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One-pot comparative analysis and characterization of young and old mice brain lysates

ABSTRACT: A comparison of different energetics based techniques for the characterization of young and old mice brain cell lysates. The techniques of stability of proteins from rates of oxidation (SPROX), thermal proteome profiling (TPP), Limited Proteolysis (LiP) and conventional expression level analyses were compared and the relative advantages and disadvantages are discussed.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain, Epithelial Cell

SUBMITTER: Morgan Bailey

LAB HEAD: Michael C. Fitzgerald

PROVIDER: PXD036324 | Pride | 2024-05-23

REPOSITORIES: pride

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Dataset's files

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			Action	DRS
	MouseTPP-OP_06-13-2020_01.msf	Msf
	MouseTPP-OP_06-13-2020_01.raw	Raw
	MouseTPP-OP_06-13-2020_02.raw	Raw
	MouseTPP-OP_06-13-2020_03.raw	Raw
	SPROX_enriched_1.msf	Msf

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1 - 5 of 18

Publications

Comparative Analysis of Protein Folding Stability-Based Profiling Methods for Characterization of Biological Phenotypes.

Bailey Morgan A MA Tang Yun Y Park Hye-Jin HJ Fitzgerald Michael C MC

Journal of the American Society for Mass Spectrometry 20230220 3

Recently, a new suite of mass spectrometry-based proteomic methods has been developed that enables evaluation of protein folding stability on the proteomic scale. These methods utilize chemical and thermal denaturation approaches (SPROX and TPP, respectively) as well as proteolysis strategies (DARTS, LiP, and PP) to assess protein folding stability. The analytical capabilities of these technique have been well-established for protein target discovery applications. However, less is known about th ...[more]

PMID: 36802530

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One-pot comparative analysis and characterization of MCF-7 and MCF-10A cell lines

Project description:A comparison of different energetics based techniques for the characterization of two mammalian breast cell lines, MCF-7 a luminal A breast cancer cell line and MCF-10A a normal human breast cell line. The techniques of stability of proteins from rates of oxidation (SPROX), thermal proteome profiling (TPP), and conventional expression level analyses were compared and the relative advantages and disadvantages are discussed.

2023-02-22 | PXD036268 | Pride

Clemastine Fumarate in Plasmodium

Project description:The binding target for clemastine fumarate in Plasmodium Falciparum 3D7 strain is identified by two techniques: SPROX and TPP.

2020-02-21 | PXD014308 | Pride

Utilization of Stability-based Proteomics for the Investigation of Structured RNA-protein Interactions

Project description:The stability of proteins from rates of oxidation (SPROX), thermal protein profiling (TPP) methods as well as triple helix pulldowns with LC-MS/MS readouts are used to identify the protein targets of three RNA ligands, the MALAT1 triple helix (TH), a viral stem loop (SL), and an unstructured RNA (PolyU) in LNCaP nuclear lysate. This work establishes a novel platform for the global discovery and interrogation of RNA-protein interactions that is generalizable to numerous biological contexts and RNA targets.

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One-pot analysis on cyclosporine A binding

Project description:Investigate if one-pot analysis works on several energetic-based proteomics method on cyclosporine A.

2021-09-08 | PXD014309 | Pride

MManassantin A iTRAQ-SPROX Drug Mode-of-Action

Project description:Manassantin A is a natural product that has been isolated from the perennial herb Saururus chinensis Baill and the aquatic plant Saururus cernuus. Manassantin A has been shown to possess potent hypoxia inducible factor 1 alpha (HIF-1α) inhibitory activity in a cell-based assay screen of thousands of natural products. Manassantin A holds promise as an anti-cancer drug since it has been shown to selectively target tumor cells over normal cells. Due to the complex biological pathways involved in cancer and hypoxia, it is difficult to determine the mode-of-action by which manassantin A inhibits HIF-1. While some of the biological activities of manassantin A have been discovered in various cell-based activity assays, the molecular basis of manassantin A’s biological activities is not well characterized. The proteins in a hypoxic MDA-MB-231 cell lysate were screened for interactions with manassantin A using large scale experiments to uncover novel manassantin A interactions that lead to the drug’s HIF-1 inhibition and anti-cancer activity. Two energetics-based approaches were utilized in this manassantin A mode-of-action study: iTRAQ-SPROX and SILAC-Pulse Proteolysis. In these energetics-based approaches, protein stability is measured using the chemical denaturant dependence of either a methionine oxidation reaction (iTRAQ-SPROX) or a thermolysin protease digest (SILAC-Pulse Proteolysis). Using boh of these techniques, the stability of proteins in the absence and presence of excess manassantin A was monitored to assess ligand-induced protein stability changes.

2016-07-12 | PXD002304 | 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

Metal-induced Protein Precipitation in E. coli

Project description:Using a range of copper we have utilized Metal-induced Protein Precipitaiton (MiPP) technique to elucidate the copper midpoints of the proteins found in an E. coli proteome. Using bottem-up proteomics techniques and TMT-10 plex isobaric mass-tags we were able to use MiPP to classify proteins based off of their sensitivity to copper.

2023-01-03 | PXD035711 | Pride

Alterations in lipid profile of the aging kidney identified by MALDI imaging mass spectrometry

Project description:During aging, the kidney undergoes functional and physiological changes that are closely affiliated with chronic kidney disease (CKD). There is increasing evidence supporting the role of lipid or lipid-derived mediators in the pathogenesis of CKD and other aging-related diseases. To understand the role of lipids in various metabolic processes during kidney aging, we conducted MALDI imaging mass spectrometry (MALDI-IMS) analysis in kidneys harvested from young (2 months old, n=3) and old mice (24 months old, n=3). MALDI-IMS analysis showed an increase in ceramide level and a decrease in sphingomyelin (SM) and phosphatidylcholine (PC) levels in kidneys of old mice. The increased expression of cPLA2 and SMPD1 protein in aged kidney was confirmed by immunohistochemistry and western blot analysis. Our MALDI-IMS data showed the altered distribution of lipids in aged kidney as indicative of aging-related functional changes of the kidney. Combined analysis of MALDI-IMS and IHC confirmed lipidomic changes and expression levels of responsible enzymes as well as morphological changes.

2019-06-10 | PXD013860 | Pride

Discovery of Age-Related Protein Folding Stability Differences in the Mouse Brain Proteome

Project description:Described here is the application of thermodynamic stability measurements to study age-related differences in the protein folding and stability of proteins in a rodent model of ageing. Using the Stability of Proteins from Rates of Oxidation (SPROX) technique the thermodynamic stability profiles were generated for 807 proteins in brain cell lystaes from mice, aged 6- (n=7) and 18-months (n=9). The biological variability of the protein stability measurements was low and within the experimental error of SPROX. A total of 83 protein hits were detected with age-related stability differences in the brain samples. Remarkably, the large majority of the brain protein hits were destabilized in the old mice, and the hits were enriched in proteins that have slow turnover rates (p <0.07). Furthermore, 70% of the hits have been previously linked to ageing or age-related diseases. These results help validate the use of thermodynamic stability measurements to capture relevant age-related proteomic changes, and they establish a new biophysical link between these proteins and ageing.

2016-12-22 | PXD005184 | Pride

Plants encode a general siRNA suppressor that is induced and suppressed by viruses

Project description:Small RNAs play essential regulatory roles in genome stability, development and stress responses in most eukaryotes. Plants encode DICER-LIKE (DCL) RNaseIII enzymes, including DCL1, which produces miRNAs, and DCL2, DCL3 and DCL4, which produce diverse size classes of siRNA. Plants also encode RNASE THREE-LIKE (RTL) enzymes that lack DCL-specific domains and whose function is largely unknown. Small RNA sequencing in plants over-expressing RTL1 or RTL2 or lacking RTL2 revealed that RTL1 over-expression inhibits the accumulation of all types of small RNAs produced by DCL2, DCL3 and DCL4, indicating that RTL1 is a general suppressor of plant siRNA pathways. By contrast, RTL2 plays minor, if any, role in the small RNA repertoire. In vivo and in vitro assays revealed that RTL1 prevents siRNA production by degrading dsRNA before they are processed by DCL2, DCL3 and DCL4. The substrate of RTL1 cleavage is likely long perfect (or near-perfect) dsRNA, consistent with the RTL1-insensitivity of miRNAs, which derive from short imperfect dsRNA. RTL1 is naturally expressed only weakly in roots, but virus infection strongly induces its expression in leaves, suggesting that RTL1 induction is a general strategy used by viruses to counteract the siRNA-based plant antiviral defense. Accordingly, transgenic plants over-expressing RTL1 are more sensitive to TYMV infection than wild-type plants, likely because RTL1 prevents the production of antiviral siRNAs. However, TCV, TVCV and CMV, which encode stronger suppressors of RNA silencing (VSR) than TYMV, are insensitive to RTL1 over-expression. Indeed, TCV VSR inhibits RTL1 activity, suggesting that inducing RTL1 expression and dampening RTL1 activity is a dual strategy used by viruses to establish a successful infection. These results reveal another level of complexity in the evolutionary battle between viruses and plant defenses. Flower sRNA profiles in diverse conditions involving RTL1 and RTL2

2013-09-10 | E-GEOD-49866 | biostudies-arrayexpress

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