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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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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 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

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2019-09-12 | PXD013960 | 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

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

Human Platelet PIP3 Signalosome

Project description:Platelets are small, anucleate cells that play an essential role in haemostasis, but can contribute critically to the pathogenesis of cardiovascular disease. Platelets express all four Class I Phosphoinositide 3-kinase (PI3K) isoforms, with previous work revealing important roles for these enzymes in regulating platelet priming, activation and stable thrombus formation. Despite this, detailed mechanistic understanding of how these lipid kinases orchestrate these roles in platelets is limited. Class I PI3Ks predominantly regulate cell function through their catalytic product, the signalling phospholipid phosphatidylinositol-3,4,5-trisphosphate (PIP3), which coordinates the localisation and/or activity of a diverse range of binding proteins. The complete repertoire of these Class I PI3K effectors in platelets remains unknown, hampering current understanding of Class I PI3K-mediated regulation of platelet function. We therefore conducted a global, unbiased screen for PIP3-binding proteins in human platelets using affinity capture coupled to high resolution proteomic mass spectrometry. Thus, we present the first in depth analysis of the PIP3 signalosome of human platelets, revealing an extensive PIP3 interactome, including many proteins previously uncharacterised in this cell type. This work provides important new insights into how Class I PI3K shapes human platelet function.

2017-06-06 | PXD003777 | 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

Genome-wide microRNA analysis of reticulocytes isolated from patients with sickle cell disease

Project description:MiRNAs are non-protein-coding small RNA molecules negatively regulating gene through inhibition of mRNA. The characteristics of microRNA expression patterns obtained from sickle cell diseases are not clearly elucidated. In this study, we recruited 12 children and adults suffering from sickle cells diseases with high HbF fetal hemoglobin (HbF) group (6 subjects) and lower HbF (6 subjects). The peripheral blood mononuclear cell (PBMN) were processed using a MACS column with magnetic anti-CD71 antibody to isolate reticulocytes according to the manufacturer’s instructions (Miltenyi Biotec, San Diego, CA). After data normalization, 327 miRNA were identified by PCA (principal component analysis) as differentially expressed in low HbF group compared to high HbF groups.

2018-03-03 | GSE111356 | GEO

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2018-04-26 | PXD009291 | Pride

Transcriptome of subsets of human haematopoietic stem cells from non-mobilised peripheral blood of healthy donors

Project description:Integration of index sorting and single cell functional assays identified two functionally distinct subsets of phenotypic haematopoietic stem cells and multipotent progenitors (HSC/MPPs) in the peripheral blood (PB) from healthy individuals. CD71- HSC/MPPs from PB are multipotent and can repopulate the NSG xenograft model. CD71+ HSC/MPPs are a subset of phenotypic HSC/MPPs that is uniquely restricted to give rise to erythroid and megakaryocytic lineages, and expands in conditions with chronic stimulation of platelet production (e.g. frequent platelet donation, idiopathic thrombocytopenic purpura, essential thrombocythaemia). Here, we report the bulk transcriptomes of pools of 20 cells from CD71- HSC/MPPs (CD19- CD38- CD45RA- CD34lo CD71-) and CD71+ HSC/MPPs (CD19- CD38- CD45RA- CD34lo CD71+). Altogether the data shows the transcriptional similarities and differences between both subsets. It shows, that the unique erythroid/megakaryocytic lineage-priming of CD71+ HSC/MPPs is already initiated at transcriptional level, and that CD71+ HSC/MPPs differ from CD71- HSC/MPPs with regards to their protein synthesis/metabolic pathways.

2020-05-01 | GSE131409 | GEO

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