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Barley newly synthesized ribosomal proteome: E.coli control samples

ABSTRACT: Ribosomal proteome profiling control for project: Plants synthesize new ribosomal proteins and recycle others in cold-acclimated ribosomes and these in turn synthesize a differential proteome in barley root meristems.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Escherichia Coli

SUBMITTER: Federico Martinez-Seidel

LAB HEAD: Berin A. Boughton

PROVIDER: PXD032938 | Pride | 2024-05-23

REPOSITORIES: Pride

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			Action	DRS
	201009_Federico_FMS-1225_1.raw	Raw
	201009_Federico_FMS-1225_2.raw	Raw
	201009_Federico_FMS-1225_3.raw	Raw
	allSpectra.HCD.FTMS.iso_0.apl	Other
	allSpectra.HCD.FTMS.iso_1.apl	Other

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Publications

Streamlining Protein Fractional Synthesis Rates Using SP3 Beads and Stable Isotope Mass Spectrometry: A Case Study on the Plant Ribosome.

Gentry-Torfer Dione D Murillo Ester E Barrington Chloe L CL Nie Shuai S Leeming Michael G MG Suwanchaikasem Pipob P Williamson Nicholas A NA Roessner Ute U Boughton Berin A BA Kopka Joachim J Martinez-Seidel Federico F

Bio-protocol 20240505 9

Ribosomes are an archetypal ribonucleoprotein assembly. Due to ribosomal evolution and function, r-proteins share specific physicochemical similarities, making the riboproteome particularly suited for tailored proteome profiling methods. Moreover, the structural proteome of ribonucleoprotein assemblies reflects context-dependent functional features. Thus, characterizing the state of riboproteomes provides insights to uncover the context-dependent functionality of r-protein rearrangements, as the ...[more]

PMID: 38737506

Similar Datasets

Barley newly synthesized ribosomal proteome

Project description:Plants synthesize new ribosomal proteins and recycle others in cold-acclimated ribosomes and these in turn synthesize a differential proteome in barley root meristems.

2024-11-16 | PXD032923 | Pride

E.coli Ribosome protein composition

Project description:Ribosomes are essential macromolecular complexes conducting protein biosynthesis in all domains of life. Cells can have heterogeneous ribosomes, i.e. ribosomes with various ribosomal RNA (rRNA) and ribosomal protein (r-protein) composition. However, the functional importance of heterogeneous ribosomes has remained elusive. One of the possible sources for ribosome heterogeneity is provided by paralogous r-proteins. In E. coli, ribosomal protein bL31 has two paralogs: bL31A encoded by rpmE and bL31B encoded by ykgM. This study investigates phenotypic effects of these ribosomal protein paralogs using bacterial strains expressing only bL31A or bL31B. We show that bL31A confers higher fitness to E. coli under lower temperatures. In addition, bL31A and bL31B have different effects on translation reading frame maintenance and apparent translation processivity in vivo as demonstrated by dual luciferase assay. In general, this study demonstrates that ribosomal protein paralog composition (bL31A versus bL31B) can affect cell growth and translation outcome.

2020-10-20 | PXD018123 | Pride

Quantitative analysis of newly synthesized proteins.

Project description:Measuring proteome response to perturbations is critical for understanding the underlying mechanisms involved. Traditional quantitative proteomic methods are limited by the large numbers of proteins in the proteome and the mass spectrometer's dynamic range. A previous method uses the biorthogonal reagent azidohomoalanine (AHA), a methionine analog, for labeling, enrichment and detection of newly synthesized proteins (NSPs). Newly synthesized AHA proteins can be coupled to biotin via CuAAC-mediated click chemistry and enriched using avidin-based affinity purification. The combination of AHA-mediated NSP labeling with metabolic stable isotope labeling allows quantitation of low-abundant, newly secreted proteins by mass spectrometry (MS). However, the resulting multiplicity of labeling complicates NSP analysis. We developed a new NSP quantification strategy, called HILAQ (heavy isotope-labeled azidohomoalanine quantification), that uses a heavy isotope-labeled AHA molecule to enable NSP labeling, enrichment, identification and quantification. In addition, the AHA-peptide enrichment used in HILAQ improves both the identification and quantification of NSPs over AHA-protein enrichment. Here, we provide a description of the HILAQ method that includes procedures for (i) pulse-labeling and harvesting NSPs; (ii) addition of biotin by click reaction; (iii) protein precipitation; (iv) protein digestion; (v) enrichment of AHA-biotin peptides by NeutrAvidin beads and four-step elution; (vi) MS analysis; and (vii) data analysis for the identification and quantification of NSPs by ProLuCID and pQuant. We demonstrate our HILAQ approach by identifying NSPs from cell cultures, but we anticipate that it can be adapted for applications in animal models. The whole protocol takes ~6 d to complete.

| S-EPMC7422752 | biostudies-literature

Identify the newly synthesized proteins by P. aeruginosa after engulfment by host immune cells by pulsed-SILAC proteomics

Project description:To identify the newly synthesized proteins by P. aeruginosa after engulfment by host immune cells, we employed the pulsed-SILAC proteomics approach to label the bacterial proteome with L-lysine-13C6 15N2 (Lys8) and L-lysine (Lys0) before and after entering host cells, respectively. The PAO1△lysA mutant which failed to synthesize lysine was cultured in ABTG medium 37 supplemented with stable isotope-containing amino acid L-lysine-13C6 15N2 (Lys8). The Lys8 labeled bacteria were then harvested and used to infect the RAW264.7 cells which were cultured in DMEM containing regular L-lysine (Lys0). After 4 h infection, RAW264.7 cells were lysed, and the intracellular bacteria were harvested and analyzed by LC-MS/MS. For the control DMEM group, labeled bacteria were diluted in pure DMEM medium. By using this pulsed-labelling approach, the newly synthesized proteins can be selectively tagged with Lys0 during the infection.

2023-04-18 | PXD041601 |

Vulnerability of newly synthesized proteins to proteostasis stress.

Project description:The capacity of the cell to produce, fold and degrade proteins relies on components of the proteostasis network. Multiple types of insults can impose a burden on this network, causing protein misfolding. Using thermal stress, a classic example of acute proteostatic stress, we demonstrate that ?5-10% of the soluble cytosolic and nuclear proteome in human HEK293 cells is vulnerable to misfolding when proteostatic function is overwhelmed. Inhibiting new protein synthesis for 30?min prior to heat-shock dramatically reduced the amount of heat-stress induced polyubiquitylation, and reduced the misfolding of proteins identified as vulnerable to thermal stress. Following prior studies in C. elegans in which mutant huntingtin (Q103) expression was shown to cause the secondary misfolding of cytosolic proteins, we also demonstrate that mutant huntingtin causes similar 'secondary' misfolding in human cells. Similar to thermal stress, inhibiting new protein synthesis reduced the impact of mutant huntingtin on proteostatic function. These findings suggest that newly made proteins are vulnerable to misfolding when proteostasis is disrupted by insults such as thermal stress and mutant protein aggregation.

| S-EPMC4893652 | biostudies-literature

Fishing for newly synthesized proteins with phosphonate-handles.

Project description:Bioorthogonal chemistry introduces affinity-labels into biomolecules with minimal disruption to the original system and is widely applicable in a range of contexts. In proteomics, immobilized metal affinity chromatography (IMAC) enables enrichment of phosphopeptides with extreme sensitivity and selectivity. Here, we adapt and combine these superb assets in a new enrichment strategy using phosphonate-handles, which we term PhosID. In this approach, click-able phosphonate-handles are introduced into proteins via 1,3-dipolar Huisgen-cycloaddition to azido-homo-alanine (AHA) and IMAC is then used to enrich exclusively for phosphonate-labeled peptides. In interferon-gamma (IFNγ) stimulated cells, PhosID enabled the identification of a large number of IFN responsive newly synthesized proteins (NSPs) whereby we monitored the differential synthesis of these proteins over time. Collectively, these data validate the excellent performance of PhosID with efficient analysis and quantification of hundreds of NSPs by single LC-MS/MS runs. We envision PhosID as an attractive and alternative tool for studying stimuli-sensitive proteome subsets.

| S-EPMC7320153 | biostudies-literature

Synthesis and Antimicrobial Activity of Newly Synthesized Nicotinamides.

Project description:Antioxidants are promising compounds with antimicrobial activity against drug-resistant pathogens, especially when combined with conventional antimicrobials. Our study aimed to characterize the structure of nicotinamides synthesized from nicotinic acid and thiocarbohydrazones and to evaluate their antibacterial and antifungal activity. Seven nicotinic acid hydrazides (NC 1-7) were synthesized using mono-thiocarbohydrazones with hydroxyl group substituents, along with quinolone, phenolic, and pyridine rings known for their antimicrobial activity. The in vitro antimicrobial activity of NC 1-7, at concentrations ranging from 0.001 to 1 mM, was tested against Staphylococcus aureus (ATCC 6538), Enterococcus faecalis (ATCC 29212), Pseudomonas aeruginosa (ATCC 27853), Klebsiella pneumoniae (NCIMB 9111), and Candida albicans (ATCC 24433) using the broth microdilution method per EUCAST 2024 guidelines. Microorganism survival percentages were calculated based on optical density, and target fishing using the PharmMapper database identified potential molecular targets. The results showed that P. aeruginosa was most susceptible to the compounds, while C. albicans was the least susceptible. NC 3 significantly inhibited P. aeruginosa and K. pneumoniae growth at 0.016 mM, while higher concentrations were required for S. aureus, E. faecalis, and C. albicans. NC 5 was most effective against gram-positive bacteria at 0.03 mM. Only NC 4 completely inhibited C. albicans below 1 mM. NC 3, with the lowest concentration for 50% growth inhibition (0.016-0.064 mM), showed promising antibacterial potential against specific AMR-related proteins (bleomycin resistance protein, HTH-type transcriptional regulator QacR, and streptogramin A acetyltransferase), suggesting that this class of compounds could enhance or restore the activity of established antibiotics.

| S-EPMC11359232 | biostudies-literature

Crystal structures of three newly synthesized flavanone hydrazones

Project description:The crystal structures of racemic mixtures of three new flavanone-hydrazones are reported: (±,E)-N′-[5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-ylidene]-2-(naphthalen-1-yl)acetohydrazide ethyl acetate monosolvate, (±,E)-N′-[5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-ylidene]-4-hydroxybenzohydrazide ethanol monosolvate and (±,E)-N′-(6-methoxy-2-phenylchroman-4-ylidene)-2-(naphthalen-1-yloxy)acetohydrazide. All three hydrazones are in the E isomeric form and exhibit a pucker at the chiral carbon atom. The naringenin-derived hydrazones both show intramolecular hydrogen bonding between the hydrazone nitrogen atom and a hydroxy group on the chromane ring. The crystal structures of racemic mixtures of three new flavanone-hydrazones in the centrosymmetric space group (P

| S-EPMC9993913 | biostudies-literature

Global gene expression profiling using newly synthesized RNA [mock and LPS]

Project description:Innate stimulation with TLR ligands leads to the activation of various genes in macrophages and various populations of these cells may exhibit different responses. Here wanted to delineate and characterize these transcriptional responses in newly established, self-renewing, in vitro grown non-transformed lines (MPI cells) and bone marrow derived macrophages We used microarrays to detail the global programme of gene expression Newly synthesized RNA from independently established MPI lines and bone marrow derived macrophages was extracted and hybridized to Affymetrix microarrays

2013-06-08 | E-GEOD-47530 | biostudies-arrayexpress

Global gene expression profiling using newly synthesized RNA [mock and LPS]

2013-06-08 | GSE47530 | GEO