Shotgun proteomics of cell lysates and absolute quantification of E. coli, Z. mobilis, C. thermocellum glycolytic proteins
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Eclipse
ORGANISM(S): Clostridium Thermocellum Zymomonas Mobilis (ncbitaxon:542) Escherichia Coli (ncbitaxon:562)
SUBMITTER: 
Daniel Amador-Noguez Joshua Coon
PROVIDER: MSV000098743 | MassIVE | Wed Aug 06 07:57:00 BST 2025
REPOSITORIES: MassIVE
Similar Datasets
Project description:By reporting molar abundances of proteins, absolute quantification determines their stoichiometry in complexes, pathways or networks and also relates them to abundances of non-protein biomolecules. Typically, absolute quantification relies either on protein- specific isotopically labelled peptide standards or on a semi-empirical calibration against the average abundance of peptides chosen from arbitrary selected standard proteins. Here we developed a generic protein standard FUGIS (Fully unlabelled Generic Internal Standard) that requires no isotopic labelling, synthesis of standards or external calibration and is applicable to proteins of any organismal origin. FUGIS is co-digested with analysed proteins and enables their absolute quantification in the same LC-MS/MS run. By using FUGIS, median based absolute quantification (MBAQ) workflow provides similar quantification accuracy compared to isotopically-labelled peptide standards and outperforms methods based on external calibration or selection of best ionized reporter peptides (Top3 quantification) with a median quantification error less than 15%
2021-11-30 | PXD026886 | Pride
Project description:Clostridium thermocellum is a Gram-positive, anaerobic, thermophilic bacterium that ferments cellulose into ethanol. It is a candidate industrial consolidated bioprocess (CBP) biocatalyst for lignocellulosic bioethanol production. However, C. thermocellum is relatively sensitive to ethanol compared to yeast. Previous studies have investigated the membrane and protein composition of wild-type and ethanol tolerant strains, but relatively little is known about the genome changes associated with the ethanol tolerant C. thermocellum strain. In this study, C. thermocellum cultures were grown to mid-exponential phase and then either shocked with the supplementation of ethanol to a final concentration of 3.95 g/L (equal to 0.5% [v/v]) or were untreated. Samples were taken pre-shock and 2, 12, 30, 60, 120, 240 min post-shock for multiple systems biology analyses. The addition of ethanol dramatically reduced the C. thermocellum growth and the final cell density was approximately half of the control fermentations, with concomitant reductions in substrate consumption in the treated cultures. The response of C. thermocellum to ethanol was dynamic and involved more than six hundred genes that were significantly and differentially expressed between the different conditions over time and every functional category was represented. Cellobiose was accumulated within the ethanol-shocked C. thermocellum cells, as well as the sugar phosphates such as fructose-6-P and cellobiose-6-P. The comparison and correlation among intracellular metabolites, proteomic and transcriptomics profiles as well as the ethanol effects on cellulosome, hydrogenase glycolysis and nitrogen metabolism are discussed, which led us to propose that C. thermocellum may utilize the nitrogen metabolism to bypass the arrested carbon metabolism in responding to ethanol stress shock, and the nitrogen metabolic pathway and redox balance may be the key target for improving ethanol tolerance and production in C. thermocellum.
2012-08-16 | GSE25236 | GEO
Project description:Clostridium thermocellum is a Gram-positive, anaerobic, thermophilic bacterium that ferments cellulose into ethanol. It is a candidate industrial consolidated bioprocess (CBP) biocatalyst for lignocellulosic bioethanol production. However, C. thermocellum is relatively sensitive to ethanol compared to yeast. Previous studies have investigated the membrane and protein composition of wild-type and ethanol tolerant strains, but relatively little is known about the genome changes associated with the ethanol tolerant C. thermocellum strain. In this study, C. thermocellum cultures were grown to mid-exponential phase and then either shocked with the supplementation of ethanol to a final concentration of 3.95 g/L (equal to 0.5% [v/v]) or were untreated. Samples were taken pre-shock and 2, 12, 30, 60, 120, 240 min post-shock for multiple systems biology analyses. The addition of ethanol dramatically reduced the C. thermocellum growth and the final cell density was approximately half of the control fermentations, with concomitant reductions in substrate consumption in the treated cultures. The response of C. thermocellum to ethanol was dynamic and involved more than six hundred genes that were significantly and differentially expressed between the different conditions over time and every functional category was represented. Cellobiose was accumulated within the ethanol-shocked C. thermocellum cells, as well as the sugar phosphates such as fructose-6-P and cellobiose-6-P. The comparison and correlation among intracellular metabolites, proteomic and transcriptomics profiles as well as the ethanol effects on cellulosome, hydrogenase glycolysis and nitrogen metabolism are discussed, which led us to propose that C. thermocellum may utilize the nitrogen metabolism to bypass the arrested carbon metabolism in responding to ethanol stress shock, and the nitrogen metabolic pathway and redox balance may be the key target for improving ethanol tolerance and production in C. thermocellum. A thirty array study using total RNA recovered from wild-type cultures of Clostridium thermocellum at different time points of 0, 12, 30, 60, 120, and 240 min post-inoculation with 3.95 g/L [0.5% (v/v)] treatment compred to that of control without ethanol supplementation. Two biological replicates for treatment and control condition.
2012-08-16 | E-GEOD-25236 | biostudies-arrayexpress
Project description:Clostridium thermocellum is a Gram-positive, anaerobic, thermophilic bacterium that ferments cellulose into ethanol. It is a candidate industrial consolidated bioprocess (CBP) biocatalyst for lignocellulosic bioethanol production to produce bioethanol directly from cellulosic biomass. However, few transcriptomic studies have been reported so far for C. thermocellum using biomass as carbon source. In this study, samples were taken from exponential and stationary phases of C. thermocellum cells growing in MTC media with pretreated switchgrass as carbon source, and transcriptomic profiling change of C. thermocellum during different growth phase was investigated using both expression array and tiling array. This study will help the understanding of gene expression of C. thermocellum using cellulosic biomass as carbon source and the knowledge will facilitate future metabolic engineering effort for strain improvement.
2014-05-08 | GSE26926 | GEO
Project description:To determine protein concentration in cells, we performed spike-in DIA experiments.
Isotopically labeled concatemers (for mTOR-related signaling proteins) were spiked into lysates from p18Rev cells (each 20 mg) with four three-fold serial dilutions from 5,000 fmol. The lysates were subjected to digestion with trypsin. For absolute quantification of proteins, the digests containing isotopically labeled recombinant proteins were separated on a column (EV1106, Evosep) using a pre-programmed gradient (15 samples per day) at a flow rate of 200 nL/min using the Evosep One system (Evosep).
2024-05-23 | PXD042413 | JPOST Repository
Project description:Background: The ability of Clostridium thermocellum ATCC 27405 wild-type strain to hydrolyze cellulose and ferment the degradation products directly to ethanol and other metabolic byproducts makes it an attractive candidate for consolidated bioprocessing of cellulosic biomass to biofuels. In this study, whole-genome microarrays were used to investigate the expression of C. thermocellum mRNA during growth on crystalline cellulose in controlled replicate batch fermentations. Results: A time-series analysis of gene expression revealed changes in transcript levels of ~40% of genes (~1300 out of 3198 ORFs encoded in the genome) during transition from early-exponential to late-stationary phase. K-means clustering of genes with statistically significant changes in transcript levels identified six distinct clusters of temporal expression. Broadly, genes involved in energy production, translation, glycolysis and amino acid, nucleotide and coenzyme metabolism displayed a decreasing trend in gene expression as cells entered stationary phase. In comparison, genes involved in cell structure and motility, chemotaxis, signal transduction and transcription showed an increasing trend in gene expression. Hierarchical clustering of cellulosome-related genes highlighted temporal changes in composition of this multi-enzyme complex during batch growth on crystalline cellulose, with increased expression of several genes encoding hydrolytic enzymes involved in degradation of non-cellulosic substrates in stationary phase. Conclusions: Overall, the results suggest that under low substrate availability, growth slows due to decreased metabolic potential and C. thermocellum alters its gene expression to (i) modulate the composition of cellulosomes that are released into the environment with an increased proportion of enzymes than can efficiently degrade plant polysaccharides other than cellulose, (ii) enhance signal transduction and chemotaxis mechanisms perhaps to sense the oligo-saccharide hydrolysis products, and nutrient gradients generated through the action of cell-free cellulosomes and, (iii) increase cellular motility for potentially orienting the cellsM-bM-^@M-^Y movement towards positive environmental signals leading to nutrient sources. Such a coordinated cellular strategy would increase its chances of survival in natural ecosystems where feast and famine conditions are frequently encountered. Total RNA was extracted from the cell pellets and the reverse transcribed cDNA was hybridized to oligo-arrays containing duplicated probes representing ~90% of the annotated ORFs in C. thermocellum ATCC27405 genome.Dual-channel dye swap experimental design was used to analyze the time-course of gene expression during cellulose fermentation using two biological replicate fermentation. The 6hr sample as the reference, to which all other time-point samples (8, 10, 12, 14, 16hr) were compared.
2011-05-27 | E-GEOD-29554 | biostudies-arrayexpress
Project description:Clostridium thermocellum is a Gram-positive, anaerobic, thermophilic bacterium that ferments cellulose into ethanol. It is a candidate industrial consolidated bioprocess (CBP) biocatalyst for lignocellulosic bioethanol production to produce bioethanol directly from cellulosic biomass. However, few transcriptomic studies have been reported so far for C. thermocellum using biomass as carbon source. In this study, samples were taken from exponential and stationary phases of C. thermocellum cells growing in MTC media with pretreated switchgrass as carbon source, and transcriptomic profiling change of C. thermocellum during different growth phase was investigated using both expression array and tiling array. This study will help the understanding of gene expression of C. thermocellum using cellulosic biomass as carbon source and the knowledge will facilitate future metabolic engineering effort for strain improvement. [HX12 expression array]: A eleven array study using total RNA recovered from wild-type cultures of Clostridium thermocellum at different growth phase of T2 and T3 with switchgrass as carbon source. Two biological replicates used for each phase. [3Plex tiling array]: A six array study using total RNA recovered from wild-type cultures of Clostridium thermocellum at different growth phase of T2 and T3 with switchgrass as carbon source. Two biological replicates used for each phase.
2014-05-08 | E-GEOD-26926 | biostudies-arrayexpress
Project description:Differential RNA-Seq analyses to investigate the basis for metabolic inhibition of Clostridium thermocellum M1570 by xylose. The M1570 strain was developed in the C. thermocellum DSM 1313 Δhpt background strain. Lactate dehydrogenase (Ldh) and phosphotransacetylase (Pta) genes are deleted (Argyros DA, Tripathi SA, Barrett TF, Rogers SR, Feinberg LF, Olson DG, Foden JM, Miller BB, Lynd LR, Hogsett DA, Caiazza NC: High ethanol titers from cellulose by using metabolically engineered thermophilic, anaerobic microbes. Appl Environ Microbiol 2010, 77:8288-8294 )
2016-10-31 | GSE74918 | GEO
Project description:To systematically evaluate the effect of embryo lysates feeding, we conducted RNA sequencing (RNA-seq) on day 7 N2 worms treated with embryo lysates. Our findings revealed that embryo lysates affected the expression of genes related to fatty acid metabolism, degradation of branched-chain amino acids, and lysosome functions. Notably, worms treated with embryo lysates showed increased fatty acid degradation gene expression and decreased fatty acid elongation gene expression.
2024-09-07 | GSE265886 | GEO