FIELD OF THE INVENTION

The present invention generally relates to methods for identifying and/or isolating at least one foetal primitive nucleated red blood cell. In particular, the invention relates to a method of identifying at least one foetal primitive nucleated red blood cell in a sample by detecting at least one membrane protein specific to the foetal primitive nucleated red blood cell.

BACKGROUND TO THE INVENTION

Currently, prenatal diagnosis of chromosomal and single gene disorders rely on foetal cells obtained by invasive procedures such as amniocentesis, chorionic villous sampling (CVS) or foetal blood sampling (FBS) for cytogenetic and/or molecular analysis. These invasive tests carry a small but significant risk of foetal miscarriage. On the one hand this limits the uptake of the diagnostic test out of fear of foetal loss, and on the other hand causes the demise of an otherwise healthy foetus. Non-invasive methods to diagnose the foetal genetic condition by enriching and analyzing foetal ceils and foetal DNA that circulate in maternal blood have been studied.

Of the foetal cells that enter the first trimester maternal circulation, primitive foetal nucleated red blood cell (FPN BC) is the preferred target cell. This is because of its short life-span and hence it is unlikely to persist from a previous pregnancy, unlike the situation with foetal lymphocyte where this phenomenon could be the basis for a misdiagnosis. First-trimester FPNRBC contain Epsilon-globin 0, an ideal foetal cell identifier which is highly specific as expression declines after the first trimester.

In humans, foetal primitive nucleated red blood cells (FPNRBCs, foetal primitive erythroblasts, first trimester foetal nucleated blood cells (FNRBCs)) generated in the yolk sac mesoderm remain the predominant blood cell type in the embryonic circulation until 10 weeks post- conception. Studies on this cell type in humans have been limited owing to limited access to pure populations of these cells for laboratory investigations; only recently has it been shown that these cells may enucleate within the first trimester human placenta, suggesting that may be terminally differentiated. Primitive erythroblasts differ from foetal definitive erythroblasts not only in their anatomical site of origin, but also in the types of haemoglobins contained within them. Adult anucleate red blood cells (AARBCs, adult red blood cells (RBCs)) are smaller, discoid, readily deformable cells that are produced in the long bone marrow. Owing to their ready availability, these cells have been extensively studied in recent years. Using mass spectrometry, AARBC membrane and cytoplasmic proteins have been characterized, and differences demonstrated between mouse and human AARBCs.

Enrichment of first trimester FPNRBC from maternal blood for non-invasive prenatal diagnosis has been a difficult task due to the lack of unique antibodies against its surface proteins. While WBCs can be separated using anti-CD45 antibody from maternal blood samples, separation of FPNRBCs from overwhelming adult RBCs has been the challenge. The success of non-invasive prenatal diagnosis using first trimester FPNRBCs from maternal blood depends on the enrichment of these rare cells (one cell amongst a million nucleated maternal cells).

The goal of isolating and analyzing foetal DNA from as little as one FPNRBC recovered from amongst a million nucleated maternal cells is possible with the use of automated micromanipulation, laser capture microscopy systems and downstream analysis of foetal cell with single cell whole genomic amplification coupled with array CGH technologies. Therefore, it is not inconceivable that very small numbers of foetal cells (-20 cells) enriched from maternal blood from an on-going euploid pregnancy may actually be sufficient for non-invasive prenatal diagnosis.

Accordingly, there is a need in the art for a method for detecting and/or isolating FNRBCs and provide methods as potential reliable approaches for future NIPD using FNRBCs present in maternal blood.

SUMMARY OF THE INVENTION

According to one aspect of the invention, there is provided a method for identifying and/or isolating at least one foetal erythroblast, the method comprising: detecting the expression of at least one foetal erythroblast specific marker selected from the group consisting of neutral amino acid transporter B (SLC1A5), solute carrier family 3 (activators of dibasic and neutral amino acid transport) member 2 isoform A (SLC3A2), splice isoform A of chloride channel protein 6, transferrin receptor protein 1, splice Isoform 3 of Protein GPR107 precursor, Olfactory receptor 11H4, Splice Isoform 1 of Protein C9orf5, Cleft lip and palate transmembrane protein 1, BCG induced integral membrane protein BIGM103, antibacterial protein FALL-39 precursor, CAAX prenyl protease 1 homolog, splice isoform 2 of synaptophysin-like protein, vitamin K epoxide reductase complex subunit 1-like protein 1, splice isoform 1 of Protein C20orf22 (ABHD12), Hypothetical protein DKFZp564K247 (Hypoxia induced gene 1 protein) (IPI Accession No. IPI00295621), Hypothetical protein DKFZp586C1924 (IPI Accession No. IPI00031064), ALEX3 protein variant, hypothetical protein MGC14288 (IPI Accession No. IPI00176708), protein with IPI Accession No. IPI00639803, and protein with IPI Accession No. IPI00646289; wherein detection of the marker indicates the presence of the foetal erythroblast.

According to other aspects of the invention, there is also provided a marker or identifying foetal erythroblast selected from the foetal erythroblast specific markeraccoding to any aspect of the present invention, a method of diagnosing at least one prenatal disorder in an individual using at least one foetal erythroblast specific marker, an antibody or antigen binding fragment thereof that is capable of binding to at least one foetal erythroblast specific marker, and a kit for identifying and/or isolating foetal erythroblast in a sample.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is histological images of FPNRBCs and AARBCs stained with Wright's stain showing (A) FPNRBCs (nucleated); (B) AARBCs without nuclei.

Figure 2 is a Venn diagram of FPNRBC proteins identified in organic solvents MeOH and TFE.

Figures 3A-B are graphs showing the locations and function of 133 FPNRBC membrane proteins. Figure 4 is a Venn diagram of membrane proteins with potential surface domains in AARBCs and FPNRBCs.

Figure 5 is images of validation of unique membrane proteins of FPNRBCs by reverse transcriptase-PCR (RT-PCR).

Figure 6A is images of immunohistochemistry of membrane proteins on FPNRBCs and AARBCs. Figure 6B is box plot showing the statistical significance (*) of intensities of immunoreaction by antibodies.

Figure 6C is a bar graph showing the statistical significance (*) of staining intensity of immunoreaction by antibodies.

Figure 6D is images of immunohistochemistry of membrane proteins on FPNRBCs and AARBCs. Figure 7A is images of immunohistochemistry of membrane proteins on FPNRBCs and AARBCs in pre-sort and post-sort fraction with NAT-B marker.

Figure 7B and 7C are bar graphs showing the percentage FPNRBCs in NAT-B positive fraction, NAT-B negative fraction pre-sort and post-sort. Figure 8 is a table presenting data on proteins identified based on single peptides from TFE and eOH extractions and ion score.

Figure 9 is a table presenting peptide sequences for proteins identified from TFE and MeOH extractions.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Bibliographic references mentioned in the present specification are for convenience listed in the form of a list of references and added at the end of the examples. The whole content of such bibliographic references is herein incorporated by reference.

Reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" as used herein thus usually means "at least one".

The term "comprising" is herein defined as "including principally, but not necessarily solely". Furthermore, the term "comprising" will be automatically read by the person skilled in the art as including "consisting of". The variations of the word "comprising", such as "comprise" and "comprises", have correspondingly varied meanings.

The term "fragment" is herein defined as an incomplete or isolated portion of the full sequence of a protein which comprises the active/binding site(s) that confers the sequence with the characteristics and function of the protein. In particular, it may be shorter by at least one amino acid. More in particular, the fragment comprises the binding site(s) that enable the protein to bind to at least one marker of the present invention.

The term "antigen binding fragment" is herein defined as an incomplete or isolated portion of the full sequence of an antibody which comprises the active/binding site(s) that confers the sequence with the characteristics and function of the antibody. In particular, it may be shorter by at least one amino acid. More in particular, the fragment comprises the binding site(s) that enable the antibody to bind to at least one marker of the present invention.

The term "erythroblast" as used herein refers to a red blood cell having a nucleus. In particular, an erythroblast refers to a nucleated precursor cell from which a reticulocyte develops into an erythrocyte. "Erythroblast" may be used interchangeably with a "Normoblast" and refers to a nucleated red blood cell, the immediate precursor of an erythrocyte. For example, the erythroblast may be of mammalian origin. In particular, the erythroblast may be a primitive or human foetal erythroblast. The term "foetal primitive nucleated red blood cell (FPNRBC)" is herein defined as cells generated in the yolk sac mesoderm that remain as the predominant blood cell type in the embryonic circulation until 10 weeks post-conception. The term "FPNRBC" may be used interchangeably with foetal primitive erythroblasts or first trimester foetal nucleated red blood cells (FNRBCs)).

The phrase "adult anucleate red blood cells (AARBCs, adult red blood cells (RBCs))" is herein defined as cells that are relatively smaller as compared to FPNRBC, discoid, readily deformable and produced in the long bone marrow. The term AARBCs may be used interchangeably with "adult red blood cells (RBCs)".

The term "mammalian" is herein defined as a mammalian individual, in particular, a primate for example a human being. For purposes of research, the subject may be a non-human. For example the subject may be an animal suitable for use in an animal model, e.g., a pig, horse, mouse, rat, cow, dog, cat, cattle, non-human primate (e.g. chimpanzee) and the like.

The term "sample" as used herein refers to a subset of tissues, cells or component parts (for example fluids) that may include, but are not limited to, maternal tissue, maternal blood, cord blood, amniocenteses, chorionic villus sample, foetal blood, and/or foetal tissue/fluids. In particular, foetal tissue may be trophoblast tissue, placental tissue or a combination thereof. The sample as used in the present invention may have been previously subjected to a density gradient purification including, but not limited to, Ficoll gradient and Percoll gradient.

The term "CD45 negative" as used herein refers to any cell that expresses no signal or is negative for native, recombinant or synthetic forms of the CD45 molecule/ marker. The presence of CD45 expression on a cell in a sample may be determined using any immunostaining method known in the art and using any anti-CD45 reagent. Any cells positively stained with anti-CD45 reagent may be excluded as these may include CD45 positive white blood cells (WBC). The term "nucleated" as used herein refers to a cell that has a nucleus. Nucleated cells may be distinguished from red blood cells which are not nucleated based on any nuclear staining known in the art.

The term "prenatal disorder" as used herein refers to diseases or conditions in a foetus or embryo before it is born. The prenatal disorder may be selected from the group consisting of a chromosomal disorder, a genetic disorder, or a combination thereof. In particular, the prenatal disorder may be selected from the non-limiting group consisting of Down Syndrome, Edwards Syndrome, Patau Syndrome, a neural tube defect, spina bifida, cleft palate, Tay Sachs Disease, sickle-cell anemia, thalassemia, cystic fibrosis, fragile X syndrome, spinal muscular atrophy, myotonic dystrophy, Huntington's Disease, Charcot-Marie-Tooth disease, haemophilia, Duchenne muscular dystrophy, mitochondrial disorder, Hereditary multiple exostoses, osteogenesis imperfecta disorder, a combination thereof and the like.

At present, enrichment of FPNRBCs from maternal blood has been a challenge because of their rarity in maternal circulation and the lack of surface specific antigens for immunocellsorting of these cells. CD71 and GPA are commonly used to enrich these cells from maternal blood: as such use of CD71 may result in loss as this surface antigen is expressed only on -68% of FPNRBCs and GPA binds to both AARBCs and FNRBCs making analyses of enriched sample difficult because of a very high background of AARBCs.

Cell surface membrane proteins have an integral role in maintaining health: when altered structurally or functionally, they are responsible for the more commonly known diseased states such as spherocytosis and sickle cell disease, and also the less commonly recognized conditions such as elliptocytosis, familial pseudohyperkalaemia, dehydrated hereditary stomatocytosis and membrane -defects in β-thalassemia. Knowledge about cell membrane proteins and their functions in health and disease could lead to understanding mechanisms of disease processes such as the invasion of the malaria parasite into human erythrocytes and the possibility of developing therapeutic interventions.

In contrast to the large amount of information already available on the AARBC membrane proteome, no information is currently available on the proteome of human foetal primitive erythroblasts. Only very limited data on their cell surface antigens such as CD71 and Glycophorin A and some information on their cytoplasmic haemoglobin are known. The knowledge on the membrane proteome of the FPNRBC may be useful in two ways: to facilitate a deeper understanding of primitive erythropoiesis in humans, and to identify specific surface antigen(s) for the enrichment of ε-globin-positive foetal primitive erythroblasts from maternal blood for non-invasive prenatal diagnosis. It has been suggested that the ε-globin-positive foetal primitive erythroblast is the ideal foetal cell type for non-invasive prenatal diagnosis and identification of unique membrane proteins on either FPNRBC or AARBC may be exploited for non-invasive prenatal diagnosis in the future. Differences between human FPNRBCs and AARBCs are disclosed herein. Accordingly, there is a need in the art to provide markers that facilitate the identification and/or isolation of FPNRBCs.

The inventors of the present application made the first attempt to explore unique membrane proteins of FPNRBCs. They identified unique surface proteins with transmembrane domains that may be useful as markers for the separation of human FPNRBCs from adult RBCs by immuno-cell sorting protocols. Antibodies against these proteins may enable the immuno-cell sorting.

According to an aspect of the present invention, there is provided a method for identifying at least one foetal erythroblast the method comprising: detecting the expression of at least one foetal erythroblast specific marker selected from the group consisting of neutral amino acid transporter B (SLC1A5), solute carrier family 3 (activators of dibasic and neutral amino acid transport) member 2 isoform A (SLC3A2), Splice Isoform A of Chloride channel protein 6, Transferrin receptor protein 1, Splice Isoform 3 of Protein GPR107 precursor, Olfactory receptor 11H4, Splice Isoform 1 of Protein C9orf5, Cleft lip and palate transmembrane protein 1, BCG induced integral membrane protein BIGM103, Antibacterial protein FALL-39 precursor, CAAX prenyl protease 1 homolog, Splice Isoform 2 of Synaptophysin-like protein, Vitamin K epoxide reductase complex subunit 1-like protein 1, Splice Isoform 1 of Protein C20orf22 (ABHD12), Hypothetical protein DKFZp564K247 (Hypoxia induced gene 1 protein) (IPI Accession No. IPI00295621), Hypothetical protein DKFZp586C1924 (IPI Accession No. IPI00031064), ALEX3 protein variant, Hypothetical protein MGC14288 (IPI Accession No. IPI00176708), protein with IPI Accession No. IPI00639803 and protein with IPI Accession No. IPI00646289; wherein detection of the marker indicates the presence of the foetal erythroblast. In particular, the foetal erythroblast is of mammalian origin. More in particular, the foetal erythroblast is of human origin. A brief description of individual foetal erythroblast specific markers, on their location, physiological roles (including those related to human foetal development), and diseases related to their mutations is provided below. The identification of foetal erythroblast specific marker will facilitate the identification and isolation of FNRBCs from maternal blood, and thus provides for a reliable approach for future NIPD using FNRBCs present in maternal blood.

Brief description of the foetal erythroblast specific markers Amino acid transporters

Transporters of cells and organelles regulate the uptake and efflux of important compounds such as sugars, amino acids, nucleotides, ions and drugs. Solute carrier (SLC) series of transporters include genes encoding passive transporters, ion transporters and exchangers.

Two amino acid transporting SLC proteins (SLC1A5 and SLC3A2) were identified unique to plasma membrane of foetal primitive erythroblasts. SLC1A5, neutral amino acid transporter B°, is a Na ⁺ dependent transporter of SLC1 family expressed in kidney and intestine. SLC1A5 amino acid transport protein identified in foetal erythroblasts belongs to ASCT2 system which can transport glutamine and asparagine with high affinity, and neutral amino acids methionine, leucine and glycine with low affinity. SLC3A2 (CD98hc) is the heavy chain of the hetero-dimeric protein 4F2 (CD98). CD98 as a hetero-dimer, is involved in amino acid transportation where the substrate specificity varies with the nature of the light chain. Different domains of CD98hc are necessary for association with light chains. Studies on the amino acid transport in human placenta is correlated well with the expression of mR As of CD98hc, and a possible role for these proteins in materno-foetal transfer of amino acids and iodo-thyronines is also suggested. CD98hc is found to be co- localized with θ β ₃ integrin to promote adhesion and motility of extravillous trophoblasts suggesting the functional importance of CD98hc in human foetal development.

There is evidence for amino acid transport in matured human red blood cells too; It has been previously demonstrated that a Na+ dependent amino acid transport system, and recently, CD98hc associated with L-type amino acid transporter 1 (LAT1) or LAT2 light chain may be involved in the cellular uptake of S-nitroso-L-cysteine into human adult red blood cells. However, the absence of CD98hc in mass spectrometric studies of AARBCs may probably be due to smaller peptides generated during MS.

Anion transporters Chloride channel (Clc) genes (Clcl-10) are expressed in all phyla from bacteria to man. Clc mediated anion transport is considered to be the main function of most of the Clc proteins. Three isoforms of Clc6 are known. Mutations in CIc genes have been implicated in various human diseases such as myotonia, renal salt loss, deafness, urinary protein loss, kidney stones, osteoporosis, blindness, and lysosomal storage disease. Recent studies in animal models suggest that Ccl6 may predominantly reside intracellular^ in endosomes. In AARBCs, in addition to a small chloride channel other inorganic ion transporters, such as urea transporter- B (SLC14A1) and bicarbonate/chloride exchanger (SLC4A1, Band 3), are known to be functional.

Binding proteins

Membrane receptors which can bind hormones, growth factors and metabolites are important for cellular growth and function. Transferrin receptor protein 1, Splice isoform 2 of protein GPR107 precursor, and olfactory receptor 11H4 were identified as being unique to primitive foetal erythroblasts. Transferrin receptor was initially identified on maturing erythroid cells and placenta. Iron is an essential requirement for the synthesis of haemoglobin in all stages in erythroid cells to where iron is transported by the transferrin receptor which, however, is absent in AARBCs as it is lost from reticulocytes as they become mature. Guanine nucleotide binding protein (G protein) coupled receptors (GPCRs) have 7 transmembrane helices and are expressed on cell surface, and bind to almost all of the known neurotransmitters and hormones released synaptically or those that are secreted into the circulatory system controlling organ functions. G-proteins are predominant intracellular molecules that bind and link GPCRs to second messenger systems such as adenyl cyclase, phospholipases, and ionic conductance channels. GPCRs are targets for 40% of all approved drugs and are the main focus of intense pharmaceutical research due to their key roles in cell physiology and disease, and the presence of GPR107 in foetal erythroblasts does not exclude the possibility for potential research using this cell type for foetal therapy.

Olfactory receptor (OR) is the largest mammalian gene family that codes for odorant receptors. Identification of one of the ORs (OR family H subfamily 11) in primitive foetal erythroblasts supports the earlier reports of an OR in hematopoietic cells and tissues: low level expression of OR-mRNA in human erythroleukemia and myeloid cell lines, and in tissues containing cells of erythroid lineage, such as human bone marrow and foetal liver were reported by Feingold and his colleagues. There is evidence for the expression of OR in non-olfactory testicular tissue; in humans, expression of hOR 17-4 and its functional role in sperm chemotaxis is known. In addition, human prostate specific G-protein coupled receptor (PSGR) with properties characteristic of an olfactory receptor was also observed in olfactory zone and the medulla oblongata (human), liver (rat) and in brain and colon (mouse).

Catalytic

CAAX prenyl endopeptidase also known as FACE, farnesylated protein-converting enzyme, is important for prenylation of CAXX motif containing eukaryotic proteins for their function and membrane targeting. FACE-1 and FACE-2 are two human enzymes expressed in several tissues, for example, leukocytes, ovary, testis, kidney and placenta. Prelamin-A is the substrate for FACE-1 and mutations in prelamin A cleavage site or FACE-1 enzyme have been documented in genetic diseases such as Hutchinson-Gilford progeria and mandibuloacral dysplasia. The identification of CAAX prenyl protease 1 homologue, an integral membrane protein containing seven transmembrane domains in foetal erythroblasts, as in other human tissues, indicates a possible house-keeping role for this enzyme in the processing of prenylated proteins.

Vitamin K epoxide reductase complex subunit 1 like protein (VKORCILI), identified in the present disclosure, is the first report in a human erythroid cell type membrane protein whose sub-cellular location is not yet defined. VKORC1 was reported to be warfarin-sensitive. Vitamin K-dependent clotting factor deficiency type 2 (VKCFD2) in humans showing warfarin resistance is the result of mutation in VKORC1. Foetal warfarin syndrome (warfarin embryopathy) due to warfarin exposure during pregnancy is well known. It has also been suggested that rare polymorphisms and interethnic differences in VKORC1 determines warfarin requirement. Signaling pathway

Splice isoform 1 of Protein C90RF5 identified in primitive foetal erythroblasts is annotated to be involved in signalling pathways. A novel human transcript CG-2 (C90RF5) was isolated from the familial dysautonomia candidate region on 9q31 and its expression was seen in human adult and foetal tissues such as brain, lung, liver and kidney. C90RF5 was also found to be upregulated in prostrate cancer where the role for this gene is unknown.

Vesicle recycling

Synaptophysin-like protein, pantophysin, an isoform of synaptophysin identified in primitive erythroblasts was annotated to be located in plasma/vesicle membrane. It is highly conserved and considered as a novel pre-synaptic marker for neurons and neuroendocrine (NE) cells. Pantophysin is localized in cytoplasmic micro-vesicles of various secretory, shuttling, and endocytotic recycling pathways and are co-localized with synaptophysin in transfected non- neuroendocrine and neuroendocrine cells and in neuroendocrine tissues. Non-neuronal distribution of pantophysin in epithelial, muscle tissues and fibroblasts has already been documented.

Antimicrobial proteins Expression of BCG induced integral membrane protein BIGM 103 (BCG induced gene in monocyte, clone 103) in foetal erythroblasts is novel. This protein was first identified from cDNA library prepared from monocytes induced with BCG cell wall. BIGM103 has sequence similarity with Zip-like family of proteins and matched with hZIP2 and hZIPl and is predicted to possess zinc transporter and metallo-protease activities. A possible role in phagocytosis- mediated elimination of microbial components in macrophages and dendritic cells has also been suggested. FALL39 identified in foetal erythroblasts is one of the antimicrobial peptides of neutrophil granules such as Azurocidin (CAP-37) and CAP-57. FALL39 was also identified from human bone marrow and testis. Contrary to the microbicidal function, a novel pro- tumorigenic role for mature FALL-39 (hCAP-18/LL-37) was also demonstrated in ovarian cancer, through activation of matrix metalloproteinases, and there is evidence for strong association between leukocyte infiltration and cancer progression.

Proteins with no known function but candidates for research related to foetal development.

Cleft lip and palate transmembrane protein 1— To date, no functional role for CLPTM 1 is defined. CLPTM 1 is reported to be homologous with Cisplatin Resistance Related gene-9, and observed to be more expressed in clinical samples resistant to chemotherapy in breast cancer. Clinically, folate deficiency is known to be associated with cleft lip and/or palate and autoantibodies against folate receptors are reported to be present in mothers of children with cleft lips. Folate is an important vitamin for several metabolic pathways including those leading to the synthesis of nucleic acids, and are considered vital during infancy and pregnancy. Functional role for CLPTM 1 in foetal erythroblast plasma membrane needs further investigation.

Hypoxia-inducible gene 1 protein, (HIG1 domain family member 1A, HIGD1A) is one of the genes expressed during hypoxia. HIGD1A gene expression was reported in human hematopoietic stem/progenitor cells and in human cervical cells cultured under hypoxic conditions. HIG1 expression in cytoplasmic vesicles and mitochondria appears to be induced by both hypoxia and tumour micro environmental stressor such as glucose deprivation. In humans, the normal foetal development depends on the availability of oxygen and nutrients to the foetus. Identification of HIGDIA protein expression in primitive foetal erythrobiasts, but not in adult erythrocytes, correlates with the relatively hypoxic environment of the placenta as compared to that of adult blood circulation.

Others Identification of ALEX3 protein variant in foetal erythrobiasts is unique. The genes for ALEXl, ALEX2 and ALEX3 are localized in human X chromosome. Significantly reduced or loss of mRNA expression of ALEXl and ALEX2 in epithelial carcinomas (human lung, prostate, colon, pancreas, and ovarian carcinomas) but not in cell lines from other types of tumours leads to a speculation that ALEX genes may play a role in suppression of tumours originating from epithelial tissue.

Reports on protein expression or functional identity of five of the identified proteins of foetal erythrobiasts (with at least one transmembrane domain) are not available in any other cell/tissue; they are, Hypothetical protein DKFZp586C1924, 8 kDa protein, 25Kda protein, Hypothetical protein GC14288, and Splice Isoform 1 of Protein C20orf22 (ABHD12). Protein databases searches (UniProtKB/Swiss-Prot) did not reveal much information for these proteins. Recently, mRNA expression of Hypothetical protein DKFZp586C1924(TMEM 126A) in human foetal and adult tissues and immuno-localization in mouse mitochondria have been reported.

According to another aspect of the invention, there is provided a method for identifying at least one foetal erythroblast comprising detecting the expression of at least one foetal erythroblast specific marker selected from the group consisting of neutral amino acid transporter B (SLC1A5), solute carrier family 3 (activators of dibasic and neutral amino acid transport) member 2 isoform A (SLC3A2), Splice Isoform A of Chloride channel protein 6, Transferrin receptor protein 1, Splice Isoform 3 of Protein GPR107 precursor, Olfactory receptor 11H4, Splice Isoform 1 of Protein C9orf5, Cleft lip and palate transmembrane protein 1, BCG induced integral membrane protein BIGM103, antibacterial protein FALL-39 precursor, CAAX prenyl protease 1 homolog, splice isoform 2 of synaptophysin-like protein, and splice isoform 1 of Protein C20orf22 (ABHD12), wherein detection of the marker indicates the presence of the foetal erythroblast. In particular, the detecting comprises detecting the expression of at least one foetal erythroblast specific marker selected from the group consisting of splice isoform 1 of Protein C20orf22 (ABHD12), Splice Isoform 3 of Protein GPR107 precursor, Olfactory receptor 11H4, and ALEX3 protein variant. Alternatively, the foetal erythroblast specific marker may be detected by an antibody, antigen binding fragment thereof, or the like. In particular, the antibody may be polyclonal or monoclonal. A person skilled in the art would understand that any molecular or com ound capable of recognizing and/or binding to the foetal erythroblast specific marker can be used to detect the foetal erythroblast specific marker.

According to another aspect of the invention, there is provided a method of isolating at least one foetal erythroblast from a sample, the method comprising: (a) contacting the sample with at least one antibody or antigen binding fragment thereof that is capable of binding to at least one marker selected from the group consisting of neutral amino acid transporter B (SLC1A5), solute carrier family 3 (activators of dibasic and neutral amino acid transport) member 2 isoform A (SLC3A2), Splice Isoform A of Chloride channel protein 6, Transferrin receptor protein 1, Splice Isoform 3 of Protein GPR107 precursor, Olfactory receptor 11H4, Splice Isoform 1 of Protein C9orf5, Cleft lip and palate transmembrane protein 1, BCG induced integral membrane protein BIGM103, Antibacterial protein FALL-39 precursor, CAAX prenyl protease 1 homolog, Splice Isoform 2 of Synaptophysin-like protein, Vitamin K epoxide reductase complex subunit 1-like protein 1, Splice Isoform 1 of Protein C20orf22 (ABHD12), Hypothetical protein DKFZp564K247 (Hypoxia induced gene 1 protein) (IPI Accession No. IPI00295621), Hypothetical protein DKFZp586C1924 (IPI Accession No. IPI00031064), ALEX3 protein variant, Hypothetical protein MGC14288 (IPI Accession No. IPI00176708), protein with IPI Accession No. IPI00639803 and protein with IPI Accession No. IPI00646289; and (b) isolating the foetal erythroblast that binds to the antibody or antigen binding fragment thereof from the sample.

In particular, the antibody may be a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody or a combination thereof. More in particular, the foetal erythroblast that binds to the antibody is isolated from the sample using immunomagnetic separation, flow cytometry or a combination thereof.

The isolation of the mammalian nucleated foetal cell from the sample may be performed using, but not limited to, a micromanipulator or any system that allows individual picking of a foetal cell. In particular, the foetal cell may be a mammalian foetal erythroblast. More in particular, the foetal cell may be a primitive or human foetal erythroblast.

Density gradients and flow sorting methods may be employed to enhance enrichment and purity of foetal erythroblasts from maternal blood. According to yet another aspect of the invention, there is provided a method of diagnosing at least one prenatal disorder in an individual, the method comprising: a. identifying at least one foetal erythroblast in a sample of the individual according to the method described above; b. isolating the foetal erythroblast; and c. determining at least one genetic marker associated with the prenatal disorder in the foetal erythroblast. In particular, the prenatal disorder may be selected from the group consisting of Down Syndrome, Edwards Syndrome, Patau Syndrome, a neural tube defect, spina bifida, cleft palate, Tay Sachs disease, sickle-cell anemia, thalassemia, cystic fibrosis, fragile X syndrome, spinal muscular atrophy, myotonic dystrophy, Huntington's disease, Charcot-Marie-Tooth disease, haemophilia, Duchenne Muscular Dystrophy, mitochondrial disorder, hereditary multiple exostoses and osteogenesis imperfecta disorder. More in particular, the sample may be selected from the group consisting of maternal tissue, maternal blood, cord blood, amniocytes, chorionic villus sample, foetal blood, and foetal tissue. In particular, the method may be carried out in vitro.

According to an aspect of the invention, there is provided a marker for identifying foetal erythroblast selected from the group consisting of neutral amino acid transporter B (SLC1A5), solute carrier family 3 (activators of dibasic and neutral amino acid transport) member 2 isoform A (SLC3A2), Splice Isoform A of Chloride channel protein 6, Transferrin receptor protein 1, Splice Isoform 3 of Protein GP 107 precursor, Olfactory receptor 11H4, Splice Isoform 1 of Protein C9orf5, Cleft lip and palate transmembrane protein 1, BCG induced integral membrane protein BIGM103, Antibacterial protein FALL-39 precursor, CAAX prenyl protease 1 homolog, Splice Isoform 2 of Synaptophysin-like protein, Vitamin K epoxide reductase complex subunit 1-like protein 1, Splice Isoform 1 of Protein C20orf22 (ABHD12), Hypothetical protein DKFZp564K247 (Hypoxia induced gene 1 protein) (IPI Accession No. IPI00295621), Hypothetical protein DKFZp586C1924 (IPI Accession No. IPI00031064), ALEX3 protein variant, Hypothetical protein MGC14288 (IPI Accession No. IPI00176708), protein with IPI Accession No. IPI00639803 and protein with IPI Accession No. IPI00646289. There is further provided an antibody or antigen binding fragment thereof that is capable of binding at least one marker according to the present invention.

Also provided is a kit for use in a method of identifying and/or isolating foetal erythroblast according to any aspects of the present invention. EXAMPLES

Standard molecular biology techniques known in the art and not specifically described were generally followed as described in Sambrook and Russel, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (2001). The foregoing describes preferred embodiments, which, as will be understood by those skilled in the art, may be subject to variations or modifications in design, construction or operation without departing from the scope of the claims. These variations, for instance, are intended to be covered by the scope of the claims.

Example 1 Material and methods

An in-depth literature search was conducted on the presence and functional roles of unique plasma membrane proteins of FPNRBCs in various human tissues and cells, including that of foetus (trophoblasts/placenta). Short description of these proteins, on their location, physiological roles (including those related to human foetal development), and diseases related to their mutations have been provided above, together with available data on similar functions in AARBCs.

FPRNBCs can be separated from WBCs in maternal blood by negative depletion of CD45 positive cells, and if suitable surface antigen known on FPNRBCs available, these ideal cells for non-invasive prenatal diagnosis can be enriched from AARBCs. Membrane proteins of FPNRBCs were profiled by mass spectrometry, and compared this profile with that of the AARBC membrane proteome as known in the art to identify unique surface membrane proteins of FPNRBCs which are absent in AARBCs.

Membrane proteins of FPNRBCs profiled by mass spectrometry may be compared to known membrane proteome of AARBC. A shot-gun proteomics approach, two-dimensional liquid chromatography coupled with MALDI-TOF/TOF-MS (2D-LCMS/MS) was used to characterize the membrane proteome of foetal primitive erythroblasts. This is the first report on the membrane proteome of the foetal primitive erythroblasts. Details of all 273 proteins identified are provided including their annotated sub-cellular locations, molecular functions and number of transmembrane domains. 133 (48.7%) proteins were membrane proteins, of which 37 were plasma membrane proteins. Unique, surface membrane proteins of FPRNBCs were identified by comparing the data of the present study with membrane proteins of AARBCs to identify common, and 12 plasma membrane proteins with transmembrane domains and 8 proteins with transmembrane domains but without known sub-cellular location were identified as unique-to-FPNRBCs. Except for the transferrin receptor, all other 19 unique-to-FPNRBC membrane proteins have never been described in red blood cells. Reverse-transcriptase PCR (RT-PCR) and immunocytochemistry validated the 2D-LCMS/MS data. The findings provide potential surface antigens for separation of FPNRBCs from maternal blood for non-invasive prenatal diagnosis, and help understand the biology these rare cells. Proteomic analyses of FPNRBCs had not been attempted previously owing to the difficulty to obtain sufficient number of cells. Access to placental villi from patients undergoing termination of pregnancy enabled to pool cells for 2D-LC S/MS analysis. In addition, the extraction of membrane proteins is yet another challenge in proteomics; recovery of more membrane proteins (48.7% of total) from a limited sample (5X10 ⁷ cells) than those from AARBCs using similar protocol is encouraging, which also explains the structural complexity of these nucleated cells.

Sub-cellular localization and molecular functions annotated for most of the proteins of FPNRBCs are novel for this cell type. Identified FPNRBC membrane proteins show diverse physiological functions varying from transport, catalytic, binding to structural, while about 32% were transport and/or catalytic. Among the membrane proteins, most were identified from mitochondria (48 proteins) and plasma membrane (37 proteins).

Tissues

Placental tissue collection from women undergoing elective first trimester surgical termination of pregnancy was approved by the Institutional Review Board, and all patients gave written informed consent.

Extraction of FPNRBCs from placental villi

FPNRBCs were extracted from placental villi, and AARBCs were prepared from volunteer blood sample. Placental tissues were collected at the termination of pregnancy (7 ^* ° to 9 ⁺³ weeks amenorrhoea). FPNRBCs were extracted from placental villi as per protocol known in the art. Placental villi were digested in trophoblast digestion buffer (146.3ml HBSS containing 0.182g trypsin and 3.75ml lM Hepes (Gibco'-lnvitrogen-Life-Technologies, NY, USA) for 30min at 37°C in a shaking-water-bath, and digestion was stopped using foetal calf serum (Pierce, IL, USA) (5ml/45ml digestion buffer). Single cell suspensions were centrifuged (3000rpm, 20°C, lOmin). Red cell pellets containing FPNRBCs were suspended in PBS, and separated using Percoll 1083 (GE Healthcare, Uppsala, Sweden) (3000rpm, 20 °C, 20min). FPNRBC purity was determined by basic staining of cytospun slides. Samples were stored for membrane preparation (if purity >90% FPNRBCs) in HES buffer (20mM HEPES, pH 7.4, ImM EDTA and 250mM sucrose) with protease-inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) at -80°C. Morphologies of FPNRBCs and AARBCs are shown in Figure 1. Bright field images were captured using 20x/0.40PhP objective lens of CKX41 Olympus microscope. Bar represents 10 μιη.

Membrane protein preparation and digestion Membranes from pooled FPNRBCs (5xl0 ⁷ cells) were prepared as described in the art. Cells stored in HES buffer were lysed by thawing and sonication, and ultra-centrifuged at 100,000xgf 4°C (lh) to obtain the membrane pellet which was then washed using high pH solution (0.1M Na ₂ C0 ₃ , pHll), and twice with Milli-Q water. Proteins were extracted from FPNRBC membranes using methanol (MeOH)/50mM NH HC0 ₃ (60:40,vol/vol), and protein reduction, alkylation and digestion were carried out as described by Blonder et al. Tryptic digestion was carried out using sequencing grade modified trypsin (Promega, Southampton, UK). Digested sample was centrifuged and the pellet washed in MeOH solution (60%MeOH in 50mM NH4HCO3) twice. Supernatants were pooled (MeOH-derived digests), while the pellet was re- suspended in Trifluoroethanol (TFE)/50mM NH ₄ HC0 ₃ (50:50vol/vol) and the proteins extracted were then diluted 10 times with 50mM NH4HCO3 for a second trypsin digestion to obtain supernatants (TFE-derived digests). Both digests were lyophilized and stored at -80°C.

Two-dimensional Liquid Chromatography and Mass Spectrometry (2D- LCMS/MS)

2D-LCMS/MS was essentially the same described earlier by us (Zhang et al., 2007). Lyophilized digests were re-suspended in solvent [(98%H ₂ 0, 2% acetonitrile (CAN) and 0.05% trifluoroacetic acid (TFA)], and after centrifugation supernatants were separated using an Ultimate-Dual-HPLC system (Dionex, Sunnyvale, CA, USA). All samples were first separated on a strong cation exchange (SCX) column (300μηι i.d., x 15 cm, packed with ΙΟμιη POROS 10S) and eluted fractions were captured on the PepMap trap column (300μηη i.d x 1mm, packed with 5μιη C18 100 A), and eluted by gradient elution to a reversed- phase column (Monolithic Capillary Column, 200μ ^η ι i.d., x 5cm). LC fractions were mixed with matrix-assisted laser desorption/ onization (MALDI) matrix (7 mg/ml a-cyano-4-hydroxycinnamic acid and 130μg/ml ammonium citrate in 75% CAN) at a flow rate of 5.4 μΙ/min through a 25nl mixing- tee (Upchurch Scientific, Oak Harbor, WA, USA) before being spotted onto 192-well stainless steel MALDI target plates (AB SCIEX, Foster City, CA, USA), at a rate of one well per 5s, using a Probot Micro Fraction collector (Dionex).

Samples on the MALDI target plates were analyzed using an ABI 4700 Proteomics Analyzer (AB SCIEX) with a MALDI source and time of flight analyzer TOF/TOF™ optics. For MS analysis, typically 1000 shots were accumulated for each sample well. Tandem-MS_( MS/MS) analyses were performed using nitrogen, at collision energy of 1 kV and a collision gas pressure of ~3.0xl0 ^"7 Torr. 3000 to 6000 shots were combined for each spectrum depending on the quality of the data. Database searching

MASCOT search engine (v2.0; Matrix Science) was used to search tandem mass spectra. GPS Explorer™ software (v3.6; AB SCIEX) was used to create and search files with the MASCOT search engine for peptide and protein identifications. The International Protein Index (IPI) human protein database (v3.10) was used for the search of tryptic peptides and 57478 entries were searched. All MS/MS spectra from the LC runs were combined for the search. Cysteine carbamidomethylation, N-terminal acetylation and pyroglutamination, and methionine oxidation were selected as variable modifications. Two missed cleavages were allowed. Precursor error tolerance was set to 200ppm and MS/MS fragment error tolerance was 0.4Da.

Estimation of False Positive Rate The false positive rate was calculated by comparing the search results from a randomized database versus the actual database. The minimum ion score C.I. percent such that no more than 5% false discovery rate (FDR) was achieved and was used as the cut-off threshold at the peptide level. All the proteins identified from random database search were single peptide- matched. Proteins identified by this method from IPI human database were colour coded as red, green or black: those red coloured proteins are matched to at least two peptides and hence are statistically confident (FDR is zero); proteins that are green coloured are identified by single peptide where match scores are higher than the highest score in the decoy database and essentially the FDR is zero; black coloured proteins were identified based on single peptide match fall within the set threshold of 5% FDR. Top ranked peptides with Best Ion scores > 33 and > 36 for TFE and MeOH extractions, respectively, were included for analysis as peptides counted for each protein. All the MS/MS spectra were further validated manually. Annotation

Sub cellular and functional categories of the identified proteins were obtained based on annotations of Gene Ontology using GoFig.

(http://udgenome.ags.udel.edu/gofigure/index.html). Swiss-prot and TrE BL data base were also used for functional annotation of unique proteins of FPNRBCs. The number of transmembrane domains (TMD) of the identified proteins was predicted using TMHMM Server (v2.0) (http://www.cbs.dtu.dk/services/TMHMM/).

Evaluation of the identified unique proteins a) Reverse transcriptase PCR (RT-PCR) for mRNA expression of unique proteins RNA extraction-RNA from FPNRBCs was isolated using an RNeasy Mini Kit (Qiagen, Germany) according to manufacturer's instructions. Briefly, FPNRBCs (3xl0 ⁶ cells) were resuspended in 350μΙ lysis buffer and passed through QIAshredder spin column. The lysate was mixed with 350μΙ of 70% ethanol and pipetted onto an RNeasy mini column, and centrifuged at 15000xg for 15sec. RNA trapped in the column was washed using 350μΙ buffer RWl and incubated with ΙΟμΙ of DNase in 70μΙ RDD buffer at room temperature for 15min. RNA was then washed twice with 350μΙ of buffer RWl and once with 500μΙ buffer RPE and recovered by the addition of 50μΙ RNase-free water onto the column and centrifugation at 15000xg for lmin.

RT-PCR- DNA template was synthesised using Sensiscript RT Kit (Qiagen, Germany). Briefly, 5μΙ of RNA was mixed with oligo-dT, RNase inhibitor, dNTP mix and RNase-free water (as per manufacturer's instructions) and incubated at 70°C for 5min and chilled on ice. RT buffer and RT enzyme were added to the mixture and incubated at 25°C (15min), 42°C (60min) and 72°C (15min), and cooled on ice. PCR mixture contained 5μΙ cDNA, lxPCR buffer, ImM dNTP, 8mM MgCI ₂ , 2.5U Taq polymerase and 0.6μΜ primers. Denatured (94°C 2min) mixture was amplified by 45 cycles of 94°C for 15 sec, ~60°C (depends on primer pairs) for 15 sec, 72°C for lmin. A final extension at 72°C for 4min was performed for each gene. RT control (no enzyme in RT step) and PCR control (Water-blanks) were also included. PCR products were separated by electrophoresis in a 2% agarose gel, stained with ethidium bromide (0.5g/ml) and visualized under UV light. The images were captured using a digital imager (Alpha Innotech Corp., San Leandro, CA). Primer pairs (Sigma-Proligo) used for the amplification for individual gene are listed in Table 1. Table 1 - Primer pairs used in mRNA expression studies by RT-PCR

b) Localisation of unique proteins on FPNRBCs by alkaline phosphatase immunocytochemistry

8 commercially available antibodies against unique proteins of FPNRBCs annotated to be on plasma membrane, and also in other membranes or unique proteins with unknown subcellular location were used to localize their antigens in both FPNRBCs and AARBCs: Neutral amino acid transporter B (SLC1A5) (Chemicon-lnternational, Temecula, CA, USA), Solute carrier family 3, member 2, isoform A (SLC3A2), Olfactory receptor 11H4 (OR11H4) and Antibacterial protein FALL-39 precursor (Catheiicidin antimicrobial peptide, CAP-18) (all from Abeam, Cambridge, UK), Cleft lip and palate transmembrane proteinl (CLPTM1), Armadillo Repeat- Containing X-linked protein 3 (ARMCX3/ALEX3), and CAAX prenyl proteasel homolog (FACE1) (all from Novus-Biologicals, Littleton, CO), and Chloride channel protein 6 (CLCN6) (Santa-Cruz Biotechnology,lnc, CA, USA). Cells were fixed for lOmin either with 4% paraformaldehyde for SLC1A5, SLC3A2, OR11H4, CLCN6, CLPTMl, ARMCX3 or ice-cold methanohacetone (1:1) for CAP-18 and FACEl; Following steps were common for all slides: Briefly, nonspecific binding was inhibited with diluted goat serum (Sigma-Diagnostics,MO,USA) (1:10 in PBS) for 120min which was followed by incubation with respective primary-antibodies (1:100) for 60min at room temperature or overnight at 4°C. Slides were then incubated with corresponding mouse or rabbit biotinylated secondary-antibody (1:100) for 60min (Vector-Laboratories, CA, USA). This was followed by incubation with streptavidin conjugated alkaline phosphatase (Vector- Laboratories) (1:100). Immunoreaction was detected with freshly prepared Vector-Blue- substrate (Vector-Laboratories) for lOmin in dark. All incubations were performed in a humidifying chamber at room temperature and washes between incubations were in 1XPBST (5min). Slides were rinsed in water and nuclei stained with nuclear-fast-stain (lOmin), slides were rinsed in water and dehydrated with 100% ethanol (30secs each). Air dried slides were mounted with Vectashield (Vector-Laboratories) and analysed by light microscopy. The staining intensity for each antibody tested was calculated as described by Lehr et aL. Mean pixel intensities calculated from the luminosity histogram function on Adobe Photoshop CS4 software (Adobe Systems, Mountain View, CA) were compared for statistical significance.

Isolation ofFPNRBCs in spiked blood samples

Spiked model mixtures (1x105 FNRBCs in 2 ml peripheral blood) were sorted by CD45 depletion (Magnetic associated cell sorting) and NAT-B positive selection. The enriched mixture was tested for FPNRBCs recovery by haemocytometer, cytospun onto slides and identified by Wright staining.

Statistical analysis

Mean staining intensities (Mean ± SD) between FPNRBCs and AARBCs were compared using Mann-Whitney U test (GraphPad Prism software, GraphPad Prism Inc, CA). Differences were considered significant when P values were < 0.05.

FPNRBC Membrane proteins

Cell membrane protein extraction is challenging because many of these proteins have hydrophobic side chains. Furthermore, the significant quantity of protein needed for detailed proteomic analysis restricts studies on limited-access cells such as the human FPNRBCs. To overcome these difficulties, cell membrane protein material harvested from several trophoblastic villi were collected and pooled, and developed a protocol for maximal cell membrane protein recovery. Two organic solvents, MeOH and TFE, were used and recovered both hydrophilic and hydrophobic proteins using pooled samples of FPNRBCS. A total of 273 proteins were identified, with 144 recovered in MeOH and 199 proteins recovered in TFE digests respectively, while 70 proteins were common to both (Table 2; Figure 2). Only 26% of total proteins identified were recovered from both the solvents. The recovery of proteins may be enhanced by the sequential use of both solvents with limited sample (5X10 ⁷ cells).

As FPNRBCs are nucleated, and also contain other organelles, protein identification found not only plasma membrane proteins, but also membrane proteins from the nucleus, mitochondria, endoplasmic reticulum, Golgi, microsomes and peroxisomes. Location annotation of identified proteins

A total of 273 proteins were identified, and their locations within the cell annotated (Table 3): 133 were membrane proteins (Table 3) while 132 were non-membrane proteins including 16 that have been described as exclusively cytoplasmic (Table 4). Locations of the remaining 8 are as yet unclassified (Table 5). Sub-cellular localization and functional categories of the identified proteins were obtained based on the annotations of Gene Ontology using GoFig. (http://udgenome.ags.udel.edu/gofigure/index.html). Swiss-prot and TrEMBL data base were also used for the functional annotations of unique proteins of FPNRBCs. Sub-cellular localizations of the 133 membrane proteins were analyzed: of these proteins, 37 were noted to localize to the plasma membrane, 48 mitochondrial membranes, 10 endoplasmic reticular membranes, and the remaining 38 membrane proteins were annotated to be localized in more than one location of the cell (Figure 3A).

Functional annotation of membrane proteins

Molecular functions of the 133 membrane proteins identified are detailed in the Figure 3B. Some proteins were noted to have more than one function. Most were transport proteins (16.54%), 15.79% were both transport and catalytic, 9,77% catalytic, 9.02% binding, 6.77% binding and catalytic, 5.26% binding and transport, 7.51% binding/catalytic/transport, 3.76% binding/signal transduction/catalytic, 3.00% each for binding/signal transduction, and structural, 9.02% unclassified and 10.53% other functions. Proteins with transmembrane domains Transmembrane domains (TMDs) of all the proteins are provided in the Table 2. The number of predicted transmembrane domains in the identified membrane proteins varied from 0 to 15: NADH dehydrogenase subunit 5 was found to possess the maximum number of TMD. Plasma membrane proteins of primitive FPNRBCs with at least one TMD (25 proteins) and the plasma membrane proteins known to be present on other membranes as well (14 proteins) are presented in Tables 6 and 7, respectively.



Table 5 - Proteins of FPNRBCs with transmembare domain but location unknown

Single peptide based identification of proteins

Colour coding of proteins based on the number of peptides for their identification shown in Table 2 indicated that only 23 of 273 total proteins were black coloured that were identified based on single peptide match which fall within the set threshold of 5% FDR, and the rest were red (< 2 peptides) or green coloured (by single peptide) where FDR was zero. Proteins identified based on single peptides from TFE and MeOH extractions, their peptide sequence and ion score are presented in Figures 7 and 8. Owing to the sample limitation of FPNRBCs, replicate mass-spectrometry analysis with more than the one pooled sample was not carried out.

Comparison of plasma membrane proteins of FPNRBCs and AARBCs to identify unique membrane proteins

Mass spectrometry-based identification of membrane proteins of AARBCs have so far been reported by only a few studies including ours. From the published literature, a comprehensive list of all AARBC membrane proteins identified by mass spectrometry to date was curated. In the final- list, only those candidates annotated as membrane proteins by gene ontology using GoFig. were included. Redundant entries were removed by manually comparing the sequences of all membrane proteins. A total of 299 non-redundant AARBC membrane proteins were finally short-listed (data not shown); Out of this, 202 were short-listed to include only membrane proteins with known- and potential surface domains (e.g. membrane-associated extracellular proteins and integral membrane proteins) (Table 8). Membrane proteins of FPNRBCs were compared manually with this final list of AARBC membrane proteins to identify both common and unique membrane proteins. Table 6- Plasma membrane proteins of FPNRBCs

No Protein description IPI Accession # TMD Sub-cellular location

1 Splice Isoform 1 of Protein C9orf5 IPI00607576 14 Plasma membrane

2 Solute carrier family 2, facilitated glucose transporter member I IPI00220194 12 Plasma membrane

3 Rhesus blood group, CcEe antigens, isoform 1 IPI00465155 12~ Plasma membrane

4 Equilibrative nucleoside transporter 1 IPI00550382 11 Plasma membrane

5 Band 3 anion transport protein IPI00022361 11 Plasma membrane

6 ATP-binding cassette hatf-transporter IPI00465442 9 Plasma membrane

7 Neutral amino acid transporter B IPI00019472 9 Plasma membrane

Splice Isoform XB of Plasma membrane calcium-transporting Plasma membrane

8 ATPase 4 IPI00217 I69 8

9 Olfactory receptor 11 H4 IPIGO168S81 7 Plasma membrane

10 Splice Isoform 3 of Protein GPR107 precursor IPI00184474 7 Plasma membrane

11 BCG induced integral membrane protein B1GM103 IPI00034208 7 Plasma membrane

12 Sodium/potassium-transporting ATPase beia-3 chain IPI00008167 1 Plasma membrane

13 Hypothetical protein FLJ31S42 IPI00043429 6 Plasma membrane

14 Cleft lip and palate transmembrane protein 1 IPI00396411 5 Plasma membrane

15 Splice Isoform A of Chloride channel protein 6 IPI00180121 3 Plasma membrane

16 Leukocyte elastase precursor IPI00027769 1 Plasma membrane

Solute carrier family 3 (activators of dibasic and neutral amino

17 acid transport), member 2 ^■ ·:-. ΙΡΙ005544Θ1 1: Plasma membrane

18 Thioredoxin-like protein KIAA1162 precursor IPI00100247 1 Plasma membrane

19 Aquaporin 1 splice variant 2 IPI00428490 1 Plasma membrane

20 Kell blood group glycoprotein IPI00220459 1 Plasma membrane

21 Erythrocyte band 7 integral membrane protein IPI00219682 1 Plasma membrane

22 Splice Isoform Glycophorin D of Glycophorin C IPI00218128 1 Plasma membrane

23 Stromal cell-derived receptor-1 beta IPI00018311 1 Plasma membrane

2 Transferrin receptor protein 1 IPI00022462 1 Plasma membrane

25 Antibacterial protein FALL-3S precursor IPI00292532 1 Plasma membrane

Table 7 - Plasma membrane proteins of FPNRBCs known to be present on other membranes

Protein description IPI Accession # TMD Sub-cellular location

CDNA PSEC0252 fis, clone NT2RP3003258, highly

similar to Likely ortholog of mouse embryo IPI00301100 11 Plasma membrane/ER Membrane Splice Isoform 1 of Vacuolar proton translocating

ATPase 116 kDa subunit a isoform 1 IPI00552514 7 Plasma membraneA esicle membrane

CAAX prenyl protease 1 homolog IPI00027180 7 Plasma membrane/ER Golgi membrane

Splice Isoform 2 of Synaptophysin-like protein IPI00335277 3 Plasma membraneA esicle membrane

Plasma membrane ER/Microsome

Microsomal glutathione S-transferase 3 IPI00639812 3 membrane

PRA1 family protein 3 IPI00007426 3 Plasma membrane/ER Membrane

Thioredoxin domain containing protein 1 precursor IPI00395887 3 Plasma membrane/ER Membrane

17 kDa protein IPI00642218 3 Plasma membrane/ER Membrane

IPI00021766

Splice Isoform 1 of Reticulon 4 1 Plasma membrane/ER Membrane

Suppressor of actin 1 IPI00022275 2 Plasma membrane/ER/Golgi membrane

Plasma membraneA esicle

Vesicle-associated membrane protein 2 IPI00553138 1 membrane/Synapse

Membrane associated progesterone receptor

component 2 IPI00005202 1 Plasma membrane/Microsome membrane

Vesicular integral-membrane protein VIP36 precursor IPI00009950 1 Plasma membrane/ER/Golgi membrane

Calnexin precursor IPI00020984 1 Plasma membrane/ER Membrane

Table 8 - Comprehensive AARRBC membrane proteins with potential surface domain(s)

(Ad acent roteins in colour were identifisd from same eptide but in different studies and were countsd as one protein) Splice Isoform 4 of Q04656 Copper-transporting

IPI00028610 ATPase 1 7 Golgi. Plasma membrane

Splice Isoform 2 or 1 of Q9H3Z4 DNAJ homolog

IPI00023780 subfamilv C member 5 1 Membrane associated protein

IPI00165394 DC-TM4F2 protein 4 Integral membrane protein

IPI00550523 DKFZP564J0863 protein 2 Unclassified

IPI00154755 Down syndrome cell adhesion molecule 2 1 Type I membrane protein

IPI00215964 Splice Isoform 1 of Duffy antigen/chemokine receptor 7 Integral membrane protein

Splice Isoform 2 of Q16570 Duffy antigen/chemokine

IPI00002940 receptor 7 Integral membrane protein

IPI00432050 Duodenal cytochrome b sequence coverage:10% 1 Integral membrane protein

Gi 13376257 Duodenal cytochrome b 6 Inteqral membrane protein

1PI00004065 Ecto-ADP-nbosyltransferase 4 precursor 0 Integral membrane protein

IPI00010341 Eosinophil granule major basic protein precursor 0 Extracellular

I I00550382 Equiiibrative nucleoside transporter 1 11 Integral membrane protein

ΙΡΙ00Θ47116 Erythroblast membrane-associated protein 1 Integral membrane protein

IPI00044556 Erythroid membrane-associated protein 2 Membrane associated protein

IPI00302538 EVIN2 8 Inteqral membrane protein

IPI0021689O Similar to expressed sequence AA536743 2 Integral membrane protein

IPI00022418 Splice Isoform 1 Of Rbronectin precursor 0 Integral membrane protein; Extracellular

Galactosylgalactosylxylosylprotein 3-beta-

IPI00221205 glucuronosyltransferase 2 1 Integral membrane protein; Golgi

IPI0D465431 Galectin-3 0 Extracellular binding RBC

1PIQQ010477 Splice Isoform Long of 000182 Galectin-9 0 Extracellular

IPI00306419 gene rich cluster, C3f gene 7 Unclassified

IPI00298800 Glycophorin A precursor 2 Type I membrane protein

Gi 3529077 Similar to Glycophorin A 2 Type I membrane protein

Gi106140 Glycophorin A 2 Type I membrane protein

I I00384414 Glycophorin Erik l-IV precursor 1 Inteqral membrane protein

Gi4504229 Glycophorin C. isoform 1 1 Inteqral membrane protein

Splice Isoform Glycophonn C of P04921 Glycophorin

IPI00026299 C 1 Integral membrane protein

1PI00218128 Splice Isoform Glycophorin D of Glycophorin C 1 Integral membrane protein

IPI00023542 Gp25L2 protein 2 Integral membrane protein

Gi9295192 HGTD- 1 Integral membrane protein qi18552304 Hypothetical protein XP 092517 1 Integral membrane protein

IPI00029002 Hypothetical protein 4 Integral membrane protein

IPI00031697 Hypothetical protein 5 Unclassified

IPI00032825 Hypothetical protein CGI-109 precursor 1 Integral membrane protein

IPI00383828 Hypothetical protein DKFZp564J0863 2 Unclassified

1PI00178934 Hypothetical protein 327024.1 1 Inteqral membrane protein

1PI00030236 Hypothetical protein DKFZp564D0478 3 Integral membrane protein

IPI00100199 Hypothetical protein DKFZp564E227 6 Integral membrane protein

IPI00032013 Hypothetical protein DKFZp762A227 11 Integral membrane protein

IPI00022300 Hypothetical protein FLJ14347 1 Unclassified

IPI0O442O30 Hypothetical protein FLJ16766 7 Integral membrane protein

IPI00043429 Hypothetical protein FLJ31842 6 Integral membrane protein

IPI00167359 Hypothetical protein FLJ40269 2 Inteqral membrane protein

IPI00171004 Hypothetical protein MGC34680 12 Integral membrane protein

1PI00003441 Hypothetical protein ORF9 precursor 1 Integral membrane protein

IPI00171421 Hypothetical protein PSEC0098 1 Unclassified

IPI00332161 Ig gamma- 1 chain C region 0 Extracellular binding RBC

IPI00385058 Ig kappa chain C region 0 Extracellular binding RBC

Gi87863 tq heavy chain V-V reaion 0 Extracellular

P05107 Integrin beta-2 precursor 1 Integral membrane protein

Splice Isoform Long of intercellular adhesion

IPI00000118 molecule-4 precursor 1 Integral membrane protein

Splice Isoform Short of Intercellular adhesion

IPI00396335 molecule-4 precursor 0 Membrane

Gi2134798 B-CAM protein 1 Integral membrane protein

Intermediate conductance calcium-activated

IPI00032466 potassium channel protein 4 5 Integral membrane protein

IPI00415077 ton transporter protein 9 Integral membrane protein

IPI00001754 Junctional adhesion molecule 1 precursor 2 Type I membrane protein JWA protein regulates intracellular concentrations of

89 IPI0Q007426 taurine and glutamate 3 Integral membrane protein

90 IPI00220459 Kell blood group glycoprotein 1 Integral membrane protein

91 IPI00001952 KIAA0830 protein 3 Integral membrane protein

92 IPI00022275 KIAA0851 protein 2 Integral membrane protein

93 IPI00002230 KIAA1363 protein 1 Integral membrane protein

94 IPI00298860 Lactotransferrin precursor 0 Extracellular binding BC

95 P42702 Leukemia inhibitory factor receptor 1 Integral membrane protein

96 IPI00027769 Leukocyte elastase precursor 1 Extracellular

Splice Isoform OA3-293 of Leukocyte surface antigen

IPI00216514 CD47 precursor 0 Integral membrane protein

Splice Isoform OA3-323 of Leukocyte surface antigen

97 IPI00374740 C047 precursor 6 Inteqral membrane protein

98 1PI00000059 LFA-3 2 Integral membrane protein

99 IPI00031397 Long-chain-fatty-acid-CoA ligase 3 1 Type III membrane protein

Splice Isoform Short of Q9U U0 Long-chain-tatty-

100 IPI00218718 acid-CoA liaase 6 0 Type III membrane protein

Low affinity immunoglobulin gamma Fc region

101 IPIQ0023858 receptor lll-B precursor 1 Intearal membrane protein

1PI00002406 Lutheran blood group glycoprotein precursor 1 Type I membrane protein

IPI00328869 Lutheran blood group 1 Integral membrane protein

Lutheran blood group glycoprotein isoform 2

102 IPI00554618 precursor 1 Integral membrane protein

103 Gi18589892 Similar to Lutheran blood group 0 Intearal membrane protein

Splice Isoform Short of Lymphocyte function-

104 IPI00219549 associated antigen 3 precursor 2 Integral membrane protein

105 IPI00019038 Lysozyme C precursor 0 Extracellular

Membrane associated progesterone receptor

106 1PI00005202 component 2 1 Integral membrane protein

107 IPI00026111 Membrane protein 2 Unclassified

108 (PI00020896 Membrane transport protein XK 9 Inteqral membrane protein

109 IPI000 0292 Mesenchymal stem cell protein DSCD75 I Unclassified

110 1PI006398 I2 Microsomal glutathione S-transferase 3 3 Integral membrane protein; Microsome

111 IPI00024650 Monocarboxylate transporter 1 11 Integral, membrane protein

Splice Isoform Delexon-17 of P33527 Multidrug

112 IPI00008338 resistance-associated protein 1 16 Integral membrane protein

113 IPI00006675 Mullidrurj resistance-associated protein 4 11 Integral membrane protein

114 1PI00385383 Multidrug resistance-associated protein 5 11 Integral membrane protein

115 IPI00027409 Myeloblasts precursor 0 Extracellular

116 IPI00021983 Splice isoform 1 of Q92542 Nicastrin precursor 0 Type I membrane protein

117 IPI00217600 Neuropathy target esterase 1 Unclassified

Splice Isoform 2 of Q14697 Neutral alpha-

118 IPI00011454 cilucosidase AB precursor 1 ER; Goiai

Splice Isoform 2 of Large neutral amino acids

119 IPI00479732 transporter small subunit 3 11 Integral membrane protein

120 IPI00513701 Novel protein o Integra] membrane protein

121 IPI00009507 Splice isoform 1 of Q16563 Pantophysin 3 Integral membrane protein

122 IPI00021075 PB39 12 Integral membrane protein

123 IPI00020124 Phosphatidyiinositol 4-kinase type II 0 Integral membrane protein

Phosphatidytinositol-4-phosphate 5-ktnase type II

IPI00009688 alpha 0 Integral membrane protein

124 Gi1730569 Phosphatidylinositol-4-phosphate 5 kinase, type 111 0 Integral membrane protein

125 IPI00005181 Phospholipid scramblase 1 0 Type II membrane protein

126 IPI00016776 Phospholipid scramblase 4 0 Type II membrane protein

Splice Isoform B of P20020 Plasma membrane

127 IPI00021695 calcium-transporting ATPase 1 7 Integral membrane protein

Splice Isofomi XB of Plasma membrane calcium-

IPI00217169 transporting ATPase 4 8 Integra] membrane protein

Splice Isofomi XD of P23634 Plasma membrane

128 IPI00012490 calcitim-transportinq ATPase 4 8 Integral membrane protein

Splice Isoform Delta of Poliovirus receptor related

129 IPI00003648 protein 1 precursor 2 Integral membrane protein

130 IPI0O02467O Polyposis locus protein 1 2 Integral membrane protein

131 IPI00029507 Potassium channel subfamily K member 5 6 Integral membrane protein

132 S6409316 Presenilm-associated protein 2 Integral membrane protein 133 IPI00022974 Prolactin 0 Extracellular binding RBC

134 IPI00033075 Protein BAT5 2 Integral membrane protein

135 IPI0Q010796 Protein disulfide-isomerase precursor 0 ER lumen, extracellular region

136 IPI00006093 Protein FAM38A 25 Unclassified

137 IPI00006072 Protein transport protein SEC61 gamma subunit 1 Integral membrane protein

138 IPI00290452 RECS1 protein homolog 7 Integral membrane protein

IPI00028946 Reticulon protein 3 3 Intearal membrane protein

IPI00555783 Reticulon 3 isoform a variant 3 Membrane: Extracellular

IPI00398795 RTN3-A1 3 Integral membrane protein

139 IPI001 7423 PREDICTED: similar to Reticulon protein 3 1 Integral membrane protein

140 IPI00298289 Splice Isoform 2 Of Reticulon 4 1 Integral membrane protein

141 IPI00039665 R blood CE group antigen polypeptide 12 Integral membrane protein

IPI00329565 RhD protein 10 Integral membrane protein

!PI00478119 Rhesus blood group D antigen 10 Integral membrane protein

142 Gi10800054 Rh blood D group antigen polypeptide 10 Integral membrane protein

143 ©2765839 Rhesus D category VI type III protein 12 Integral membrane protein

144 IPI00024094 Rhesus blood group-associated glycoprotein 11 Integral membrane protein

145 1PI00465155 Rhesus blood group, CcEe antigens, isoform 1 12 Integral membrane protein

Splice Isoform RHVIII of P18577 Blood group

146 IPI00221017 Rh(CE) polypeptide 10 Integra membrane protein

Hypothetical protein FLJ45640 (Rhesus blood group,

147 IPI00444375 CcEe antigens) 10 Integral membrane protein

148 IPI00166865 Similar to RIKEN cDNA 1500009 05 gene 1 Unclassified

149 IPI00373867 PREDICTED, similar to RIKEN cDNA C730027E14 1 Integral membrane protein

150 IPI0005631D Secretory carrier-associated membrane protein 4 4 Integra! membrane protein

151 IPI00025257 Semaphorin 7A precursor 0 integral membrane protein

152 IPI00022434 Serum albumin precursor 0 Extracellular

153 IPI00219755 Signal peptidase complex subunit 1 2 Inteqral membrane protein

154 Q64689 Alpha-2,8-siaivltransferase 8C 1 Inner cell membrane

Splice Isoform 2 of Sodium channel protein type !

155 IPI00216029 alpha subunit 19 Integral membrane protein

Splice Isoform of Sodium/potassium-transporting

156 IPI00006482 ATPase alpha- chain 10 Integral membrane protein

Sodium/potassium-transporting ATPase alpha-2

157 IPI00003021 chain precursor 8 Integral membrane protein

Splice Isoform Short of Sodium/potassium-

158 IPI00414005 transporting ATPase alpha-1 chain precursor 4 Integral membrane protein

Solute carrier family 1 (giutamate transporter),

159 IPI00100081 member 7 7 Integra! membrane protein

160 IP 100301180 Solute carrier family 12 member 5 12 Integral membrane protein

161 IPI00299186 solute carrier family 19 member 1 isoform b 9 Inteqral membrane protein

Solute carrier family 2, facilitated glucose transporter,

162 IPI00003909 member 3. or 14 10 Integral membrane protein

Solute carrier family 2, facilitated glucose transporter,

163 IPI00027281 member 4 12 Integral membrane protein

Solute carrier family 2, facilitated glucose transporter,

IPI00220194 member 1 12 integral membrane protein

GiP11166 Glucose transporter type I 12 Integral membrane protein

164 Gi3387905 Glucose transporter glycoprotein 8 Integra membrane protein

Splice Isoform 1 of Q9Y666 Solute carrier family 12

165 IPI00008616 member 7 11 Integral membrane protein

Solute carrier family 27 (fatty acid transporter),

166 IPI00021089 member 4 2 Inteqral membrane protein

Solute carrier famiiy29 (nucleoside transporters):

67 IPI00 12547 member 1 11 Integral membrane protein

168 IPI00005547 Solute earner family 40, member 1 10 Integral membrane protein

169 IPI00301100 Solute carrier family 43, member 3 11 Integral membrane protein

170 IPI00377081 Stomatin 1 C toskeleton

IPI00219682 Stomatin isoform a 1 Integral membrane protein

Erythrocyte band 7 integral membrane protein

171 P27 05 (stomatin) (protein 7.2B) Integral membrane protein

IPI0001 1578 Stromal cell-denved receptor- 1 alpha 1 Extracellular, integral membrane protein

1 2 IPI000 8311 Stromal cell-derived receptor-1 beta 1 Extracellular: Integral membrane protein

173 IPI00399142 Surfeit 4 2 Integral membrane protein, ER membrane 174 1PI00029730 Svntaxin 4 1 Type IV membrane protein

IPI00289876 Syntaxin 7 1 Type IV membrane protein

175 1PI00552913 Splice Isoform 2 of Svntaxin-7 0 Membrane; Cytoplasmic

Splice Isoform I of P14209 T-cel! surface

176 IPI00253036 glycoprotein E2 precursor 2 Integral membrane protein

77 P3689 TGF-beta receptor type I precursor 2 Inteqral membrane protein

Thioredoxin domain containing protein 1 precursor

178 1PI00395887 Protein disulfide-isomerase A6 precursor 3 ER lumen

179 IPIOO 100247 Thioredoxin-like protein KIAA1162 precursor 1 Type I membrane protein

Thrombospondin 1 precursor; glycoprotein IV, also in

180 IPI00296099 mature RBCs 0 Extracellular region

181 IPI00028642 Splice Isoform 1 Of Thyrotropin receptor precursor 0 Integral membrane protein

182 IPI00007052 TPR repeat containing protein 1 Integral membrane protein

183 IPI00394781 Transmembrane protein 24 1 Integral membrane protein

184 IPI00028055 Transmembrane protein Tmp21 precursor 2 Type I membrane protein

185 IPI00332278 Splice Isoform 2 of Transmembrane protein 55B 2 Integral, membrane protein

186 IPI00220272 Triad in 1 Integral membrane protein

UDP-glucose:glycoprotein glucosyltransferase

187 1PI00024466 1 precursor 1 Integral membrane protein

Uncharacterised hematopoietic stem/progenitor cells

188 IPI00020515 protein MDS032 1 Type II membrane protein

189 1PI00007061 UPF0198 protein CGi-141 3 Integral membrane protein

190 IPI00298337 Urea transporter, erythrocyte 8 Integral membrane protein

191 1PI00018855 Vacuolar ATP synthase 16 kDa proteolipid subunit 4 Integral membrane protein

Splice Isoform 1 of Vacuolar proton translocating

192 IPI00552514 ATPase 116 kDa subunit a isoform 1 7 Integral membrane protein

193 IPI00006865 Vesicle trafficking protein SEC22b 1 Type IV membrane protein

Vesicle-associated membrane protein-associated

194 IPI001 0692 protein A isoform 2 1 Type IV membrane protein

Vesicle-associated membrane protein-associated

195 1PI00374657 protein A isoform 1 1 Membrane: Cytoskeleton

Splice Isoform 1 of 095292 Vesicle-associated

196 IPI00006211 membrane protein associated protein B/C 1 Type IV membrane protein

197 Gi7657675 Vesicle-associated membrane protein 2 1 Type IV membrane protein

198 IPI00009950 Vesicular integral-membrane protein VIP36 precursor 1 Type I membrane protein

199 P56703 WNT-3 proto-oncogene protein [precursor] 1 Integral membrane protein

Splice Isoform 2 of Zinc finger DHHC domain

200 IPI00216069 containing protein 3 4 Integral membrane protein

201 IPI00002483 Zinc transporter 1 6 Integral membrane protein

202 Gi5902116 Zona pelluctda binding protein 1 Integral membrane protein

Membrane proteins common to both AARBCs and FPNRBCs

31 proteins were common to both cell types. These included: structural proteins such as the erythrocyte band 7 integral-membrane protein, ankyrin, spectrin, dematin, Protein 4.1; proteins with transport function such as band 3, aquaporin, calcium-transporting ATPase, sodium/potassium-transporting ATPase, solute carrier family 2, facilitated glucose transporter, member 1; and plasma membrane binding proteins like Kell blood group glycoprotein (CD238).

Plasma membrane proteins unique to FPNRBCs

A comparison of membrane proteins with potential surface domains (as annotated) indicated that only 31 proteins were common membrane proteins to AARBCs and FPNRBCs. It was further revealed that 20 proteins were unique to FPNRBCs, and 171 unique to AARBCs, respectively (Figure 4). Among membrane proteins unique to FPNRBCs, 9 proteins were annotated as being present only on plasma membranes, and 3 others were noted to be present on plasma membranes as well as on ER/Golgi/vesicle membranes (Table 9); but, for 8 other membrane proteins found unique to FPNRBCs, the exact sub-cellular localization was not available (Table 10).

Table 9 - Unique membrane proteins of FPNRBCs with transmembrane domain

No Protein description IPI Accession # TMD Sub-cellular location Molecular function

1 Neutral amino acid transporter B (SLC IA5) IPI0001 472 9 Plasma membrane Transporter-Ammo acid

Solute carrier family 3 {activators of dibasic and 1 Transporter-Amino acid

2 neutral amino acid transport), member 2, isoform A IPI00554481 Plasma membrane

(SLC3A2)

3 Splice Isoform A of Chloride channel protein 6 IPI00 I80 I21 Q Plasma membrane Transporter-Chloride ion

4 Transferrin receptor protein 1 IPI00022462 1 Plasma membrane Binding and transport -Iron

5 Splice Isoform 3 of Protein GPR107 precursor IPI00184474

7 Plasma membrane Binding receptor -Hormone and neurotransmitter

6 Olfactory receptor 11 H4 IPI00168981 7 Plasma membrane Binding receptor-Odor

7 Splice Isoform 1 of Protein C9orf5 IPI00607576 14 Plasma membrane Signaling pathways fl Cleft lip and palate transmembrane protein 1 IPI0Q396411 5 Plasma membrane Unknown

9 BCG induced integral membrane protein BIGM103 IPI00034208 7 Plasma membrane Antimicrobial

1 Plasma membrane Antibacterial

10 Antibacterial protein FALL-39 precursor IPI00292532

/Extracellular

7 Plasma/ER/Golgi

11 CAAX prenyl protease 1 rtomolog IPIOOD27180 Catalytic

membrane

5277 3 Plasma/Vesicle Vesicle recycling

12 Splice Isoform 2 of Synaptophysin-like protein IPI0033

membrane

Table 10 - Unique membrane proteins of FPNRBCs with transmembrane domain but location unknown

No Protein description IPI Accession # TN1D Sub-cellular location Molecular function

Vitamin K epoxide reductase complex subunit 1- Unclassified Membrane Catalytic

1 IPI00166079

like protein 1 (potential)

Unclassified Membrane Catalytic

2 Splice Isoform 1 of Protein C20orf22 IPI00394779

(by similarity)

Hypothetical protein DKFZp5<34K247 (Hypoxia

Unclassified Membrane Unclassified

3 induced gene I protein) 1PI0Q2S562 I

(potential)

4 Hypothetical protein DKFZp586C 1924

IPI00031064 Unclassified Membrane Unclassified

(potential)

Unclassified Single pass unclassified

ALEX3 protein variant IPI00604615

membrane (potential)

Hypothetical protein MGC14288 Unclassified Membrane Unclassified

6 1PI00176708

(potential)

8 kDa protein Unclassified

7 IPI00639803 Unclassified

25 kDa protein I I00646289 1 Unclassified Unclassified

Membrane proteins unique to FPNRBCs fall mainly under broad functional groups such as (a) transporter proteins: neutral amino acid transporter B, solute carrier family 3 (activators of dibasic and neutral amino acid transport), splice isoform A of chloride channel protein 6 (chloride ion transport); (b) binding proteins: transferrin receptor protein, splice isoform 3 of Protein GPR107 precursor, olfactory receptor 11H4; and (c) catalytic proteins: CAAX prenyl protease 1 homolog, Vitamin K epoxide reductase complex subunit 1-like protein 1 (VKORC1 LI), Splice Isoform 1 of Protein C20orf22 (ABHD12). Reverse transcriptase PCR (RT-PCR) to confirm expression of unique membrane proteins within FPNRBCs

FPNRBCs from trophoblastic villi were obtained and all were used to perform the mass spectrometry experiments. To determine if the proteins identified as unique to FPNRBCs were indeed expressed within FPNRBCs, extracted total RNA from FPNRBCs was used to perform an RT-PCR. mRNA expression of unique proteins of FPNRBCs using total RNA extracted from FPNRBCs and by RT-PCR using primers specific for genes tested (Table 1). The mRNA expression of 23 proteins including 13 proteins unique to FPNRBCs was evaluated(Figure 5). In Figure 5, the RT control sample contains no RT enzyme. In the PCR control sample, water was added in place of template. The top panel of Figure 5 showed the expression of haemoglobin epsilon chain (HBE1,), haemoglobin gamma-2 chain (HBG2), solute carrier family 4 member 1 (SLC4A1,); solute carrier family 39 member 8 (SLC39A8); chloride channel protein 6 (CLCN6); Azurocidin precursor (AZU1); vitamin K epoxide reductase complex subunit 1-like protein 1 (VKORC1L1); protein GPR107 precursor (GPR107); neutral amino acid transporter B (SLC1A5); Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The bottom panel of Figure 5 showed the expression of solute carrier family 3 member 2 (SLC3A2), isoform A; solute carrier family 22 member 11 (SLC22A11), isoform 2; antibacterial protein FALL-39 precursor (CAMP); vesicle- associated membrane protein 2 (VAMP2); transferrin receptor protein 1 (TFRC); cleft lip and palate transmembrane protein 1 (CLPTM1); CAAX prenyl protease 1 homolog; ATP6VOA1 (ZMPSTE24), vacuolar proton translocating ATPase 116 kDa subunit a isoform 1 (ATP6V0A1); steroid dehydrogenase homolog (HSD17 i2); solute carrier family 43 member 3 (SLC43A3); synaptophysin-like protein (SYPL1); and protein C9orf5 (C9orf5). mRNA expression of all the unique proteins on FPNRBCs tested, except olfactory receptor 11H4 (OR11H4), was detected. The absence of amplification of olfactory receptor could probably be due to the low levels of mRNA accumulated as suggested by Feingold and his colleagues.

Immunocytochemical localization of unique FPNRBC proteins

In situ localization of the putative unique FPNRBC proteins was thought to be more informative than western blotting because the location of plasma membrane, cytoplasmic and nuclear proteins could be readily visualized. These were compared to AARBCs. Intensities of immunostaining for the five antibodies tested, FACE-1, SLC1A5, CAP-18, ARMCX3 and OR11H4 were significantly higher (< 0.001) on FPNRBCs than on AARBCs; in contrast, anti-CLCN6 antibody stained AARBCs much more intensely than FPNRBCs (< 0.001). There was no significant difference in the staining between FPNRBCs and AARBCs for CLPTM1 and SLC3A2 (Figures 6A-B). The Bar represents 10 μηι. Bright field images were captured using 20x/0.7 UPlan APO objective lens of BX61 Olympus microscope with Evolution™MP colour Media Cybernetics CCD camera linked to Image-Pro Discovery software. In Figure 6B, Mean pixel intensities calculated from the luminosity histogram function on Adobe photoshop CS4 software (Adobe Systems, Mountain View, CA) were compared for statistical significance. Mean staining intensity values and intensity of immunoreaction are inversely related. Intensities of immunostainirtg of four out of eight antibodies tested were significantly higher (p<0.05; Figures 6C and D). Antibodies which are significantly more intense are antibodies towards markers FACE-1, SLC1A5 (NAT-B), ALEX3 (ARMCX3) and CLCN6. In Figure 6C, mean pixel intensities calculated from the luminosity histogram function on Adobe photoshop CS4 software (Adobe Systems, Mountain View, CA) were compared for statistical significance. Mean staining intensity values and intensity of immunoreaction are inversely related. Test used was Mann-Whitney Test, p<0.05 is considered significant.

In Figure 6D, FPNRBCs extracted from placental villi are relatively larger and identified by the presence of nuclei stained red by nuclear fast stain. FPNRBCs and AARBCs are shown in first and second panels respectively; negative control was carried out by omitting the primary antibody and positive control were run in all experiments.

FPNRBCs recovery with anti-NAT-B antibody

To test the possibility of sorting FPNRBCs using any of the markers found in the present disclosure, adult blood sample was spiked with FPNRBCs. Spike recovery of FPNRBCs using NAT-B (SLC1A5) marker was about 62.5% (Figure 7B). Sort results are further validated immunohistochemically (Figure 7A). Immunohistochemical study showed successful recovery of FPNRBCs using NAT-B marker.

Identification of 133 membrane proteins from various sub-cellular locations with different functions would help to explore the importance of FPNRBC in medicine. 132 non-membrane proteins including a few known cytoplasmic proteins (for example, haemoglobin chains ε,γ,δ) are also provided. Proteomic analyses of FPNRBCs had not been attempted previously owing to the difficulty to obtain sufficient number of cells. Access to placental villi from patients undergoing termination of pregnancy enabled to pool cells for 2D-LCMS/MS analysis. In addition, the extraction of membrane proteins is yet another challenge in proteomics; recovery of more membrane proteins (48.7% of total) from a limited sample (5X10 ⁷ cells) than those from AARBCs using similar protocol is encouraging, which also explains the structural complexity of these nucleated cells.

Sub-cellular localization and molecular functions annotated for most of the proteins of FPNRBCs are novel for this cell type, which may be useful for protein/developmental/structural biologists, pathologists, haematologists and others. Identified FPNRBC membrane proteins show diverse physiological functions varying from transport, catalytic, binding to structural, while about 32% were transport and/or catalytic. Among the membrane proteins, most were identified from mitochondria (48 proteins) and plasma membrane (37 proteins).

Unique membrane proteins of FPNRBCs were identified to be potential candidates as surface antigens for future separation of this cell type by antibody based techniques. A list of human AARBC membrane proteins prepared based on publications was used for comparison of membrane proteins of FPNRBCs with that of AARBCs: 12 membrane proteins annotated to be in plasma membranes and eight without known sub-cellular locations were found to be unique to FPNRBCs. Proteins with transmembrane domains without known sub-cellular location and molecular function may contain novel antigens of biological significance. This comparison also revealed that 171 proteins are unique to AARBCs which are not found in the data set of FPNRBCs.

A few proteins were found to be common in both the cell types, which included major structural and transport proteins of plasma membrane such as band 3, erythrocyte band 7, facilitated glucose transporter (SLCA2A1), Kell blood group glycoprotein (CD238), aquaporin, ATP-binding cassette half-transporter 1 and glycophorin C, suggesting similar functions for these proteins in FPNRBCs as of their adult counterpart.

In the present disclosure, plasma membrane proteins which are developmental-stage specific to immature red cells but not to AARBCs, such as transferrin receptor and ferritin heavy chain were identified unique to FPNRBCs; similarly, absence of leukocyte specific antigen in the data set a/so confirms the purity of the samples used. Indirect validation of unique proteins of FPNRBCs by mRNA expression analysis using RT-PCR revealed the presence of all candidates tested except the olfactory receptor (OR11H4); and the reason for the failure of this protein may probably be due to the low level of the template present in the sample. RT-PCR results for unique proteins confirm their identifications by mass spectrometry. Such validation is not possible for AARBCs as they are mature cells without nuclei or RNA.

Proteomic identification followed by confirmation of their expression in tissues and cells by immunological techniques has been an useful tool in areas such as biomarker discovery, drug discovery and disease biology for example, tumour heterogeneity studies in bladder cancer. Stronger expression levels of unique proteins of FPNRBCs as identified by immunostaining for four of eight antibodies (FACE-1, SLC1A5, CAP-18 and OR11H4) on these cells compared to AARBCs, do support their mass spectrometry identifications. However, expression of chloride channel protein (CLCN6) was found to be opposite (stronger in AARBCs) and two other proteins (SLC3A2 and CLPTM1) did not reveal any difference in their immunostaining in the present study, and such observations may probably be due to the specificity and reactivity of the antibodies used or due to the expression levels and the isoforms of proteins identified. As mentioned earlier, FACE-1 and CAP-18 are also annotated to be present in other locations in addition to their presence in the plasma membrane.

Potential surface antigens for separation of FPNRBCs from maternal blood for non-invasive prenatal diagnosis were identified: these cells in maternal blood, can be separated easily from WBCs using leukocyte specific anti-CD 45 antibody, whereas, it is still challenging to select FPNRBCs from overwhelming AARBCs due to the absence of specific surface antigen present only in any one of these cell types. Identification of unique membrane proteins with transmembrane domains such as FACE-1, SLC1A5, CAP-18 and 0R11H4 by mass spectrometry and their intense expressions in FNRBCs, as shown by immunocytochemistry have been done. These potential candidates may be used for separation of this cell type from AARBCs by positive selection by means of immuno-cell sorting techniques such as magnetic activated cell sorting (MACS) or fluorescence activated cell sorting (FACS). Similarly, the absence of immunoreaction of the chloride channel protein in FPNRBCs may also be useful for depletion from AARBCs by such strategies. Biological significance of the unique plasma membrane proteins of FPNRBCs

Figure 8 shows the locations, and physiological roles (including those related to human foetal development), and diseases related to their mutations of the unique plasma membrane proteins of FPNRBCs. Briefly, 20 unique membrane proteins could be categorized under seven functional sub-groups: Transportes/Channel molecules: two amino acid transporting Solute Carrier (SLC) proteins, neutral amino acid transporter BO (NAT-B; SLC1A5, ATB (0), ASCT2), SLC3A2; and an anion transporter, splice isoform A of chloride channel protein 6. Binding proteins: Transferrin receptor protein 1, Splice isoform 3 of protein GPR107 precursor and olfactory receptor 11H4. Catalytic: CAAX prenyl endopeptidase also known as farnesylated protein-converting enzyme (FACE), Vitamin K epoxide reductase complex subunit 1 like protein (VKORC1L1), Splice isoform 1 of protein C20orf22 (ABHD12); Signaling pathway: Splice isoform 1 of Protein C90RF5; vesicle recycling: Pantopysin; Anti-microbial proteins: BCG induced integral membrane protein BIGM 103 (BCG induced gene in monocyte, clone 103), FALL39; Proteins with no known function: Cleft lip and palate transmembrane protein 1.

Proteins of unknown location and function- reports on protein expression or functional identity of five of the identified proteins of FPNRBCs (with at least one transmembrane domain) are not available in any other cell/tissue; they are, Hypothetical protein DKFZp586C1924, Splice isoform lof protein C20orf22 (ABHD12), Hypothetical protein MGC14288, 8 KDa protein and 25 KDa protein. Protein databases searches (UniProtKB/Swiss-Prot) did not reveal much information for these proteins.

These studies on human foetal primitive erythroblasts enables the understanding of the biology of these cells, including haemoglobin switching and regulation of their expression, and, to some extent, on the enrichment of these ideal cells from maternal blood for non-invasive prenatal diagnosis. The proteomic information on the membrane proteins of these cells would help to understand the biology and develop technology for enrichment of these cells from maternal blood for non-invasive prenatal diagnosis. Example 2

Enrichment of FPNRBCs from post-termination of pregnancy (TOP) maternal blood using anti-ASCT2 antibody following three-step enrichment protocol

10 mis of post-TOP maternal blood was collected from two patients. Blood samples were processed using three-step enrichment protocol of our laboratory. Briefly, diluted blood sample was layered over Percoll 1118 density medium and centrifuged. The interface was collected and white blood cells were depleted by magnetic activated cell sorting (MACS) using anti-CD45 magnetic beads. Cells from negative fraction were incubated with anti-ASCT2 antibody for 30 minutes and washed and again incubated with anti-rabbit IgG-magnetic beads for indirect MACS (positive) selection of FPNRBCs. 20 FPNRBCs could be recovered from each sample (Table 11).

Table 11 - Enrichment of FPNRBCs from post-TOP maternal blood using anti-ASCT2 antibody

Recovery of fetal nucleated erythroblasts from model mixture experiments using antibodies against ABHD12, GPR107, ORH114 and ALEX3

Fetal nucleated red blood cells were extracted from placental villi and stored in IMDM medium overnight. FPNRBCs and AARBCs in the sample were counted using haemocytometer. Fresh AARBCs were obtained by Ficoll-Plague centrifugation of diluted whole blood at 3,000 rpm for 20 minutes. The pelleted RBCs were collected and washed with lxPBS and also stored in IMDM medium. AARBCs were spiked into the FPNRBCs-containing tubes such that the concentration of FPNRBCs was maintained at 1-9%. Either 0.5xl0 ⁵ or lxlO ⁵ FPNRBCs (depending on the availability of FPNRBCs extracted) were used in the mixtures. Each experiment was carried out in duplicates or triplicates depending on the availability of FPNRBCs extracted. The cell mixture was pelleted by centrifuging at 2,200 rpm for 5 minutes. Supernatant was removed and appropriate volume of MACS buffer added. The concentration of antibodies for incubation with cell mixture was 1:50 for GPR107, OR11H4 and ABHD12, and 1:100 for ALEX3. After incubation at 4°C for 30 minutes, cells were washed once at 2,200 rpm for 5 minutes and the buffer supernatant was discarded. 60 μΙ of MACS buffer and 40 μΙ of anti-rabbit IgG or anti-mouse IgG beads (Miltenyi) as appropriate were added and incubated at 4°C for 30 minutes. After washing, the cells were separated using Miltenyi MS columns. The recovery of FPNRBCs from model mixture using anto-GPR107 appeared to be higher (29.4%) than that of OR11H4 and ABHD12, or ALEX3.

Table 12 - Summary of separation of FPNRBCs from model mixtures containing adult enucleated RBCs using antibodies against 4 unique surface markers of FNRBCs

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