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In planta expression of human polyQ-expanded huntingtin fragment reveals mechanisms to prevent disease-related protein aggregation

ABSTRACT: LiP-MS experiment was performed HEK293 cells which are expressing eGFP +mRFP-HTTQ74 or eGFP-SPP+mRFP-HTTQ74 to analyze structural changes in proteome level between these two conditions.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Prerana Wagle

LAB HEAD: David Vilchez

PROVIDER: PXD044409 | Pride | 2023-08-11

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	3488_GFP_report.tsv	Tabular
	3488_lipMSsequences.fasta	Fasta
	E2_ColID567_3488_01.raw	Raw
	E2_ColID567_3488_02.raw	Raw
	E2_ColID567_3488_03.raw	Raw

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In planta expression of human polyQ-expanded huntingtin fragment reveals mechanisms to prevent disease-related protein aggregation.

Project description:We overexpressed eGFP, eGFP-SPP and pmRFP-Q74 construct in HEK93 cells. We made immunoprecipitation of GFP. We compare the interactome of eGFP and eGFP-SPP.

2023-08-11 | PXD044408 | Pride

LiP-MS on Purified CAMKK1 treated with Staurosporine

Project description:Limited proteolysis coupled to mass spectrometry (LiP-MS) is a structural proteomics technique that can be used to identify the targets of small molecules. This is achieved by incubating protein with the drug of interest, which induces structural changes such as occupation of the binding site or changes and protein-protein interactions. This is followed by a limited proteolysis step in which the unspecific protease proteinase K is added to the treated protein sample. The protease preferentially cleaves accessible and flexible regions of the protein, thus generating peptides that reflect the changes in surface accessibility caused by the treatment with the small molecule. Here we treated affinity-purified CAMKK1 kinase inhibitor Staurosporine. The data were generated as part of the IMI EUbOPEN project.

2023-04-26 | PXD041089 | Pride

Proteome-wide structural changes measured with limited proteolysis-mass spectrometry: an advanced protocol for high-throughput applications.

Project description:Proteins regulate biological processes by changing their structure or abundance to accomplish a specific function. In response to any perturbation or stimulus, protein structure may be altered by a variety of molecular events, such as post translational modification, protein-protein interaction, aggregation, allostery, or binding to other molecules. The ability to probe these structural changes in thousands of proteins simultaneously in cells or tissues can provide valuable information about the functional state of a variety of biological processes and pathways. Here we present an updated protocol for LiP-MS, a proteomics technique combining limited proteolysis with mass spectrometry, to detect protein structural alterations in complex backgrounds and on a proteome-wide scale (Cappelletti et al., 2021; Piazza et al., 2020; Schopper et al., 2017). We describe advances in the throughput and robustness of the LiP-MS workflow and implementation of data-independent acquisition (DIA) based mass spectrometry, which together achieve high reproducibility and sensitivity, even on large sample sizes. In addition, we introduce MSstatsLiP, an R package dedicated to the analysis of LiP-MS data for the identification of structurally altered peptides and differentially abundant proteins. Altogether, the newly proposed improvements expand the adaptability of the method and allow for its wide use in systematic functional proteomic studies and translational applications.

2023-01-10 | PXD031627 | Pride

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Proteome-wide structural changes measured with 2 limited proteolysis-mass spectrometry: an advanced 3 protocol for high-throughput applications.

2023-01-10 | PXD031616 | Pride

CRISPRi meets metabolomics: a platform for rapid functional annotation of compound libraries

Project description:Molecular profiling of small-molecules offers invaluable insights on compound functionality and allows for hypothesis generation of targets. However, current profiling methods are either limited in the number of measurable parameters or throughput. Here, we developed a multiplexed, unbiased framework that by linking genetic to drug-induced changes in nearly a thousand metabolites allows for high-throughput functional annotation of compound libraries in Escherichia Coli. First, we generated a reference map of metabolic changes from (CRISPR) interference with 352 genes in all major essential biological processes. Next, based on the comparison of essential gene knockdown metabolic profiles with 1342 drug-induced metabolic changes we demonstrated the ability to make de novo predictions of compound functionality and revealed drugs interfering with unconventional antibacterial targets. The same framework that combines dynamic gene silencing with metabolomics we implemented in E. coli can be adapted and applied as a general strategy for comprehensive high-throughput analysis of compound functionality, from bacteria to human cells.

2021-12-16 | PXD024133 | Pride

Structural basis of calmodulin modulation of the rod cyclic nucleotide-gated channel (LiP-MS)

Project description:The aim of the project was to characterize structural changes that occur on the rod cyclic nucleotide-gated channel upon interaction with calmodulin (CaM) directly in the native membrane.

2023-04-06 | PXD038768 | Pride

Dynamic 3D proteomes reveal functional pathway alterations in situ

Project description:Many biological processes are regulated via molecular events, such as intermolecular interactions and chemical modifications, which do not affect protein levels and escape detection in classical proteomic screens. Reasoning that these events affect protein structure, we propose here that a global protein structural readout could detect many functional alterations simultaneously and in situ. We test this idea using limited proteolysis-mass spectrometry (LiP-MS), which monitors structural changes in thousands of proteins within a native-like environment. In bacteria undergoing nutrient adaptation and in yeast responding to acute stress, this global structural readout captures enzyme activity changes, allosteric regulation, phosphorylation, protein aggregation and protein complex formation, with sufficiently high resolution to identify alterations of single functional sites such as active site occupancy and binding interfaces. Comparison with prior knowledge, including flux, phosphoproteomics and metabolomics data, shows that LiP-MS captures most known enzyme functional alterations and suggests novel metabolite-protein binding events, enabling identification of a fructose-1,6-bisphosphate-based regulatory mechanism of glucose uptake in E. coli. The structural readout dramatically increases classical proteomics screen coverage and generates mechanistic hypotheses, thus better linking holistic and reductionist approaches and paving the way for a new in situ structural systems biology.

2020-11-02 | PXD022297 | Pride

Purkinje cell proteome of the cerebellum of AP-2 cKo

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2024-12-18 | PXD058847 | Pride

Discovery of the Xenon-Protein Interactome using Large-Scale Measurements of Protein Folding and Stability

Project description:The intermolecular interactions of noble gases in biological systems are associated with numerous biochemical responses, including apoptosis, inflammation, anesthesia, analgesia, and neuroprotection. The molecular modes of action underlying these responses are largely unknown. This is in large part due to the limited experimental techniques to study protein-gas interactions. The few techniques that are amenable to such studies are relatively low throughput and require large amounts of purified proteins. Thus, they do not enable the large-scale analyses that are useful for protein-target discovery. Here we report the application of Stability of Proteins from Rates of Oxidation (SPROX) and limited proteolysis (LiP) methodologies to detect protein-xenon interactions on the proteomic scale using protein folding stability measurements. Over 5,000 methionine-containing peptides and semi-tryptic peptides, mapping to ~1,500 and ~950 proteins in a yeast cell lysate, respectively, were assayed for Xe-interacting activity using the SPROX and LiP techniques. The SPROX and LiP analyses identified 31 and 60 Xe-interacting proteins, respectively, none of which were previously known to bind Xe. A bioinformatics analysis of the proteomic results revealed that these Xe-interacting proteins were enriched in those involved in ATP-driven processes. A fraction of the protein targets that were identified is tied to previously established modes of action related to xenon’s anesthetic and organoprotective properties. These results enrich our knowledge and understanding of biologically relevant xenon interactions, and the sample preparation protocols and analytical methodologies developed here for xenon should be applicable to the discovery of a wide range of protein-gas interactions in complex biological mixtures, including cell lysates.

2023-07-20 | PXD029353 | Pride