Proteomic mining of the red blood cell: focus on the membrane proteome

The plasma membrane is strategically located at the interface between the inside and the outside of the cell. Membrane proteins, as part of the plasma membrane, act as key players mediating diverse cellular functions including, but not limited to, metabolite and ion transport, intercellular communication, cell adhesion and cell movement [1].

The main function of the red blood cell (RBC) is to mediate O₂/CO₂ exchange between cells/tissues and lungs and this is only achieved thanks to the morphology and mechanical deformability of the RBC membrane, which is responsible for the RBC’s capability to perfuse across vessels and capillaries along its 120-day journey. The RBC membrane possesses other distinctive features, such as high elasticity (with little increase on surface area) and robustness (stronger than steel in terms of structural resistance) [2]. These unique properties result from a composite structure in that cholesterol and phospholipids, which compose the plasma membrane envelope, are anchored to a 2D elastic network of skeletal proteins through transmembrane proteins embedded in the lipid bilayer [2,3]. Both the appropriate function of integral membrane proteins and their interaction with the cytoskeleton are vital for the maintenance of RBC structural stability and RBC shape. Many RBC disorders are caused by malfunctioning membrane proteins [4,5]. Hence, studying the membrane proteome is important for comprehending the biology of disease states in the quest for novel biomarkers. Consequently, it is also important at the pharmacological level as many successful drugs known to date target membrane proteins modulating the proteome’s activity [6].

Since its very beginning, the study of membrane proteomes has been a major challenge. In 1974, a review was published concerning the organization of the human RBC membrane, compiling several studies and hypothesizing on a possible arrangement for the most abundant RBC integral membrane proteins and interactors [7]. Given their importance, it is not surprising that membrane proteins have been studied by a variety of biochemical techniques. One of those techniques is 2D gel electrophoresis (2DE), which paved the way to the proteomics era [8]. In 1978, 2 years after the application of the O’Farrell 2DE system to the study of membrane proteins [9,10], over 200 spots were resolved from human RBC membranes [11]. However, the application of 2DE to the study of membrane proteins was far from being ideal, owing to the unique characteristics of membrane proteins, such as their amphiphilic nature (poor solubility in the aqueous buffers used for isoelectric focusing), high isoelectrical points (pIs can be higher than the upper limit of the immobilized pH gradient strips) and low abundance, which greatly hinder detection through 2DE [1,12–14]. To overcome the solubility issue, Rosenblum et al. used different concentrations of urea, NP-40 detergent and mercaptoethanol to detect about 600 spots using silver staining [15]. However, the real improvement on the methods/studies presented in the large majority of the publications released until the early 1990s relied on the number of additional spots and the reproducibility of the new/modified methodologies rather than identification and classification of hypothetical new proteins found. One must bear in mind that before the development of an analytical technique for naïve peptide/protein identifications, in this case, peptide mass fingerprint [16–20], proteins could only be identified by means of targeted approaches, such as comigration with known proteins or immunoblotting, a more sensitive technique [21]. Therefore, the first ‘serious’ proteomic study on RBC membranes (i.e., the first study where modern mass spectrometry [MS] and database searching in the postgenomic era was employed) was the one performed by Low and coworkers in 2002 [22]. Using 1DE and 2DE, silver staining and in-gel trypsin digestion of selected spots followed by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF)-MS, the authors were able to identify 84 unique proteins: 59 proteins were identified by 2DE and 44 by 1DE (19 proteins were common to both approaches). In addition, several isoforms were found in the study. The first indepth study on the RBC proteome was conducted in 2004, where cytoplasmic and membrane fractions were further fractionated and resulting subfractions were then analyzed (and classified) resulting in the identification of 181 unique proteins, 91 of which were identified from the membrane fraction [23]. In 2005, Tyan et al. were able to identify 272 proteins using a trypsin-immobilized chip for protein digestion prior to 2D high-performance liquid chromatography–electrospray ionization (2D-HPLC-ESI)-MS/MS [24]. In the same year, Bruschi et al. presented an approach to improve the analysis of high-molecular-weight proteins in 2DE [25]. The authors used diluted Immobiline™ gels combined with sample delipidation generating gels with more than 500 spots, including filamentous proteins such as spectrins and ankyrins and integral membrane proteins as bands 3, 4.1 and 4.2 [25]. Still, in 2005, Kakhniashvili et al. used 2D fluorescence difference gel electrophoresis (2D-DIGE) to compare the RBC membrane profile of one sickle cell disease patient to one healthy individual and found 49 differentially expressed spots using a threshold of 2.5-fold. Selected spots were further analyzed by LC-MS after in-gel trypsin digestion to identify 44 protein forms from 22 unique proteins [26]. The same strategy was employed to investigate the therapeutic action of hydroxyurea in sickle cell disease [27]. In 2006, Pasini and colleagues published the most complete study on the human RBC membrane proteome known to date [28]. By combining sample preparation techniques and top-quality MS instruments, such as quadrupole time-of-flight (Q-TOF) and Fourier transform–ion cyclotron resonance (FT-ICR), they were able to identify 314 membrane proteins (and also 252 soluble proteins). A very promising gel-based approach for analysis of membrane proteins is 2D blue-native/SDS-PAGE. This novel approach was applied to the study of the RBC membrane by van Gestel et al. who were able to detect 146 spots, from which 524 unique proteins were identified by LC-MS. These data were compared with two other comprehensive datasets produced by Pasini et al.[28] and Bosman et al.[29] and it was exciting to observe that only 112 from a total of 1431 unique proteins were commonly identified in the three studies. In addition, the authors were able to use blue-native/SDS-PAGE in combination with CyDye labeling to quantitatively analyze samples from healthy volunteers and a patient suffering from congenital anemia [30], an approach that can potentially be used for biomarker discovery. Noteworthy, although only targeted to cytoplasmic proteins, is the study carried out by Roux-Dalvai et al., since the use of the new technology of peptide ligand libraries was responsible for the identification of as many as 1578 proteins from a highly purified preparation of RBCs.

Datasets from some of the studies herein presented were gathered on a minireview paper [31], but unfortunately the authors have not provided information on the accession numbers, therefore making it difficult to determine how many proteins were found to present and also to compare newly obtained datasets with the data collected so far. Indeed, no review produced to date compiles the information concerning protein identifications (and classification) together with the correspondent accession number (e.g., UniProt, International Protein Index) in the different studies, including very interesting recently published reviews [32–35].

Conclusion & future outlook

The RBC proteome was believed to be a simple entity when taking into account that they are enucleated cells and lack internal organelles and protein synthesis machinery. But in the past decade, the knowledge of the RBC proteome has increased dramatically and changed this picture. This was owing not only to the development of sample preparation techniques (e.g., fractionation, depletion/enrichment), but mostly to the use of sophisticated mass spectrometers, appropriate search algorithms and to comprehensive human protein databases. In order to entirely comprehend the action of the RBC, and particularly the different roles played by its membrane, it is necessary to identify every single protein, their structure, function, post-translational modifications, interactions, location and abundance in the cell. RBC membrane proteome revelation will have a tremendous impact in medicine, including hot topics as transfusion medicine [33,36,37] and malaria. Malaria, which is caused by a eukaryotic protist of the genus Plasmodium, is responsible for the death of about three million people worldwide [38]. Malaria parasites first invade hepatocytes of the human host before traveling into the blood to infect RBCs. As circulating infected RBCs are removed in the spleen, Plasmodium falciparum (responsible for 80% of malaria cases and 90% deaths from malaria) exhibits adhesion proteins at the RBC surface, causing RBCs to attach to the blood vessels. These surface adhesion proteins, such as P. falciparum erythrocyte membrane protein 1, PfEMP1, are exposed to the immune system and would, therefore, be an easy target. But remarkably, the parasites stay one step ahead of the immune system by presenting extreme diversity in PfEMP1 isoforms: there are over 60 variations of the protein within a single parasite and virtually limitless versions within parasite populations [39]. Furthermore, there are other RBC proteins of particular interest, such as glucose-6-phosphate dehydrogenase or Duffy antigens (used by Plasmodium vivax to enter the cell), whose expression deficiency in RBCs results in increased protection against P. vivaxand severe malaria [40–42]. These facts alone present major challenges for clinicians and researchers, and set important cases for the continued detailed study of the RBC membrane proteome. But clinical proteomics applications are far from being limited to diseases that directly affect the RBC; other diseases that indirectly provoke alterations in the RBC are no less important to be used for diagnostic purposes. For instance, when RBCs are depleted of critical enzymes needed for intermediary metabolism and antioxidant activity, it results in oxidation of critical membrane proteins, lipids and hemoglobin that lead to distortion and rigidity of the RBC membrane.

It is also important to acknowledge that RBCs are one of the most abundant cells in humans and are involved in numerous processes through the interplay with other blood cells and endothelial cells. Moreover, with a lifespan as long as 4 months, they potentially accumulate modifications on their proteins (surface and membrane will likely be the more affected ones) that can indirectly report the underlying features of a specific pathology, ultimately before the symptoms were ever manifested.

Financial & competing interests disclosure

The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.