Browse
Submit Data
Databases
API
Help

Dataset Information

301 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

A whole proteome inventory of background photocrosslinker binding

ABSTRACT: Affinity based protein profiling (AfBPP) is a widely applied methodology for target identification of bioactive molecules. Photocrosslinkers such as benzophenone, diazirine and aryl azide irreversibly link the molecule of interest to its dedicated target protein upon irradiation with UV light. Despite their prevalent application in chemical biology little is known about photolinker-specific off-targets affecting the reliability of results. Here we investigate background protein labelling in intact human cell lines via gel-free quantitative proteomics. Characteristic off-targets were identified for each photo-reactive group and compiled in a comprehensive inventory (photome). In a proof of principle study, H8, an established inhibitor of protein kinase A (PKA), was equipped with a diazirine moiety and revealed, by alignment with the photo-background, unprecedented insight into its in situ proteome targets. Taken together, our findings provide an experimental guideline to surmount photoprobe ambiguity and focus results on biologically relevant binders.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: Matthias Stahl

LAB HEAD: Stephan Axel Sieber

PROVIDER: PXD004009 | Pride | 2016-12-05

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Similar Datasets

Whole proteome and Secretome analysis of Staphylococcus aureus ClpXP mutant strains

Project description:Staphylococcus aureus represents an opportunistic pathogen which utilizes elaborate quorum sensing mechanisms to precisely control the expression and secretion of virulence factors. Previous studies indicated a role of the ClpXP proteolytic system in controlling pathogenesis. While detailed transcriptome data for ClpP and ClpX is available, corresponding studies on the proteome, secretome and metabolome level are largely lacking. In order to decipher the functional roles of ClpP and ClpX more globally we utilized S. aureus deletion mutants of the corresponding genes for in-depth proteomic LC-MS/MS analysis. These studies were complemented by a ClpP active site mutant strain to monitor changes solely depending on the presence and not the activity of the protein. A comparison of these strains with wild type revealed e.g. downregulation of virulence, purine/pyrimidine biosynthesis, iron uptake and stress response. Correspondingly, a reduction of a subset of purine and pyrimidine metabolite levels independently confirmed these results. Interestingly, comparison between the ClpP knockout and ClpP active site mutant strains revealed characteristic differences in UMP biosynthesis and the staphyloferrin B pathway, suggesting that the presence of the protein, although inactive, is important for maintaining these processes. These results are not only of fundamental importance to understand the cellular role of ClpXP but also have implications for the development of novel virulence inhibitor classes.

2021-03-12 | PXD018932 | Pride

A chemical probe to monitor parkinsonism-associated protein DJ-1 in live cells

Project description:Oxidative stress is a contingent trigger of Parkinson`s disease (PD). DJ-1, a protein involved in oxidative stress sensing, gained major attention when mutations in its gene were identified in PD patients. Although the study of the corresponding protein function is still in its infancy, a crucial oxidation sensor at a conserved cysteine was linked to the disease. Inspired by inhibition studies with a bacterial homolog of DJ-1 we here screen several amino-epoxycylcohexenones and identify a chemical probe that exhibits specificity for the human protein in various cell lines. The probe selectively labeled the oxidation sensor via nucleophilic attack of the conserved cysteine onto the epoxide ring. This covalent addition occurred only in the reduced state. Whole proteome studies in HeLa, A549 and SHSY5Y cell lines confirmed strong enrichment of solely reduced DJ-1 which was diminished by increasing oxidative stress. The probe thus facilitates the first selective in situ monitoring of DJ-1 in its reduced state and enables studying the function of this important biomarker in dependence of oxidative stress.

2018-07-17 | PXD009444 | Pride

Tranylcypromine specificity for monoamine oxidase is limited by promiscuous protein labelling and lysosomal trapping

Project description:Monoamine oxidases MAOA and MAOB catalyze important cellular functions such as the deamination of neurotransmitters. Correspondingly, MAO inhibitors are used for the treatment of severe neuropsychiatric disorders such as depression. A commonly prescribed drug against refractory depression is tranylcypromine, however, the side effects are poorly understood. In order to decipher putative off-targets, we synthesized two tranylcypromine probes equipped with either an alkyne moiety or an alkyne-diazirine minimal photocrosslinker for in situ proteome profiling. Surprisingly, LC-MS/MS analysis revealed low enrichment of MAOA and relatively promiscuous labeling of proteins. Photoprobe labeling paired with fluorescent imaging studies revealed lysosomal trapping which could be largely reverted by the addition of lysosomotropic drugs.

2020-10-20 | PXD018580 | Pride

mRNA profiling of MEST knockdown in human adipocyte precursor cells at early stage (day 3) of adipocyte differentiation

Project description:The Mesoderm-specific transcript homolog protein MEST is a member of the alpha/beta hydrolase fold family that is expressed in adipose tissue, adipocytes as well as their precursor cells. To get an idea about potential functions of MEST in adipocyte development, we knocked down MEST in human Multipotent Adipose-Derived Stem (hMADS) cells, induced adipocyte differentiation by a chemically defined medium and performed gene expression profiling at the early stage of adipocyte differentiation. Two-condition experiment, hMADS cells at day 3 after induction of adipocyte differentiation, comparison of cells transfected with an siRNA targeting MEST (siMEST) to cells transfected with a control siRNA (siC). Biological replicates: 3, indepently grown and harvested. On each array, one biological replicate of siMEST-transfected cells was directly compared to one biological replicate os siC-transfected cells (serving as reference sample). All hybridizations were repeated with reversed dye assignment (dye-swap) as technical replicates.

2015-06-24 | E-GEOD-63132 | biostudies-arrayexpress

mRNA profiling of white hMADS cells converted in brite adipocytes and transfected with mimic miR-125b-5p

Project description:miR-125b-5p is a well known miRNA already describded in several forms of cancer. miR-125b-5p is expressed in adipose tissue, adipocytes as well as their precursor cells. We aim to invest the role of miR-125b-5p in white adipocytes conversion into brite adipocytes. To get an idea about putative targets of miR-125b-5p in adipocyte conversion, we transfected miR-125b-5p mimic in human Multipotent Adipose-Derived Stem (hMADS) cells, differenciated in white adipocytes. Gene expression profiling is performed 48h after hMADSC transfection. Two-condition experiment, hMADS cells at day 16 after conversion of white adipocytes into brite adipocytes, comparison of cells transfected with a mimic miR-125b-5p to cells transfected with a negative controle. Biological replicates: 4, indepently grown and harvested. On each array, one biological replicate of mimic miR-125b-5p transfected cells was directly compared to one biological replicate of mimic negative control transfected cells (serving as reference sample). All hybridizations were repeated with reversed dye assignment (dye-swap) as technical replicates.

2016-07-01 | E-GEOD-80816 | biostudies-arrayexpress

Global proteomics analysis reveals a role for histone variant H3.3 in the response to starvation

Project description:Periodic starvation of animals induces large shifts in metabolism, but may also influence many other cellular systems and can lead to adaption to prolonged starvation conditions. To date, there is limited understanding of how starvation affects gene expression, particularly at the protein level. Here, we have used mass spectrometry‐based quantitative proteomics to identify global changes in the C. elegans proteome due to acute starvation of young adult animals. Measuring changes in abundance of up to 7,000 proteins, we show that acute starvation rapidly alters the levels of hundreds of proteins, many involved in central metabolic pathways, highlighting key regulatory responses. Surprisingly, we also detect changes in the abundance of chromatin‐associated proteins including specific linker histones, histone variants and histone post‐translational modifications associated with the epigenetic control of gene expression. A null mutant for one of these proteins, the histone H3.3 variant HIS‐71, identified a subset of proteins whose abundance no longer varied in response to starvation. This mutant also displayed defects in starvation stress resistance and showed a reduced adult lifespan. To maximise community access to these data, they are presented in an online searchable database, the Encyclopedia of Proteome Dynamics (http://www.peptracker.com/epd/).

2015-06-17 | PXD001723 | Pride

Methylome analysis of 123 epiRILs, WT and ddm1 mutant aerial part

Project description:DNA methylation profiling in 123 epiRILs, WT and ddm1 mutant aerial part (epiRIL008 - epiRIL011 - epiRIL014 - epiRIL018 - epiRIL020 - epiRIL024 - epiRIL036 - epiRIL046 - epiRIL052 - epiRIL053 - epiRIL054 - epiRIL055 - epiRIL060 - epiRIL062 - epiRIL064 - epiRIL069 - epiRIL070 - epiRIL071 - epiRIL073 - epiRIL092 - epiRIL093 - epiRILv94 - epiRIL095 - epiRIL098 - epiRIL099 - epiRIL101 - epiRIL108 - epiRIL112 - epiRIL114 - epiRIL118 - epiRIL122 - epiRIL137 - epiRIL144 - epiRIL147 - epiRIL148 - epiRIL150 - epiRIL159 - epiRIL164 - epiRIL166 - epiRIL169 - epiRIL170 - epiRIL172 - epiRIL183 - epiRIL193 - epiRIL195 - - epiRIL202 - epiRIL208 - epiRIL215 - epiRIL216 - epiRIL218 - epiRIL222 - epiRIL225 - epiRIL229 - epiRIL232 - epiRIL238 - epiRIL244 - epiRIL252 - epiRIL257 - epiRIL258 - epiRIL260 - epiRIL262 - epiRIL275 - epiRIL276 - epiRIL277 - epiRIL297 - epiRIL305 - epiRIL315 - epiRIL323 - epiRIL326 - epiRIL333 - epiRIL340 - epiRIL344 - epiRIL350 - epiRIL356 - epiRIL362 - epiRIL361 - epiRIL363 - epiRIL366 - epiRIL368 - epiRIL371 - epiRIL375 - epiRIL391 - epiRIL393 - epiRIL394 - epiRIL400 - epiRIL408 - epiRIL410 - epiRIL425 - epiRIL432 - epiRIL434 - epiRIL437 - epiRIL438 - epiRIL439 - epiRIL454 - epiRIL458 - epiRIL466 - epiRIL467 - epiRIL471 - epiRIL473 - epiRIL477 - epiRIL480 - epiRIL488 - epiRIL492 - epiRIL493 - epiRIL494 - epiRIL495 - epiRIL497 - epiRIL500 - epiRIL503 - epiRIL506 - epiRIL508 - epiRIL523 - epiRIL538 - epiRIL539 - epiRIL556 - epiRIL558 - epiRIL559 - epiRIL561 - epiRIL567 - epiRIL570 - epiRIL572 - epiRIL573 - epiRIL579 - wt - ddm1 mutant) Keywords: Genome binding/occupancy profiling by genome tiling array 1 biological replicate in dye-swap - MeDIP-chip

2012-09-03 | E-GEOD-37284 | biostudies-arrayexpress

Genome-Scale Mapping of Escherichia coli σ54 Reveals Widespread, Conserved Intragenic Binding

Project description:We investigated the genome-wide binding sites for sigma54 and RNA polymerase in wild-type Escherichia coli MG1655.

2015-08-06 | E-MTAB-3697 | biostudies-arrayexpress

MG1655 flagellar regulator ChIP-seq (FlhD, FlhC, FliA)

Project description:We mapped the genome-wide binding of the flagellar regulators FlhD, FlhC, and FliA in FLAG-tagged derivatives of E. coli K-12 MG1655 using ChIP coupled with deep sequencing (ChIP-seq). We identify new binding sites for each factor.

2014-04-10 | E-MTAB-2457 | biostudies-arrayexpress

Methylome analysis of nerd1-1 mutant seedlings

Project description:DNA methylation profiling in nerd1-1 mutant seedlings 2 biological replicates in dye-swap - MeDIP-chip

2012-12-20 | E-GEOD-35344 | biostudies-arrayexpress