Dataset Information

ChaFRAtip - Simple, scalable and ultra-sensitive tip-based identification of protease substrates

ABSTRACT: Enrichment of N-terminal peptides using HPLC-based ChaFRADIC and the novel tip-based ChaFRAtip method.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: René Zahedi

LAB HEAD: Rene Zahedi

PROVIDER: PXD006595 | Pride | 2018-01-24

REPOSITORIES: Pride

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Publications

Simple, scalable, and ultrasensitive tip-based identification of protease substrates.

Shema Gerta G Nguyen Minh T N MTN Solari Fiorella A FA Loroch Stefan S Venne A Saskia AS Kollipara Laxmikanth L Sickmann Albert A Verhelst Steven H L SHL Zahedi René P RP

Molecular & cellular proteomics : MCP 20180122 4

Proteases are in the center of many diseases, and consequently, proteases and their substrates are important drug targets as represented by an estimated 5-10% of all drugs under development. Mass spectrometry has been an indispensable tool for the discovery of novel protease substrates, particularly through the proteome-scale enrichment of so-called N-terminal peptides representing endogenous protein N termini. Methods such as combined fractional diagonal chromatography (COFRADIC)<sup>1</sup> an ...[more]

PMID: 29358340

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Project description:High activation of the PI3K-AKT-mTOR pathway is characteristic for T-cell acute lymphoblastic leukemia (T-ALL). Here we investigated how 4EGI-1, a small inhibitor of cap-dependent translation, induces apoptosis in T-ALL clones displaying hyperactive PI3K-AKT-mTOR signaling. 4EGI-1 treatment reduced the ribosomal occupancy of transcripts that define the molecular difference between T-ALL blasts and normal thymocytes. These transcripts encoded proteins for the translational machinery, for mitochondrial metabolism as well as oncoproteins such as Cyclin D1, Bcl-2 and c-myc. Therefore, targeting translation initiation proves valuable in situations where mTOR is activated by multiple events. For each biological replicate 5 x 107 #483 T-ALL cells were treated with 100 ÂµM 4EGI-1 or DMSO. After 2 h incubation at 37Â°C cells were washed in ice-cold PBS containing cycloheximide (0.1 mg/ml), pelleted and resuspended in extraction buffer (20 mM Tris-HCl, pH 8.0, 140 mM KCl, 0.5 mM DTT, 5 mM MgCl2, 0.5 % nonidet-P40, 0.1 mg/ml cycloheximide, 10000 IU/ml dalteparin sodium) and incubated for 5 min on ice. All subsequent steps were carried out in the cold. After centrifugation for 10 min at 12000 x g, 0.4 ml supernatant was layered onto a 12 ml linear sucrose gradient (10 - 50% sucrose [w/v] in extraction buffer without nonidet-P40) and centrifuged at 4 Â°C in an SW40Ti rotor (Beckman) at 35000 rpm without brake for 120 min. The gradients were harvested and absorbance profiles at 254 nm recorded (type 11 optical unit/UA-6 detector, ISCO). 1 ml fractions were collected and supplied with 0.1 volume of 3 M sodium acetate (pH 5.2) and 1 volume of isopropanol for overnight precipitation at -20 Â°C. RNA was purified using Nucleospin RNAII tubes (Macherey & Nagel) following the manufacturerÂ´s protocol. For microarray analysis RNA from polysome fractions were pooled, precipitated by adding LiCl to a final concentration of 2 M and resolubilized in 15 Âµl of RNase-free water. RNA concentrations were determined and the samples were stored at -80 Â°C.

Dataset Information

ChaFRAtip - Simple, scalable and ultra-sensitive tip-based identification of protease substrates

Publications

Simple, scalable, and ultrasensitive tip-based identification of protease substrates.

Similar Datasets

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