This report describes an analysis of the red blood cell proteome by ion trap tandem mass spectrometry in line with liquid chromatography. Mature red blood cells lack all internal cell structures and consist of cytoplasm within a plasma membrane envelope. To maximize outcome, total red blood cell protein was divided into two fractions of membrane-associated proteins and cytoplasmic proteins. Both fractions were divided into subfractions, and proteins were identified in each fraction separately through tryptic digestion. Membrane protein digests were collected from externally exposed proteins, internally exposed proteins, “spectrin extract” mainly consisting of membrane skeleton proteins, and membrane proteins minus spectrin extract. Cytoplasmic proteins were divided into 21 fractions based on molecular mass by size exclusion chromatography. The tryptic peptides were separated by reverse-phase high-performance liquid chromatography and identified by ion trap tandem mass spectrometry. A total of 181 unique protein sequences were identified: 91 in the membrane fractions and 91 in the cytoplasmic fractions. Glyceraldehyde-3-phosphate dehydrogenase was identified with high sequence coverage in both membrane and cytoplasmic fractions. Identified proteins include membrane skeletal proteins, metabolic enzymes, transporters and channel proteins, adhesion proteins, hemoglobins, cellular defense proteins, proteins of the ubiquitin-proteasome system, G-proteins of the Ras family, kinases, chaperone proteins, proteases, translation initiation factors, and others. In addition to the known proteins, there were 43 proteins whose identification was not determined.

Adult mouse brain contains at least two distinct spectrin subtypes, both consisting of 240-kD and 235-kD subunits. Brain spectrin(240/235) is found in neuronal axons, but not dendrites, when immunohistochemistry is performed with antibody raised against brain spectrin isolated from enriched synaptic/axonal membranes. A second spectrin subtype, brain spectrin(240/235E), is exclusively recognized by red blood cell spectrin antibody. Brain spectrin(240/235E) is confined to neuronal cell bodies and dendrites, and some glial cells, but is not present in axons or presynaptic terminals.

The erythrocyte membrane skeleton composed of spectrin, actin, and several other proteins is essential for the maintenance of the erythrocyte shape, reversible deformability, and membrane structural integrity in addition to controlling the lateral mobility of integral membrane proteins. In this review, we shall give an historical development of the current model of the erythrocyte membrane skeleton. We will then describe how the experimental technology developed to study the normal membrane skeleton has paved the way for the recent identification of alterations of skeletal protein interactions in hereditary spherocytosis, hereditary elliptocytosis, and hereditary pyropoikilocytosis. We will conclude with a discussion of some of the more exciting and promising directions for future research that are currently being initiated in this vanguard field of cell biology.

We have demonstrated the existence of a spectrin-like protein in a variety of nonerythroid cultured cells. Indirect immunofluorescence studies with monospecific antispectrin IgG indicated the presence of proteins that have common antigenic determinants to spectrin in embryonic chicken cardiac myocytes, mouse fibroblast lines (3T3, simian virus 4-transformed 3T3), and rat hepatoma lines (HTC, HMOA). Two spectrin-like peptides of 240,000 and 230,000 daltons were immunoprecipitated from octyl glucoside-solubilized embryonic chicken cardiac myocytes, along with associated cytoskeletal proteins. Immunoautoradiographic characterization of the myocyte immunoprecipitate showed that only the spectrin-like 240,000- and 230,000-dalton peptides were stained with monospecific antispectrin IgG and 125I-labeled protein A. One-dimensional partial proteolytic mapping of the myocyte 240,000- and 230,000-dalton peptides showed that these peptides share substantial sequence homology with embryonic chicken erythrocyte spectrin 240,000- and 220,000-dalton peptides.

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.

IgG autoantibodies in human serum selectively bind to a glycopeptide antigen that appears on senescent and damaged cells in situ. We identified the membrane protein from which the senescent cell antigen is derived by using a phagocytosis-inhibition assay and immunoautoradiographic gel staining and electroblotting techniques. Results of the phagocytosis-inhibition assay revealed that only the purified transmembrane glycoprotein designated "band 3" and senescent cell antigen inhibited the phagocytosis of erythrocytes induced by IgG eluted from senescent erythrocytes. Purified spectrin, syndein, band 4.1, actin, glycophorin A, and intact or desialylated sialoglycoprotein periodic acid/Schiff (PAS) staining bands 1-4 containing glycophorins A, B, and C did not inhibit phagocytosis. Specific antibodies against the senescent cell antigen and erythrocyte band 3 were used to identify the membrane protein from which the senescent cell antigen is derived. Band 3-related polypeptides (MrS approximately equal to 60,000, 42,000, and 18-26,000) were identified in erythrocyte ghosts prepared in the presence of diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, and EDTA by immunoautoradiography with antiband 3. Antibodies to senescent cell antigen reacted with band 3 and the same lower Mr band 3-related polypeptides. Thus, the senescent cell antigen is immunologically related to band 3.