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NanoLC-MSMS TMT whole cell comparison of ex vivo derived macrophage subtypes.

ABSTRACT: Triplicate samples of donor matched ex vivo generated macrophage subtypes M1, M2a, M2c compared against the M0 control analysed using TMT-based 11plex quantitative proteomics.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Macrophage

SUBMITTER: Charlotte Severn

LAB HEAD: Ash Toye

PROVIDER: PXD032801 | Pride | 2023-09-04

REPOSITORIES: Pride

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NanoLC-MSMS TMT surfaceome comparison of ex vivo derived macrophage subtypes.

Project description:Triplicate samples of donor matched ex vivo generated macrophage subtypes M1, M2a, M2c compared against the M0 control analysed using TMT-based 11plex quantitative proteomics.

2023-09-04 | PXD032823 | Pride

SMS TMT surfaceome comparison of ex vivo derived M1 macrophages treated with dexamethasone

Project description:Triplicate samples of donor matched ex vivo generated macrophage subtype M1 cells treated with and without dexamethasone compared against the M0 control analysed using TMT-based 11plex quantitative proteomics.

2023-09-04 | PXD032905 | Pride

SMS TMT surfaceome comparison of ex vivo derived M1 macrophages treated with dexamethasone

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SMS TMT surfaceome comparison of ex vivo derived M1 macrophages treated with dexamethasone

2023-09-04 | PXD032967 | Pride

Enhancement of red blood cell transfusion compatibility using CRISPR-mediated erythroblast gene editing

Project description:Regular blood transfusion is the cornerstone of care for patients with red blood cell (RBC) disorders such as thalassemia or sickle cell disease. With repeated transfusion, alloimmunisation often occurs due to incompatibility at the level of minor blood group antigens. We use CRISPR-mediated genome editing of an immortalised human erythroblast cell line (BEL-A) to generate multiple enucleation competent cell lines deficient in individual blood groups. Edits are combined to generate a single cell line deficient in multiple antigens responsible for the most common transfusion incompatibilities: ABO (Bombay phenotype), Rh (Rhnull), Kell (K0), Duffy (Duffynull), GPB (S- s- U-). These cells can be differentiated to generate deformable reticulocytes, illustrating the capacity for coexistence of multiple rare blood group antigen null phenotypes. This study provides the first proof-of-principle demonstration of combinatorial CRISPR-mediated blood group gene editing to generate customisable or multi-compatible RBCs for diagnostic reagents or recipients with complicated matching requirements.

2018-04-26 | PXD009291 | Pride

Molecular identification of a BAR domain-containing coat complex for endosomal recycling of transmembrane proteins

Project description:Protein trafficking requires coat complexes that couple recognition of sorting motifs in transmembrane cargos with biogenesis of transport carriers. The mechanisms of cargo transport through the endosomal network are poorly understood. Here, we identify a sorting motif for endosomal recycling of cargos including the cation-independent mannose-6-phosphate receptor and semaphorin 4C by the membrane tubulating BAR domain-containing sorting nexins SNX5 and SNX6. Crystal structures establish that this motif folds into a β-hairpin that binds a site in the SNX5/SNX6 phox homology domains. Over sixty cargos share this motif and require SNX5/SNX6 for their recycling. These include cargos involved in neuronal migration and a Drosophila snx6 mutant displays defects in axonal guidance. These studies identify a sorting motif and provide molecular insight into an evolutionary conserved coat complex, the ‘Endosomal SNX-BAR sorting complex for promoting exit 1’ (ESCPE-1), which couples sorting motif recognition to BAR domain-mediated biogenesis of cargo-enriched tubulo-vesicular transport carriers.

2021-05-28 | PXD014927 | Pride

Ribosome heterogeneity in Drosophila melanogaster gonads through paralog-switching

Project description:Ribosomes have long been thought of as homogeneous macromolecular machines, but recent evidence suggests they are heterogeneous and could be specialised to regulate translation. Here, we have isolated ribosomal complexes and characterised their protein content across 4 tissues of Drosophila melanogaster via TMT-MS. We find that testes and ovaries contain the most heterogeneous ribosome populations, which occurs through a combination of ribosomal protein paralog-enrichment and ribosomal protein paralog-switching. We have also solved structures of ribosomes isolated from tissues by cryo-EM, revealing differences in precise ribosomal arrangement for testis and ovary 80S ribosomes. Differences in the amino acid composition of paralog pairs and their localisation on the ribosome exterior indicate paralog-switching could alter the ribosome surface, enabling different proteins to regulate translation. One testis-specific paralog-switching pair is also found in humans, suggesting this is a conserved site of ribosome heterogeneity. Overall, this work allows us to propose that mRNA translation might be regulated in the gonads through ribosome heterogeneity, providing a potential means of ribosome specialisation.

2021-07-22 | PXD026227 | Pride

Multiomic Approach Characterises the Neuroprotective Role of Retromer in Regulating Lysosomal Health

Project description:Retromer controls cellular homeostasis through regulating integral membrane protein sorting and transport and by controlling late-stage maturation of the endo-lysosomal network. Retromer dysfunction, which is linked to neurodegenerative disorders including Parkinson’s and Alzheimer’s diseases, manifests in complex cellular phenotypes, though the precise nature of this dysfunction, and its relation to neurodegeneration, remain unclear. Here, we perform the first integrated multiomics approach to provide precise insight into the impact of Retromer dysfunction on endo-lysosomal health and homeostasis within a human neuroglioma cell model. We quantify profound changes to the lysosomal proteome, indicative of broad lysosomal dysfunction and inefficient autophagic lysosome reformation, coupled with a reconfigured cell surface proteome and secretome reflective of increased lysosomal exocytosis. Through this global proteomic approach and parallel transcriptomic analysis, we provide an unprecedented integrated view of Retromer function in regulating lysosomal homeostasis and emphasise its role in neuroprotection.

2023-07-20 | PXD041323 | Pride

Characterisation of proteome of a novel Escherichia coli strain exhibiting mucoviscous phenotype.

Project description:Protein expression by E. coli 26561 during the late-exponential phase of cultures under anaerobic conditions was examined. E. coli 26561 is a multidrug resistant (MDR) and shows an unusual hyper-mucoviscous phenotype. Resistance includes ESBL (CTX-M-14) and proteome was determined with and without exposure to sub-MIC concentrations of the 3rd generation cephalosporin ceftazidime. Ceftazidime exposure was at two sub-MIC levels, specifically 0.25x MIC (samples 5-7), 0.5x MIC (samples 8 - 10); samples 1-4 provided the unexposed Control. Both whole and phospho-enriched fractions for each sample were analysed. Quantification of peptides was assessed using 10-plex TMT labelling in conjunction with an Orbitrap Fusion Tribrid. Raw data produced by the Orbitrap were processed using Max Quant 1.5.4.7 using the included Andromeda search engine. Peptides were searched against our own database of E. coli 26561 proteins which was produced from a hybrid assembly of our reads obtained from MiSeq and PacBio sequencing platforms.

2018-08-22 | PXD008339 | Pride

Analysis of oak stem lesions in Acute Oak Decline using shotgun proteomics

Project description:Acute Oak Decline (AOD) is a decline-disease currently spreading in Britain, threatening oak trees. Here, we analyze and compare the proteomes of inner bark tissue sampled from oak stems of trees symptomatic with AOD and non-symptomatic trees.

2018-02-07 | PXD004991 | Pride

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