comparison of proteomes derived from proximal bony tissue and the growing tip of antlers revealed substantial differences. Moreover several cell growth or signaling-related proteins are expressed exclusively in the growing tip, suggesting that these proteins function in the growth and differentiation of antlers. Currently, using the antler proteome maps, we are actively searching for the regulatory factor(s) that may control the antler development.     

Accentuate the Negative:Proteome Comparisons Using the Negative Proteome Database

The availability of complete genome sequence information for diverse organisms including model genetic organisms has ushered in a new era of protein sequence comparisons making it possible to search for commonalities among entire proteomes using the Basic Local Alignment Search Tool (BLAST). Although the identification and analysis of proteins shared by humans and model organisms has proven an invaluable tool to understanding gene function, the sets of proteins unique to a given model organism's proteome have remained largely unexplored. We have constructed a searchable database that allows biologists to identify proteins unique to a given proteome. The Negative Proteome Database (NPD) is populated with pair-wise protein sequence comparisons between each of the following proteomes: Homo sapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans, Saccharomyces cerevisiae, Dictyostelium discoideum, Chlamydomonus reinhardti, Escherichia coli K12, Arabidopsis thaliana and Methanoscarcina acetivorans. Our analysis of negative proteome datasets using the NPD has thus far revealed 107 proteins in humans that may be involved in motile cilia function, 1628 potential pesticide target proteins in flies, 659 proteins shared by flies and humans that are not represented in the less neurologically complex worm proteome, and 180 nuclear encoded human disease associated proteins that are absent from the fly proteome. The NPD is the only online resource where users can quickly perform complex negative and positive comparisons of model organism proteomes. We anticipate that the NPD and the adaptable algorithm which can readily be used to duplicate this analysis on custom sets of proteomes will be an invaluable tool in the investigation of organism specific protein sets.     

Accentuate the negative: proteome comparisons using the negative proteome database

The availability of complete genome sequence information for diverse organisms including model genetic organisms has ushered in a new era of protein sequence comparisons making it possible to search for commonalities among entire proteomes using the Basic Local Alignment Search Tool (BLAST). Although the identification and analysis of proteins shared by humans and model organisms has proven an invaluable tool to understanding gene function, the sets of proteins unique to a given model organism's proteome have remained largely unexplored. We have constructed a searchable database that allows biologists to identify proteins unique to a given proteome. The Negative Proteome Database (NPD) is populated with pair-wise protein sequence comparisons between each of the following proteomes: Homo sapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans, Saccharomyces cerevisiae, Dictyostelium discoideum, Chlamydomonus reinhardti, Escherichia coli K12, Arabidopsis thaliana and Methanoscarcina acetivorans. Our analysis of negative proteome datasets using the NPD has thus far revealed 107 proteins in humans that may be involved in motile cilia function, 1628 potential pesticide target proteins in flies, 659 proteins shared by flies and humans that are not represented in the less neurologically complex worm proteome, and 180 nuclear encoded human disease associated proteins that are absent from the fly proteome. The NPD is the only online resource where users can quickly perform complex negative and positive comparisons of model organism proteomes. We anticipate that the NPD and the adaptable algorithm which can readily be used to duplicate this analysis on custom sets of proteomes will be an invaluable tool in the investigation of organism specific protein sets.     

Widespread and ample peptide overlapping between HCV and Homo sapiens proteomes

Alignment of protein sequences is fundamental in analyzing homology, evolutionary events and functional relationships. Searching for the epitopic peptide platform underlying hepatitis C virus (HCV) infection and autoimmune phenomena, we have used sequence-sequence peptide matching to compare the HCV polyprotein sequence to the human proteome. The following results were obtained: (1) pentamers from HCV polyprotein have a widespread and high level of similarity to a large number of human proteins (19,605 human proteins, that is 57.6% of the human proteome); (2) remarkable similarity between the two proteomes persists even using longer peptide motifs as probes for identity scanning; (3) only a limited number of HCV pentameric fragments have no similarity to the human host, so representing molecular sequence signatures of the virus. We conclude that the widespread sharing of numerous perfect exact matches between HCV and human proteomes might explain HCV persistence in humans.     

Cystic fibrosis transmembrane conductance regulator in human and mouse red blood cell membranes and its interaction with ecto-apyrase

Elevated blood ATP and increased red blood cell (RBC) ATP transport is associated with cystic fibrosis (CF). In this report, we demonstrate the presence of the wild-type and the DeltaF508 mutant form of the CF transmembrane conductance regulator protein in RBC membranes and its putative interaction with ecto-apyrase, an ATP hydrolyzing enzyme also present in the RBC membrane. RBC membranes of control and DeltaF508 individuals and of wild-type and CF transmembrane conductance regulator-knockout mice were examined by immunoblot using several antibodies directed against different epitopes of this protein. These experiments indicated that human RBC membranes contain comparable amounts of the wild-type CF transmembrane conductance regulator protein and the DeltaF508 mutant form of the protein, respectively. CF transmembrane conductance regulator protein was also detected in wild-type mouse RBC membranes but not in the gene knockout mouse RBC membranes. Antibodies directed against ecto-apyrase co-immunoprecipitated CF transmembrane conductance regulator protein of human RBC membranes indicating a physical interaction between these two membrane proteins consistent with ATP transport and extracellular hydrolysis. We conclude that RBCs are a significant repository of CF transmembrane conductance regulator protein and should provide a novel system for evaluating its expression and function.     

A comparison of the HUPO Brain Proteome Project pilot with other proteomics studies

The pilot phase of the Brain Proteome Project (BPP), the Human Proteome Organization (HUPO) initiative that focuses on studies of the brain of both humans and mice, has now been completed. Participating laboratories studied the proteomes of two human samples derived from biopsy and autopsy as well as three mouse samples from various developmental stages. With the combined and centrally reprocessed data now available, a comparison in terms of protein identifications and project organization is made between the HUPO BPP pilot and three other proteomics studies: the HUPO Plasma Proteome Project (PPP) pilot, a proteome of human blood platelets and a recently published comprehensive mouse proteome. Finally, as any comparison between large-scale proteomics datasets is decidedly non-trivial, we also evaluate and discuss several ways to go about comparing such different result sets.     

Human autologous rosette-forming cells. III. Binding of erythrocytes from different species to the T-cell receptors for autologous red blood cells

Spontaneous rosette formation in humans is restricted to a subpopulation of the circulating T cells. We have previously shown that the interaction between lymphocytes and autologous red blood cells (auto-RBC) is not mediated by a self-recognition mechanism, since allogeneic (allo-) RBC bind to T cells through the same receptors. In this work, we have extended these observations to thymocytes. Using a mixed-rosette assay in which one type of erythrocyte was identified by FITC labeling, we have shown that almost all the thymocytes which attached auto-RBC could also fix allo-RBC. However, as for the peripheral blood lymphocytes (PBL), binding of human RBC to thymocytes occurred with varying affinities according to the erythrocyte's origin. In order to further study the specificity of the erythrocyte to lymphocyte binding in rosette formation, PBL were mixed with auto-RBC and erythrocytes of xenogeneic (xeno-) origin. Although very disparate incidences of rosettes were found according to the species from which the RBC were derived, most of the autorosetting lymphocytes also had receptors for xeno-RBC. In addition, preincubation of PBL with monoclonal antibody OKT11A (directed against the sheep RBC receptors on T cells) completely abrogated rosette formation with all the erythrocytes tested (human auto- and allo-, sheep, pig, and rabbit) except mouse RBC. Taken together these data strongly suggest that human auto- or allo-, as well as sheep or some other xeno-RBC, bind to T lymphocytes by a single receptor and that the combining sites are expressed with different densities or varying affinities depending upon the RBC's origin. Therefore, spontaneous autorosettes may represent T lymphocytes having high-affinity receptors for sheep RBC.     

Comparative plasma proteome analysis of lymphoma-bearing SJL mice

In SJL mice, growth of RcsX lymphoma cells induces an inflammatory response by stimulating V(beta)16+ T cells. During inflammation, various serum protein levels can increase (e.g., acute phase reactants) or decrease (e.g., albumin), and most of these altered proteins are thus potential biomarkers. Although blood plasma is a valuable and promising sample for biomarker discovery for diseases or for novel drug targets, its proteome is complex. To address this, we have focused on a comprehensive comparison of the plasma proteomes from normal and RcsX-tumor-bearing SJL mice using the 1D-Gel-LC-MS/MS method after removing albumin and immunoglobulins. This analysis resulted in the identification of a total of 1079 nonredundant mouse plasma proteins; more than 480 in normal and 790 in RcsX-tumor-bearing SJL mouse plasma. Of these, only 191 proteins were found in common. The molecular weights ranged from 2 to 876 kDa, covering the pI values between 4.22 and 12.09, and included proteins with predicted transmembrane domains. By comparing the plasma proteomic profile of normal and RcsX-tumor-bearing SJL mice, we found significant changes in the levels of many proteins in RcsX-tumor-bearing mouse plasma. Most of the up-regulated proteins were identified as acute-phase proteins (APPs). Also, several unique proteins i.e., haptoglobin, proteosome subunits, fetuin-B, 14-3-3 zeta, MAGE-B4 antigen, etc, were found only in the tumor-bearing mouse plasma; either secreted, shed by membrane vesicles, or externalized due to cell death. These results affirm the effectiveness of this approach for protein identification from small samples, and for comparative proteomics in potential animal models of human disorders.     

Adenosine‐to‐Inosine RNA Editing in Mouse and Human Brain Proteomes

Proteogenomics is based on the use of customized genome or RNA sequencing databases for interrogation of shotgun proteomics data in search for proteome‐level evidence of genome variations or RNA editing. In this work, the products of adenosine‐to‐inosine RNA editing in human and murine brain proteomes are identified using publicly available brain proteome LC‐MS/MS datasets and an RNA editome database compiled from several sources. After filtering of false‐positive results, 20 and 37 sites of editing in proteins belonging to 14 and 32 genes are identified for murine and human brain proteomes, respectively. Eight sites of editing identified with high spectral counts overlapped between human and mouse brain samples. Some of these sites have been previously reported using orthogonal methods, such as α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) glutamate receptors, CYFIP2, coatomer alpha. Also, differential editing between neurons and microglia is demonstrated in this work for some of the proteins from primary murine brain cell cultures. Because many edited sites are still not characterized functionally at the protein level, the results provide a necessary background for their further analysis in normal and diseased cells and tissues using targeted proteomic approaches.     

Pharmaco-proteomic study of hydroxyurea-induced modifications in the sickle red blood cell membrane proteome

Hydroxyurea (HU) is an effective oral drug for the management of homozygous sickle cell anemia (SS) in part because it increases fetal hemoglobin (HbF) levels within sickle red blood cells (RBCs) and thus reduces sickling. However, results from the Multicenter Study of HU suggested that clinical symptoms often improved before a significant increase in HbF levels occurred. This indicated that HU may be acting through the modification of additional cellular mechanisms that are yet to be identified. Hence, in this study, we focused on the analysis of the sickle RBC membrane proteome +/- HU treatment. 2D-DIGE (Two Dimensional Difference In-Gel Electrophoresis) technology and tandem mass spectrometry has been used to determine quantitative differences between sickle cell membrane proteins in the presence and absence of a clinically relevant concentration of HU. In vitro protein profiling of 13 sickle RBC membrane samples +/- 50 muM HU identified 10 statistically significant protein spots. Of these, the most remarkable class of proteins to show a statistically significant increase was the anti-oxidant enzymes-catalase, thioredoxin peroxidase and biliver-din reductase and the chaperonin containing TCP1 complex assisting in the folding of RBC cytoskeletal proteins. Interestingly, catalase immunoblots showed an increase in the acidic forms of the enzyme within sickle RBC membranes on incubation with 50 muM HU. We further identified this modification in catalase to be phosphorylation and demonstrated that HU exposed SS RBC membranes showed a 2-fold increase in tyrosine phosphorylation of catalase as compared to counterparts not exposed to HU. These results present an attractive model for HU-induced post-translational modification and potential activation of catalase in mature sickle RBCs. These findings also identify protein targets of HU other than fetal hemoglobin and enhance the understanding of the drug mechanism.     

Proteomic and bioinformatic characterization of the biogenesis and function of melanosomes

Melanin, which is responsible for virtually all visible skin, hair, and eye pigmentation in humans, is synthesized, deposited, and distributed in subcellular organelles termed melanosomes. A comprehensive determination of the protein composition of this organelle has been obstructed by the melanin present. Here, we report a novel method of removing melanin that includes in-solution digestion and immobilized metal affinity chromatography (IMAC). Together with in-gel digestion, this method has allowed us to characterize melanosome proteomes at various developmental stages by tandem mass spectrometry. Comparative profiling and functional characterization of the melanosome proteomes identified approximately 1500 proteins in melanosomes of all stages, with approximately 600 in any given stage. These proteins include 16 homologous to mouse coat color genes and many associated with human pigmentary diseases. Approximately 100 proteins shared by melanosomes from pigmented and nonpigmented melanocytes define the essential melanosome proteome. Proteins validated by confirming their intracellular localization include PEDF (pigment-epithelium derived factor) and SLC24A5 (sodium/potassium/calcium exchanger 5, NCKX5). The sharing of proteins between melanosomes and other lysosome-related organelles suggests a common evolutionary origin. This work represents a model for the study of the biogenesis of lysosome-related organelles.     

Vitamin C in mouse and human red blood cells: an HPLC assay

Although vitamin C (ascorbate) is present in whole blood, measurements in red blood cells (RBCs) are problematic because of interference, instability, limited sensitivity, and sample volume requirements. We describe a new technique using HPLC with coulometric electrochemical detection for ascorbate measurement in RBCs of humans, wild-type mice, and mice unable to synthesize ascorbate. Exogenously added ascorbate was fully recovered even when endogenous RBC ascorbate was below the detection threshold (25 nM). Twenty microliters of whole blood or 10 μl of packed RBCs was sufficient for assay. RBC ascorbate was stable for 24h from whole-blood samples at 4°C. Processed, stored samples were stable for >1 month at -80°C. Unlike other tissues, ascorbate concentrations in human and mouse RBCs were linear in relation to plasma concentrations (R=0.8 and 0.9, respectively). In healthy humans, RBC ascorbate concentrations were 9-57 μM, corresponding to ascorbate plasma concentrations of 15-90 μM. Mouse data were similar. In human blood stored as if for transfusion, initial RBC ascorbate concentrations varied approximately sevenfold and decreased 50% after 6 weeks of storage under clinical conditions. With this assay, it becomes possible for the first time to characterize ascorbate function in relation to endogenous concentrations in RBCs.     

Four stage liquid chromatographic selection of methionyl peptides for peptide-centric proteome analysis: the proteome of human multipotent adult progenitor cells

Serial application of strong cation-exchange and diagonal reversed-phase chromatography selecting methionyl peptides by stepwise shifting them from their reduced to their sulfoxide and sulfone forms generates a four-stage fractionation system, allowing high coverage analysis of complex proteome digests by LC-MALDI-MS/MS. Application to the proteome of a human multipotent adult progenitor cell line (MAPC) identified 2151 proteins with high confidence as on average four MS/MS-spectra were linked to each protein. Our dataset contains several novel, potential marker proteins that may be evaluated as affinity-anchors for isolating different adult stem cells in further studies. Furthermore, at least 2 tyrosine kinases that were previously linked to the self-renewal potential of stem cells were identified, validating the stemness of the analyzed cells. We also present data hinting at possible involvement of the ubiquitin/proteasome machinery in steering proliferation and/or differentiation of MAPC. Finally, following comparison of the MAPC proteome with proteomes of four human differentiated cell lines reveals differential usage of chromosomal information: compared to differentiated cells, MAPC do not appear to hold any preference for expressing genes located on specific chromosomes.     

Characterization of individual mouse cerebrospinal fluid proteomes

Analysis of cerebrospinal fluid (CSF) offers key insight into the status of the CNS. Characterization of murine CSF proteomes can provide a valuable resource for studying CNS injury and disease in animal models. However, the small volume of CSF in mice has thus far limited individual mouse proteome characterization. Through nonterminal CSF extractions in C57Bl/6 mice and high‐resolution 2D‐LC MS/MS analysis of individual murine samples, we report the most comprehensive proteome characterization of individual murine CSF to date. We identified a total of 566 unique proteins, including 128 proteins from three individual CSF samples that have been previously identified in brain tissue. Our methods and analysis provide a mechanism for individual murine CSF proteome analysis. The data are available in the ProteomeXchange with identifier PXD000248 ( http://proteomecentral.proteomexchange.org/dataset/PXD000248 ).     

Proteomic analysis of the Arabidopsis nucleolus suggests novel nucleolar functions

The eukaryotic nucleolus is involved in ribosome biogenesis and a wide range of other RNA metabolism and cellular functions. An important step in the functional analysis of the nucleolus is to determine the complement of proteins of this nuclear compartment. Here, we describe the first proteomic analysis of plant (Arabidopsis thaliana) nucleoli, in which we have identified 217 proteins. This allows a direct comparison of the proteomes of an important nuclear structure between two widely divergent species: human and Arabidopsis. The comparison identified many common proteins, plant-specific proteins, proteins of unknown function found in both proteomes, and proteins that were nucleolar in plants but nonnucleolar in human. Seventy-two proteins were expressed as GFP fusions and 87% showed nucleolar or nucleolar-associated localization. In a striking and unexpected finding, we have identified six components of the postsplicing exon-junction complex (EJC) involved in mRNA export and nonsense-mediated decay (NMD)/mRNA surveillance. This association was confirmed by GFP-fusion protein localization. These results raise the possibility that in plants, nucleoli may have additional functions in mRNA export or surveillance.     

Modulation of red blood cell aggregation and blood viscosity by the covalent attachment of Pluronic copolymers

Despite many years of research, the physiologic or possible pathologic significance of RBC aggregation remains to be clearly determined. As a new approach to address an old question, we have recently developed a technique to vary the aggregation tendency of RBCs in a predictable and reproducible fashion by the covalent attachment of nonionic polymers to the RBC membrane. A reactive derivative of each polymer of interest is prepared by substitution of the terminal hydroxyl group with a reactive moiety, dichlorotriazine (DT), which covalently bonds the polymer molecule to membrane proteins. Pluronics are block copolymers of particular interest as these copolymers can enhance or inhibit RBC aggregation. Pluronics exhibit a critical micellization temperature (CMT): a phase transition from predominantly single, fully hydrated copolymer chains to micelle-like structures. The CMT is a function of both copolymer molecular mass and concentration. This micellization property of Pluronics has been utilized to enhance or inhibit RBC aggregation and hence to vary low-shear blood viscosity. Pluronic-coated RBCs were prepared using reactive DT derivatives of a range of Pluronics (F68, F88, F98 and F108) and resuspended in autologous plasma at 40% hematocrit. Blood viscosity was measured at a range of shear rates (0.1-94.5 s(-1)) and at 25 and 37 degrees C using a Contraves LS-30 couette low shear viscometer. RBC aggregation and whole blood viscosity was modified in a predictable manner depending upon the CMT of the attached Pluronic and the measurement temperature: below the CMT, RBC aggregation was diminished; above the CMT it was enhanced. This technique provides a novel tool to probe some basic research questions. While certainly of value for in vitro mechanistic studies, perhaps the most interesting application may be for in vivo studies: typically, intravital experiments designed to examine the role of RBC aggregation in microvascular flow require perturbation of the suspending plasma to promote or reduce aggregation (e.g., by the addition of dextran). By binding specific Pluronics to the surface, we can produce RBCs that intrinsically have any desired degree of increased or decreased aggregation when suspended in normal plasma, thereby eliminating many potential artifacts for in vivo studies. The copolymer coating technique is simple and reproducible, and we believe it will prove to be a useful tool to help address some of the longstanding questions in the field of hemorheology.