Dataset Information

Proteome of Yki-driven intestinal stem cell tumors in Drosophila midgut

ABSTRACT: The experiment was undertaken to characterize Yki-driven intestinal stem cell tumor proteome from Drosophila adult midguts, and to identify changes in the ISC tumor proteome in flies fed on TONDU peptide, using LC MS/MS approach. esgts>UAS-yki3SA flies on day 1, day 7 of tumor induction; TONDU-peptide fed flies on day 7; and esgts>UAS-yki3SA UAS-vgTONDU on day 7 were used for proteome analysis. Briefly, gut lysate from 20 female flies was obtained in extraction buffer (6M GnHCl in 50mM Tris-Cl pH 7.4, 65 mM DTT) with 50 mM sodium acetate and protease inhibitors (1X protease inhibitor cocktail with 0.2 mM PMSF). 5 microgram of the protein samples were reduced with 5 mM TCEP, further alkylated with 50 mM iodoacetamide, and digested with Trypsin (1:50, Trypsin/lysate ratio) for 16 hours at 37 degree Celsius. Digests were cleaned using a C18 silica cartridge to remove the salt and dried using a speed vac. The dried pellet was resuspended in 5% acetonitrile, 0.1% formic acid (Buffer A). The experiment was performed using an EASY-nLC 1000 system (Thermo Fisher Scientific) coupled to a Thermo Fisher-Orbitrap Fusion mass spectrometer equipped with a nanoelectrospray ion source. 1.0 microgram of the peptide mixture was resolved using a 25 cm Thermo Easy-spray PepMap C18 column. The peptides were loaded with Buffer A and eluted with a 0 to 40% gradient of Buffer B (95% acetonitrile, 0.1% formic acid) at a flow rate of 300 nl/min for 60 min. MS data was acquired using a data-dependent top20 method dynamically choosing the most abundant precursor ions from the survey scan.

INSTRUMENT(S): Thermo Fischer-Orbitrap Fusion Tribrid

ORGANISM(S): Drosophila Melanogaster (ncbitaxon:7227)

SUBMITTER: Anjali Bajpai

PROVIDER: MSV000084841 | MassIVE | Sat Jan 25 00:10:00 GMT 2020

REPOSITORIES: MassIVE

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A <i>Drosophila</i> model of oral peptide therapeutics for adult intestinal stem cell tumors.

Bajpai Anjali A Quazi Taushif Ahmad TA Tang Hong-Wen HW Manzar Nishat N Singh Virender V Thakur Ashwani A Ateeq Bushra B Perrimon Norbert N Sinha Pradip P

Disease models & mechanisms 20200723 7

Peptide therapeutics, unlike small-molecule drugs, display crucial advantages of target specificity and the ability to block large interacting interfaces, such as those of transcription factors. The transcription co-factor of the Hippo pathway, YAP/Yorkie (Yki), has been implicated in many cancers, and is dependent on its interaction with the DNA-binding TEAD/Sd proteins via a large Ω-loop. In addition, the mammalian vestigial-like (VGLL) proteins, specifically their TONDU domain, competitively ...[more]

PMID: 32540914

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