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

ABSTRACT: Temporal profiling of proteolytic cleavage during apoptosis.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: René Zahedi

LAB HEAD: René Zahedi

PROVIDER: PXD006594 | Pride | 2018-01-24

REPOSITORIES: Pride

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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

Similar Datasets

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

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

2018-01-24 | PXD006595 | Pride

The mitochondrial CLPXP complex supports the coordination and assembly of mitochondrial and nuclear encoded protein complexes in Arabidopsis thaliana

Project description:The mitochondrial CLPXP complex supports the coordination and assembly of mitochondrial and nuclear encoded protein complexes in Arabidopsis thaliana. Protein labeling using iTRAQ reagents and application of optimized ChaFRADIC technology to enrich N-Terminal peptides.

2020-09-14 | PXD016263 | Pride

Arabidopsis thaliana N-terminal proteome,LC-MS

Project description:An extended ChaFRADIC workflow was applied to analyze the N-terminal proteome of Arabidopsis thaliana seedlings. Using iTRAQ protein labeling, a multi-enzyme digestion approach including trypsin, GluC, and subtilisin, a total of 200 μg per enzyme were used, and only 1/3 of each ChaFRADIC-enriched fraction were analyzed by LC-MS. Furthermore, our goal was to gain insights of the Met-excision dogma where initiator Met residues are cleaved posstranslationally if the second residue is small, as well as the N-end rule degradation pathway (NERD) discriminating between stabilizing/destabilizing functions of N-terminal amino acid residues in Arabidopsis. We found bona fide NERD destabilizing residues underrepresented. The list of neo N-termini from wild type samples represents an extremely helpful resource for excluding pseudo-candidates of NERD.

2015-07-21 | PXD001855 | Pride

Periostin as a blood biomarker of muscle cell fibrosis, cardiomyopathy and disease severity in myotonic dystrophy type 1

Project description:Myotonic dystrophy type 1 (DM1) is the most common form of adult-onset muscular dystrophy and is caused by an repeat expansion [r(CUG)exp] located in the 3' untranslated region of the DMPK gene. Symptoms include skeletal and cardiac muscle dysfunction and fibrosis. In DM1, there is a lack of established biomarkers in routine clinical practice. Thus, we aimed to identify a blood biomarker with relevance for DM1-pathophysiology and clinical presentation.

2023-05-02 | PXD024107 | Pride

Regulatory function of sympathetic innervation on the endo/lysosomal trafficking of acetylcholine receptor

Project description:Regulation of membrane receptors involves management of endocytosis. At the neuromuscular junction, the synapse between skeletal muscle and motoneuron, proper density of the major receptor, the acetylcholine receptor, is of utmost importance for sustaining life in context of mobility. Recent work has revealed innervation of NMJs by sympathetic neurons and destruction of them had morphological and functional consequences, suggesting influence on endocytosis. To investigate the pathways and proteins that are relevant for acetylcholine receptor turnover and affected by sympathetic signaling, proteomes of mouse hindlimb muscles from sympathectomized and saline-treated control muscles were compared. Using proteomic, Western blot, and immunofluorescence analysis in chemically sympathectomized mouse hindlimb muscles, the cause of these consequences were aimed to analyzed. This revealed extensive regulation of the proteome by the sympathetic nervous system and a possible regulatory function of the endo/lysosomal and autophagic pathway by sympathetic neuronal input. This finding might provide a new explanation to the observed benefit of sympathicomimetic treatment in several congenital myasthenic syndromes.

2021-03-01 | PXD021601 | Pride

Mild proteasomal stress improves photosynthetic performance in Arabidopsis chloroplasts

Project description:The ppi2 mutant introgressed into the Columbia-0 ecotype as previously reported. Doublemutant ppi2xrpn8a was crossed with the above referenced genotypes using heterozygous ppi2 plants. After 2 days of stratification at 4 °C the plants were grown on half-strength Murashige and Skoog (M&S) medium supplemented with 0.8% (w/v) plant agar (Duchefa) and 0.8% (w/v) sucrose under short day conditions.

2020-05-27 | PXD017126 | Pride

Combined assessment of global proteome, phosphoproteome and N-terminal protein cleavage to characterize altered platelet functions in human Scott syndrome

Project description:The Scott syndrome is a rare bleeding disorder associated with a mutation in the gene encoding anoctamin-6 (TMEM16F). After stimulation of Ca2+-mobilizing agonists, syndromatic platelets show a reduced phosphatidylserine exposure and do not form membrane blebs. Given the central role of anoctamin-6 in the platelet procoagulant response, we used quantitative proteomics to understand the underlying molecular mechanisms and the complex phenotypic changes in Scott platelets compared to control platelets. Therefore, we applied an iTRAQ-based multi-pronged strategy to quantify changes in (i) the global proteome, (ii) the phosphoproteome and (iii) proteolytic events between resting and stimulated Scott and control platelets. Our data indicate a limited number of proteins with decreased (70) or increased (64) expression in Scott platelets, among those we observed the absence of anoctamin-6 and the strong up-regulation of aquaporin-1. Furthermore, the quantification of 1,566 phosphopeptides revealed major differences between Scott and control platelets after stimulation with thrombin/convulxin or ionomycin. Finally, we quantified 1,596 N-terminal peptides in activated Scott and control platelets, 180 of which we identified as calpain-regulated, whereas a distinct set of 23 neo-N-termini was caspase-regulated. In Scott platelets, calpain-induced cleavage of cytoskeleton-linked and signaling proteins was down-regulated, in accordance with an increased phosphorylation state. Thus, multi-pronged proteomic profiling of Scott platelets provides detailed insight into their protection against detrimental Ca2+-dependent changes that are normally associated with phosphatidylserine exposure.

2016-08-24 | PXD002883 | Pride

Engineered transfer RNAs for suppression of premature termination codons

Project description:PTCs cause a multitude of human diseases and there are no established therapeutic options for their therapeutic management. Herein, we report the high-throughput cloning and identification, characterization and functional analysis of anticodon-edited tRNA which display efficacious PTC reversion in eukaryotic cells and mouse skeletal muscle. Notably, our screen identifies ACE-tRNA, in total, with the potential to repair a vast majority of known human disease-causing PTC, but this therapeutic will require overcoming tissue and delivery specific challenges. However, the engineered tRNA, once delivered, faithfully encode their cognate amino acid, thus abrogating spurious effects on downstream protein stability, folding, and trafficking, and consequently negating the need for tandem therapies involving protein folding or trafficking agents. When transfected as cDNA, ACE-tRNAs rescued multiple full-length proteins via PTC suppression; a NLuc luciferase reporter, a model protein HDH, and two disease nonsense mutations in CFTR.

2019-02-11 | PXD012581 | Pride

HEK293 cells LC-MSMS - XBP1 mitigates aminoglycoside-induced endoplasmic reticulum stress and neuronal cell death

Project description:The iTRAQ 4-plex experiment was carried out with 2 biological replicates of untreated HEK cells (114 and 116 label) and 2 biological replicates of HEK cells treated with geneticin (16 µM) for 32 h (115 and 117 label).

2015-08-10 | PXD000933 | Pride

Proteomic analysis of primary tumor and lymph metastatic tissues as well as lung cancer cells

Project description:4 pairs of matched primary versus metastatic tumor tissues from formalin-fixed, paraffin-embedded (FFPE) tissue bank,and two frozen lung tumor tissues from patients with primary orlymph metastasis. Then performed tandem mass tag (TMT) labeling and quantitative mass spectrometry analysis. A549 Cells we used was overexpressed luceiferase and designated as L0,underwent 3rounds selection in vivo to form L2, L6 that were metastatic to the spinal. We cultured L0 cells in "light" medium, L2,L6 in "medium heavy" and "heavy" respectively. Harvesting of cells and combined in 1:1:1 ratio, peptides were seperated using high-PH RPLC and analyzed by LC-MS/MS.

2022-02-17 | PXD025052 | Pride

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