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VHH D4 target identification by Mass Spectrometry

ABSTRACT: Using a synthetic library of humanized llama single domain antibodies, we identified a novel surface marker for human naturally-occuring monocyte-derived DCs. Proteomics Mass-spectrometry was used to idenitfy the target of this antibody. We found that our antibody targets an extra-cellular domain of LSP-1.

INSTRUMENT(S): TripleTOF 6600

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Cell, Monocyte

SUBMITTER: Valentin SABATET

LAB HEAD: Damarys Loew

PROVIDER: PXD015647 | Pride | 2020-05-26

REPOSITORIES: Pride

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			Action	DRS
	B0150BL.mgf	Mgf
	B0151BL.mgf	Mgf
	B0152BL.mgf	Mgf
	B0153BL.mgf	Mgf
	Design.xls	Xls

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Publications

Surface LSP-1 Is a Phenotypic Marker Distinguishing Human Classical versus Monocyte-Derived Dendritic Cells.

Moutel Sandrine S Beugnet Anne A Schneider Aurélie A Lombard Bérangère B Loew Damarys D Amigorena Sebastian S Perez Franck F Segura Elodie E

iScience 20200316 4

Human mononuclear phagocytes comprise several subsets of dendritic cells (DCs), monocytes, and macrophages. Distinguishing one population from another is challenging, especially in inflamed tissues, owing to the promiscuous expression of phenotypic markers. Using a synthetic library of humanized llama single domain antibodies, we identified a novel surface marker for human naturally occurring monocyte-derived DCs. Our antibody targets an extra-cellular domain of LSP-1, specifically on monocyte-d ...[more]

PMID: 32224433

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Evaluation of phospho-tyrosine antibodies for label-free phosphoproteomics

Project description:In the past decade multiple tyrosine kinase inhibitors (TKIs) have been implemented in standard treatment regimens for patients with cancer. Unfortunately the majority of patients develops resistance to these drugs. Reliable tools for analysis of pharmacodynamic effects and drug resistance mechanisms are therefore warranted. Phosphoproteomics has meanwhile emerged as tool for the analysis of tyrosine protein phosphorylation. These studies rely on antibodies for enrichment of tyrosine-phosphorylated peptides. Here we compared two commercially available phosphotyrosine antibodies and show that P-Tyr-1000 yields 64% more phosphopeptides than the 4G10 antibody. To investigate performance of P-Tyr-1000 in a label-free comparative phosphoproteomics analysis, a commonly used cell culture model was employed. U87 glioma cells with or without EGFRvIII mutation, lacking the extracellular ligand binding domains (exon 2 - 7) and displaying constitutive signaling activity, were analyzed with the workflow described above. U87 cells and the isogenic version with EGFRvIII were both treated with or without the TKI Erlotinib. In total 1330 phosphopeptides were detected derived from 683 phosphorylated proteins.

2015-12-09 | PXD001565 | Pride

Genome-wide identification of RNA polymerase, RpoD, and RpoS binding sites in Klebsiella pneumoniae MGH 78578

Project description:Genome-wide identification of RNA polymerase (RNAP) binding sites were performed in Klebsiella pneumoniae MGH 78578 (KP). Anti-RNAP is used to capture the RNAP in KP. ChIP-chip was performed on tiling array specifically made for KP. Comparison ChIP by anti-RNAP antibody vs ChIP by normal mouse IgG (control, mock IP)

2012-12-31 | E-GEOD-35927 | biostudies-arrayexpress

Exploring the glycosylation state of the Cryptosporidium parvum CpTSP protein family-Cman pulldowns

Project description:Cryptosporidium parvum is a zoonotic apicomplexan parasite and a common cause of diarrheal disease worldwide. The development of vaccines to prevent or limit infection remains an important goal for tackling these diarrheal diseases, which are a significant cause of infant morbidity in the developing world. The only approved vaccine against an apicomplexan parasite targets conserved adhesins possessing a thrombospondin repeat (TSR) domains. Orthologous TSR domain-containing proteins are commonplace in the apicomplexa and C. parvum possess 12 such proteins. Here, we explore the molecular evolution and conservation of these proteins and examine their abundance in C. parvum oocysts to assess the likelihood that they may be useful as vaccine candidates. We go onto examine the glycosylation states of these proteins using antibody-enabled and ZIC-HILIC enrichment techniques, which revealed that these proteins are modified with C-linked Hex and N-linked Hex5-6HexNAc2 glycans.

2023-01-12 | PXD023934 | Pride

Chromatin immunoprecipitation with p53-antibody of human osteosarcoma cell line Saos-2 infected with adenomviruses encoding GFP, or GFP with p53 H1 helix mutants EL or RE

Project description:ChIP-chip study using Saos-2 cell line infected with adenoviruses encoding GFP (Mock) or GFP together with the p53 H1 helix mutants EL or RE. DNA-protein-complexes were precipitated with monoclonal p53-antibody (clone DO-1). Mock-chromatin was immunoprecipitated in the absence of antibody.<br>Three completely independent biological replicates were performed for each antibody, obtaining the corresponding input as total genomic DNA reference. Hybridizations were performed using Affymetrix GeneChip Human Tiling 2.0R Array set (7 arrays set).<br><br> Affymetrix .BAR files and an additional processed data file containing the coordinates of the identified binding sites can be found in the FTP directory for this experiment. <A HREF="ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/MEXP/E-MEXP-1748">FTP directory</A>

2010-05-13 | E-MEXP-1748 | biostudies-arrayexpress

SvenBldDChIPSeq

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2016-07-03 | E-MTAB-2196 | biostudies-arrayexpress

ZSWIM4 regulates early embryonic patterning and BMP signaling pathway by promoting nuclear Smad1 degradation

Project description:The dorsoventral gradient of BMP signaling plays an essential role in embryonic patterning, with high BMP signal activating ventral-lateral mesoderm markers directly, and low BMP signal inducing neural tissues. The Zinc finger SWIM domain-containing protein 4 (zswim4) is expressed in the dorsal blastopore lip at the onset of Xenopus gastrula and then enriched at the forming neuroectoderm at mid-gastrula stages. Overexpression of zswim4 in Xenopus embryos causes inhibition of the anterior axis and shortened, curved body, and knockdown or knockout of zswim4 disturbed embryonic body axis formation and head development. The expression of ventral-lateral mesoderm marker genes was reduced after zswim4 overexpression and increased in embryos with zswim4 knockdown. Neural marker genes were repressed in zswim4 morphant. Mechanistically Zswim4 attenuates BMP signal through reducing protein stability of Smad1 in both Xenopus embryos and HEK293T cells. Zswim4 interacts with Smad1 and promotes ubiquitination of Smad1 in HEK293T cells. To identify the interaction partner of Zswim4 in regulating Smad1 stability, we performed SILAC based IP in HEK293T cells, and the precipitates were analyzed by Mass Spectrometry.

2024-05-22 | PXD035207 | Pride

Regulation of the endosomal SNX27 retromer by OTULIN

Project description:OTULIN specifically hydrolyzes methionine1 (Met1)-linked ubiquitin chains conjugated by LUBAC (linear ubiquitin chain assembly complex). Using mass spectrometry, we discovered an interaction of OTULIN with SNX27 (sorting nexin 27), an adaptor of the endosomal retromer complex responsible for protein recycling to the cell surface. The C-terminal PDZ binding motif (PDZbm) in OTULIN associates with the cargo-binding site in the PDZ domain of SNX27. By solving the structure of the OTU domain in complex with the PDZ domain, we demonstrate that a second interface contributes to the selective, high affinity interaction of OTULIN and SNX27. SNX27 does not affect OTULIN catalytic activity, OTULIN-LUBAC binding or Met1-linked ubiquitin chain homeostasis. However, via association OTULIN antagonizes SNX27-dependent cargo loading, binding of SNX27 to the retromer subunit VPS26 and endosome-to-membrane trafficking. Thus, we define an additional, non-catalytic function of OTULIN in the regulation of retromer assembly and protein recycling to the cell surface.

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Perilipin 5-mediated lipid droplet-mitochondria coupling determines mitochondrial capacity

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