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Analysis of the Chlamydomonas proteome with UPS1 spike-in

ABSTRACT: Benchmarking dataset for the objective evaluation of quantitative and statistical workflows.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Chlamydomonas Reinhardtii

SUBMITTER: Evan McConnell

LAB HEAD: Leslie M. Hicks

PROVIDER: PXD009694 | Pride | 2020-10-20

REPOSITORIES: pride

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			Action	DRS
	20141222_WOS5211.wiff	Wiff
	20141222_WOS5212.wiff	Wiff
	20141222_WOS5216.wiff	Wiff
	20141222_WOS5217.wiff	Wiff
	20141222_WOS5217.wiff.mtd	Wiff

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Publications

Evaluation of linear models and missing value imputation for the analysis of peptide-centric proteomics.

Berg Philip P McConnell Evan W EW Hicks Leslie M LM Popescu Sorina C SC Popescu George V GV

BMC bioinformatics 20190314 Suppl 2

<h4>Background</h4>Several methods to handle data generated from bottom-up proteomics via liquid chromatography-mass spectrometry, particularly for peptide-centric quantification dealing with post-translational modification (PTM) analysis like reversible cysteine oxidation are evaluated. The paper proposes a pipeline based on the R programming language to analyze PTMs from peptide-centric label-free quantitative proteomics data.<h4>Results</h4>Our methodology includes variance stabilization, nor ...[more]

PMID: 30871482

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Analysis of reversibly oxidized cysteines from the Chlamydomonas proteome with yeast spike-in

Project description:Benchmarking dataset for the objective evaluation of quantitative and statistical workflows.

2019-03-19 | PXD009693 | Pride

Chemical inhibition of the target of rapamycin kinase in Chlamydomonas reinhardtii leads to rapid cysteine oxidation and sustained physiological changes.

Project description:The target of rapamycin (TOR) kinase is a key metabolic regulator with a role in diverse processes, including nutritional sensing, protein translation, and autophagy. In the green alga Chlamydomonas reinhardtii, TOR maintains these responsibilities and is additionally linked to the regulation of increased triacylglycerol accumulation. In this study, we used an ATP-competitive inhibitor (AZD8055) to inactivate TOR kinase in vivo and quantified any accompanying changes in global protein abundance and reversible cysteine oxidation.

2019-10-04 | PXD014819 | Pride

Development of a maleimide-based chemical proteomic method for quantitative analysis of cysteine reactivity

Project description:Cysteine is the most intrinsically nucleophilic residue in proteins and serves as a sentinel against increasing reactive oxygen species (ROS) via reversible thiol oxidation. Despite the importance of Cys oxidation in understanding biological stress response, it remains largely unknown and a major analytical challenge to determine which protein Cys-sites are the most reactive toward ROS. Herein, we describe initial coverage and sensitivity of a chemical proteomic method to quantify site-specific Cys reactivity using a maleimide-activated, thiol-specific probe (N-propargylmaleimide, NPM).

2020-10-20 | PXD015409 | Pride

Collection of various small RNA sequencing libraries of C. Reinhardtii.

Project description:Collection of various small RNA libraries of C. Reinhardtii sequenced using Illumina sRNA sequencing. Libraries are derived from various C. Reinhardtii genotypes (CC1883, CC4350, CC125, J), ecotypes, life cycles (Vegatative, Zygote) treatments and mutants. Preparation was done over a period of several years as a continuous effort to build up a comprehensive collection.

2020-06-16 | E-MTAB-8526 | biostudies-arrayexpress

Three acyltransferases and a nitrogen responsive regulator are implicated in nitrogen starvation-induced triacylglycerol accumulation in Chlamydomonas

Project description:Three acyltransferases, DGAT1, DGTT1, and PDAT1, are induced by nitrogen starvation and are likely to have a role in TAG accumulation based on their patterns of expression. Each gene also shows increased mRNA abundance in other TAG-accumulating conditions (-S, -P, -Zn, -Fe). Insertional mutants, pdat1-1 and pdat1-2, accumulate 25% less TAG compared to the parent strain, D66+, which demonstrates the relevance of the trans-acylation pathway in Chlamydomonas. The biochemical functions of DGTT1 and PDAT1 were validated by rescue of oleic acid sensitivity and restoration of TAG accumulation in a yeast strain lacking all acyltransferase activity. Time course analysis suggest than an SBP domain transcription factor protein, whose mRNA increases precede that of other genes like DGAT1, is a candidate regulator of the N-deficiency response. An insertional mutant, nrr1-1, accumulates only 50% of the TAG compared to the parental strain in N-starvation conditions and is unaffected by other nutrient stresses, suggesting the operation of multiple signaling pathways leading to stress-induced TAG accumulation. Sampling of Chlamydomonas 2137 cultivated in TAP, N-Free TAP or TAP with varying amounts of N.

2013-01-10 | E-GEOD-34585 | biostudies-arrayexpress

yeast-UPS1 standard LC-MS/MS dataset

Project description:Proteomic workflows based on nanoLC-MS/MS data-dependent-acquisition analysis have progressed tremendously in recent years due to the technical improvement of mass spectrometers, and now allow to extensively characterize complex protein mixtures. High-resolution and fast sequencing instruments have enabled the use of label-free quantitative methods, which appear as an attractive way to analyze differential protein expression in complex biological samples. Classical label-free quantitative workflows are based either on spectral counting of MS/MS sequencing scans for each protein, or on the extraction of peptide ion peak area values in the LC-MS map composed of all the survey MS scans acquired during the chromatographic gradient. However, the computational processing of the data for label-free quantification still remains a challenge. Here, we provide a dual proteomic standard composed of an equimolar mixture of 48 human proteins (Sigma UPS1) spiked at different concentrations into a background of yeast cell lysate, that was used to benchmark several label-free quantitative workflows, involving different software packages developed in recent years. This experimental design allowed to finely assess their performances in terms of sensitivity and false discovery rate, by measuring the number of true and false-positive (respectively UPS1 or yeast background proteins found as differential). This dataset can also be used to benchmark other label-free workflows, adjust software parameter settings, improve algorithms for extraction of the quantitative metrics from raw MS data, or evaluate downstream statistical methods

2015-12-01 | PXD001819 | Pride

Examples of ribosome profiling in chlamydomonas and mouse

Project description:Ribosome profiling is a technique that permits genome-wide, quantitative analysis of translation and has found broad application in recent years. Here, we describe a modified profiling protocol and software package designed to benefit more broadly the translation community in terms of simplicity and utility. The protocol, applicable to diverse organisms, including organelles, is based largely on previously published profiling methodologies, but employs duplex-specific nuclease as a convenient, bias-free, species-independent way to reduce rRNA contamination. Our protocol typically produces high levels of triplet periodicity, facilitating the detection of coding sequences, including upstream, downstream and overlapping open reading frames (ORFs). This feature also allows the identification of an alternative ribosome conformation evident during termination of protein synthesis. To test the effectiveness of DSN in ribosome profiling, we generated Ribo-Seq libraries from mouse tissue culture cells and from the green alga Chlamydomonas reinhardtii.

2015-10-16 | E-MTAB-2934 | biostudies-arrayexpress

Phosphate Starvation in Chlamydomonas reinhardtii

Project description:Phosphorus (P) is an essential nutrient that is limiting in many environments. When P is scarce organisms employ strategies for conservation of internal stores, and to efficiently scavenge P from their external surroundings. In this study we investigated the acclimation response of Chlamydomonas reinhardtii to P deficiency, comparing the transcriptional profiles of P starved wild-type cells to the P replete condition. RNA was prepared from P-containing or P-deprived logarithmic growth phase cells and subjected to RNA-Seq analysis. During the 24 hours after the imposition of P starvation we observed that from the 407 significantly changing genes (> 2 fold change, corrected p-value < 0.05) in the wild-type 317 genes were up-regulated, in average 8.36-fold, and 90 genes were down-regulated by 3.43-fold, in average. Many of the upregulated genes encoded enzymes involved in specific responses to P starvation, including PHOX, encoding the major secreted alkaline phosphatase, and multiple putative, high-efficiency phosphate transporter genes. More general responses included the up-regulation of genes involved in photoprotective processes (LHCSR3, LHCSR1, LHCBM9, PTOX1) and genes involved in protein modification and degradation. Down-regulated mRNAs indicated an early stage of the reduction of chloroplast ribosomal proteins, which are considered to be a reservoir for P in the cell. Chlamydomonas reinhardtii strain 21 gr (CC1690, wild-type) grown in TAP medium (Harris 1989) in a rotary incubator (200 rpm) at 25 M-BM-0C in continuous light (70 M-BM-5mol m-2 s-1). For 24 hours, either 1.1 mM phosphate or 0 mM were provided with the growth media. P deprivation was achieved by washing cells twice in midlogarithmic growth phase with liquid TAP medium without P (TAP-P) and cells were resuspended at a density of 2.5 mg/ml Chlorophyll in TAP or TAP-P. Cell aliquots were collected for mRNA isolation 24 h after being transferred either to TAP or TAP-P medium.

2014-04-05 | E-GEOD-56505 | biostudies-arrayexpress

Characterization of eukaryotic DNA N6-methyladenine by a highly sensitive restriction enzyme-assisted sequencing

Project description:To interrogate single-base resolution 6mA sites in the genome-wide, we develop DA-6mA-seq (DpnI-Assisted N6-methylAdenine sequencing), an optimized sequencing method taking advantage of restriction enzyme DpnI, which exclusively cleaves methylated adenine sites. We find DpnI also recognizes other sequence motifs besides the canonical GATC restriction sites, largely expanding the application range of this method. DA-6mA-seq requires less starting material and lower sequencing depth than previous methods, but achieves higher sensitivity, providing a good strategy to identify 6mA in large genome with a low abundance of 6mA. We rebuild the 6mA maps of Chlamydomonas by DA-6mA-seq and then apply this method to another two eukaryotic organisms, Plasmodium and Penicillium. Further analysis reveals most 6mA sites are symmetric at various sequence contexts, suggesting 6mA may function as a new heritable epigenetic mark in eukaryotes. A new sequencing method is developed to detect 6mA in eukaryotes

2016-04-12 | E-GEOD-74430 | biostudies-arrayexpress

Arabidopsis thaliana spiked UPS1 standard protein mixtures

Project description:This project aims at providing a quantitative dataset from LC-MS/MS injections of a calibrated UPS1 mixture spiked in an Arabidopsis thaliana background.

2022-07-31 | PXD027800 | Pride

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