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NanoLC-MSMS Phosphoproteomics comparison of in vitro cultured/native reticulocytes and erythrocytes

ABSTRACT: Triplicate samples of donor-matched native reticulocytes (CD71+ cells), erythrocytes (CD71- cells) and in vitro culture-derived reticulocytes (derived from CD34+ cells) were analysed through qualitative phosphoproteomics to examine possible novel mechanisms underlying reticulocyte maturation.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Erythrocyte, Blood Cell, Cell Culture, Reticulocyte

SUBMITTER: Pedro Moura

LAB HEAD: Ashley Mark Toye

PROVIDER: PXD009015 | Pride | 2018-08-03

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	200916AllSampleComparison.xlsx	Xlsx
	Cone1_CD71_Minus.raw	Raw
	Cone1_CD71_Plus.raw	Raw
	Cone1_FilteredRetics.raw	Raw
	Cone3_CD71_Minus.raw	Raw

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Publications

Non-muscle myosin II drives vesicle loss during human reticulocyte maturation.

Moura Pedro L PL Hawley Bethan R BR Mankelow Tosti J TJ Griffiths Rebecca E RE Dobbe Johannes G G JGG Streekstra Geert J GJ Anstee David J DJ Satchwell Timothy J TJ Toye Ashley M AM

Haematologica 20180803 12

The process of maturation of reticulocytes into fully mature erythrocytes that occurs in the circulation is known to be characterized by a complex interplay between loss of cell surface area and volume, removal of remnant cell organelles and redundant proteins, and highly selective membrane and cytoskeletal remodeling. However, the mechanisms that underlie and drive these maturational processes <i>in vivo</i> are currently poorly understood and, at present, reticulocytes derived through <i>in vi ...[more]

PMID: 30076174

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NanoLC-MSMS TMT comparison of in vitro cultured/native reticulocytes and erythrocytes

Project description:Triplicate samples of donor-matched native reticulocytes (CD71+ cells), erythrocytes (CD71- cells) and in vitro culture-derived reticulocytes (derived from CD34+ cells) were analysed through TMT-based quantitative proteomics to examine possible protein abundance changes underlying reticulocyte maturation.

2018-08-03 | PXD009023 | Pride

NanoLC-MSMS TMT comparison of circulated and unstimulated in vitro cultured reticulocytes

Project description:Triplicate samples of unstimulated in vitro culture-derived reticulocytes (derived from CD34+ cells) and cultured reticulocytes circulated overnight in an ex vivo circulation system were analysed through TMT-based quantitative proteomics to explore the impact of shear stress on reticulocyte protein abundance.

2018-08-03 | PXD009024 | Pride

NanoLC-MSMS Phosphoproteomics comparison of in vitro cultured reticulocytes before and after passage through microfluidic capillaries

Project description:Triplicate samples (biological replicates) of in vitro culture-derived reticulocytes (derived from CD34+ cells) were analysed through qualitative phosphoproteomics before and after passage through a microfluidics capillary chip to investigate the mechanisms underlying shear stress recognition.

2019-09-12 | PXD013652 | Pride

NanoLC-MSMS Phosphoproteomics comparison of in vitro cultured reticulocytes and red blood cells before, during and after microsphiltration

Project description:Four samples of in vitro culture-derived reticulocytes (biological replicates, derived from CD34+ cells) and three samples of red blood cells (biological replicates) were analysed through qualitative phosphoproteomics before, during and after passage through a spleen-mimicking microsphiltration device in order to investigate the mechanisms underlying shear stress recognition.

2019-09-12 | PXD013960 | Pride

The genomic analysis of erythrocyte microRNA expression in sickle cell diseases

Project description:Background Since mature erythrocytes are terminally differentiated cells without nuclei and organelles, it is commonly thought that they do not contain nucleic acids. In this study, we have re-examined this issue by analyzing the transcriptome of a purified population of human mature erythrocytes from individuals with normal hemoglobin (HbAA) and homozygous sickle cell disease (HbSS). Methods and Findings Using a combination of microarray analysis, real-time RT-PCR and Northern blots, we found that mature erythrocytes, while lacking ribosomal and large-sized RNAs, contain abundant and diverse microRNAs. MicroRNA expression of erythrocytes was different from that of reticulocytes and leukocytes, and contributed the majority of the microRNA expression in whole blood. When we used microRNA microarrays to analyze erythrocytes from HbAA and HbSS individuals, we noted a dramatic difference in their microRNA expression pattern. We found that miR-320 played an important role for the down-regulation of its target gene, CD71 during reticulocyte terminal differentiation. Further investigation revealed that poor expression of miR-320 in HbSS cells was associated with their defective downregulation CD71 during terminal differentiation. Conclusions In summary, we have discovered significant microRNA expression in human mature erythrocytes, which is dramatically altered in HbSS erythrocytes and their defect in terminal differentiation. Thus, the global analysis of microRNA expression in circulating erythrocytes can provide mechanistic insights into the disease phenotypes of erythrocyte diseases. Keywords: Sickle cell disease RBC profiling The microRNA of purified erythrocytes from 7 normal and 12 HbSS individuals

2009-04-04 | E-GEOD-11060 | biostudies-arrayexpress

The human reticulocyte transcriptome

Project description:RNA from circulating blood reticulocytes was utilized to provide a robust description of genes transcribed at the final stages of erythroblast maturation. After depletion of leukocytes and platelets, Affymetrix HG-U133 arrays were hybridized with probe from total RNA isolated from blood sampled from 14 umbilical cords and 14 healthy adult humans. Keywords: Adult vs. fetal reticulocyte transcriptome comparison

2007-03-26 | GSE6236 | GEO

Reticulocyte 3 timepoints

Project description:Mouse models have proven invaluable for understanding erythropoiesis. Here, we describe an autosomal recessive inherited anemia in the mouse mutant hem6. Hematologic and transplantation analyses revealed a mild, congenital, hypochromic, microcytic anemia intrinsic to the hematopoietic system that is associated with a decreased red blood cell zinc protoporphyrin to heme ratio, indicative of porphyrin insufficiency. Iron uptake experiments showed that hem6 reticulocytes are defective in heme production, but not cellular iron uptake defects. Male hem6 mice are infertile due to defects in sperm structure and motility. Through positional cloning and BAC complementation, we identified the gene responsible for the hem6 anemia. We hypothesized that the relative deficiency in erythroid-specific mRNAs in hem6 reticulocytes might be due to decreased mRNA stability. Indeed, serial microarray analysis of reticulocytes aged in vitro showed that numerous, abundantly expressed erythroid-specific transcripts decayed at faster rates in hem6 reticulocytes compared to control reticulocytes. Furthermore, these mRNAs also have progressively shorter poly (A) tails, suggesting a mechanism for the increased rate of decay. Keywords: serial time points Reticulocyte rich blood were collected and cultured ex vivo for 24 hours, samples were collected at 0, 12,24 hours for microarray analysis. There are 3 wild type (wt) biological replicates and 5 mutant (mut) biological replicates in each time point.

2009-10-12 | E-GEOD-16174 | biostudies-arrayexpress

The genomic analysis of erythrocyte microRNA expression in sickle cell diseases

2009-04-04 | GSE11060 | GEO

Comparison of the proteome of human adult and cord erythroid cells and changes in the proteome following reticulocyte maturation.

Project description:Cord blood stem cells are an attractive starting source for the production of red blood cells in vitro for therapy because of additional expansion potential compared to adult peripheral blood progenitors, and cord blood banks usually being more representative of national populations than blood donors. Consequently it is important to establish how similar cord RBCs are to adult cells. In this study we used Multiplex TMTs combined with nanoLC-MS/MS to compare the proteome of adult and cord RBCs and reticulocytes. 2838 unique proteins were identified, providing the most comprehensive compendium of RBC proteins to date. Using stringent criteria 1674 proteins were quantified, and only a small number differed in amount between adult and cord RBC. We focussed on proteins critical for RBC function. Of these only the expected differences in globin subunits, along with higher levels of carbonic anhydrase 1 & 2 and aquaporin-1 in adult RBCs would be expected to have a phenotypic effect since they are associated with the differences in gaseous exchange between adults and neonates. Since the RBC and reticulocyte samples used were autologous, we catalogue the change in proteome following reticulocyte maturation. The majority of proteins (>60% of the 1671 quantified) reduced in abundance between 2 and 100-fold following maturation. However, ~5% were at a higher level in RBCs, localised almost exclusively to cell membranes, in keeping with the known clearance of intracellular recycling pools during reticulocyte maturation. Overall, these data suggest that with respect to the proteome there is no barrier to the use of cord progenitors for the in vitro generation of RBCs for transfusion to adults other than the expression of fetal not adult haemoglobin.

2016-03-31 | PXD003276 | Pride

Antigen discovery in circulating extracellular vesicles from Plasmodium vivax patients

Project description:Plasmodium vivax is the most widely distributed human malaria parasite with 7 million annual clinical cases and 2.5 billion people living under risk of infection. Presently, there is an urgent need to discover new antigens for vaccination as only two vaccine candidates, both based on the Duffy Binding protein, are currently in clinical trials. Extracellular vesicles (EVs) are small membrane-bound vesicles involved in intercellular communication and initially described in reticulocytes, the host cell of P. vivax, as a selective disposal mechanism of the transferrin receptor (CD71) in the maturation of reticulocytes to erythrocytes. We have recently reported the proteomics identification of P. vivax proteins associated to circulating EVs in P. vivax patients using size exclusion chromatography followed by mass spectrometry. Parasite proteins, however, were detected in two out of ten patients and only three of them with more than one unique peptide (UP). To increase the signal, we have implemented the Direct Immuno-affinity Capture (DIC) technique to enrich in EVs derived from CD71-expressing cells. Remarkably, we identified parasite proteins in all patients totaling 48 proteins (24 identified with ≥2 UP) and including several previously identified P. vivax vaccine candidate antigens (MSP1, MSP3, MSP7, MSP9, Serine-repeat antigen 1, and HSP70) as well as several membrane, cytosolic and exported proteins. Notably, a member of the Plasmodium helical interspersed sub-telomeric (PHIST-c) family, previously shown to be a main component of the caveola vesicle complexes, was robustly detected in five out six analyzed patients. Humoral immune response analysis by luminex confirmed its antigenicity. Collectively, we showed that enrichment of EVs by CD71-DIC from plasma, allows a robust identification of P. vivax proteins. This study represents a major advance in identifying new antigens for vaccination against this human malaria parasite.

2022-02-22 | PXD029439 | Pride