Dataset Information

A comprehensive evaluation of the Ewing sarcoma surface and global proteomes reveals actionable immunotherapeutic targets.

ABSTRACT: Ewing sarcoma (EwS) is the second most common bone sarcoma in children, with 1 case per 1.5 million in the United States. While the survival rate of patients diagnosed with localized disease is ~70%, this decreases to ~30% for patients with metastatic disease and only ~10% for treatment refractory disease, which remain unchanged for decades. Clearly, new therapeutic strategies are urgently needed for metastatic/refractory EwS. In this study, 19 unique EwS patient or cell line-derived xenografts were analyzed using proteomics to identify surface proteins with potential for immunotherapeutic targeting. Plasma membranes were enriched from each model via density gradient ultracentrifugation and compared to a reference standard consisting of 12 immortalized non-EwS cell lines prepared in a similar manner. In parallel, global proteome analysis was carried out on each model and reference controls to compliment the surfaceome data. All models were analyzed by Tandem Mass Tags (TMT)-based mass spectrometry to quantify identified proteins.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Ewing's Sarcoma Cell

SUBMITTER: Gian Luca Negri

LAB HEAD: Gregg Morin

PROVIDER: PXD043375 | Pride | 2023-09-29

REPOSITORIES: Pride

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			Action	DRS
	231221_U54_Sorensen_EwingWholeProteome_P1F1.raw	Raw
	231221_U54_Sorensen_EwingWholeProteome_P1F10.raw	Raw
	231221_U54_Sorensen_EwingWholeProteome_P1F2.raw	Raw
	231221_U54_Sorensen_EwingWholeProteome_P1F3.raw	Raw
	231221_U54_Sorensen_EwingWholeProteome_P1F4.raw	Raw

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Project description:FMRP loss-of-function causes Fragile X Syndrome (FXS) and autistic features. FMRP is a polyribosome-associated neuronal RNA-binding protein, suggesting that it plays a key role in regulating neuronal translation, but there has been little consensus regarding either its RNA targets or mechanism of action. Here we use high throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP) to identify FMRP interactions with mouse brain polyribosomal mRNAs. FMRP interacts with the coding region of transcripts encoding pre- and postsynaptic proteins, and transcripts implicated in autism spectrum disorders Polyribosomes were prepared from UV254-crosslinked pooled littermate FVB mouse brains as described in detail in published paper PMID 21784246. Polyribosomes were dissociated under denaturing conditions using two different protocols to disrupt ribonucleoprotein complexes. In most experiments Fmr1-null littermates were used in parallel to confirm FMRP specificity. After mild RNAse treatment to reduce the size of RNA to 60-100 nucleotides, endogenous FMRP was immunoprecipitated with one of two antibody combinations, either mixed monoclonal antibodies 7G1-1 and 2F5, or polyclonal antibody ab17722 (Abcam). The ages of the mice were P11, P13, P14, P15, and P25. In addition, to assess a different neuronal polysome-associated protein, as another control, lysates prepared from two samples, the P11 and P13 mice, were split in half and one half IPed with a human anti-Hu antisera as the neuronal Hu proteins are polysome-associated in brain. In sum, 7 FMRP HITS-CLIP experiments were performed including (1) P14 mouse polysomes prepared by Protocol 1 and IPed with 7G1-1/2F5 (2) P14 mouse polysomes prepared by Protocol 1 and IPed with ab17722 (3) P15 mouse polysomes prepared by Protocol 1 and IPed with 7G1-1/2F5 (4) P25 mouse polysomes prepared by Protocol 1 and IPed with 7G1-1/2F5 (5) P25 mouse polysomes prepared by Protocol 1 and IPed with ab17722 (6) P11 mouse polysomes prepared by Protocol 2 and IPed with ab17722 and (7) P13 mouse polysomes prepared by Protocol 2 and IPed with ab17722. FMRP-bound or Hu-bound RNA tags were cloned and submitted for high throughput sequencing on the Life Science 454 platform (samples 1-5) or the Illumina platform (samples 6 and 7 and the 2 Hu samples processed in parallel with samples 6 and 7). Full details are given in PMID 21784246.

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A comprehensive evaluation of the Ewing sarcoma surface and global proteomes reveals actionable immunotherapeutic targets.

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