Dataset Information

Quantitative proteomic analyses of Chlorella vulgaris NJ-7 and UTEX259

ABSTRACT: A total of 12 samples (2 strains, at 20 degrees Celsius and 4 degrees Celsius, each with three biological replicates) were analyzed using the quantitative proteomics approach. Algal cells were grown at 20 degrees Celsius, exposed to 4 degrees Celsius for 24 h or not, harvested and frozen in liquid nitrogen. The frozen cells were suspended, sonicated and centrifuged. Proteins in the supernatant were precipitated and subjected to trypsin digestion. After labelling with Tandem Mass Tags/Isobaric Tag for Relative Absolute Quantitation (TMT kit/iTRAQ), peptides were analyzed by liquid chromatography coupled with tandem mass spectrometry (MS/MS).

INSTRUMENT(S): Q Exactive

ORGANISM(S): Chlorella Vulgaris (ncbitaxon:3077)

SUBMITTER: Yali Wang

PROVIDER: MSV000083861 | MassIVE | Mon May 27 02:28:00 BST 2019

SECONDARY ACCESSION(S): PXD014018

REPOSITORIES: MassIVE

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Project description:This study measured the differential expression of genes from a single clonal Daphnia pulex isolate obtained from an eutrophic flooded opencast mine near Gräfenhain (Saxony, Germany) and has been kept in the laboratory since 2002. The daphniids (50 adult animals per batch) were raised in 3-L glass beakers under a 16 h:8 h L:D photoperiod. Three-quarter of the culture medium (M4) was renewed once a week, and the animals were equally fed ad libitum with algae (Desmodesmus subspicatus; SAG 53.80, Göttingen, Germany). Long-term-acclimated animals were kept at three different temperatures (i.e., 10 ± 0.5°C, 20 ± 0.3°C, and 24 ± 0.3°C) under normoxia (Po2 ≥ 20 kPa) for at least twelve weeks. Time-resolved experiments (acute heat stress) were carried out with long-term 20°C acclimated animals, which were exposed for three different time intervals (2, 4, and 8 h) to either 30 ± 0.2°C (test conditions) or 20 ± 0.3°C (control conditions). For this, 50 animals each were rapidly transferred with a sieve (mesh size: 1.2 mm) from 20°C-medium to a set of beakers containing 30°C-medium. As control, 50 animals each were transferred from 20°C-medium to another set of beakers with 20°C-medium. Only adult female animals with a body length of 2−2.5 mm (between the base of the apical spine and the anterior part of the head) and carrying parthenogenetic eggs and embryos were used for experiments. Animals were not fed during short-term exposures. During experiments, all animals were in good physical condition. After thermal acclimation or heat exposure, animals were transferred into 1.5-ml microcentrifuge tubes and shock-frozen in liquid nitrogen after removing adhering water with a tissue paper. Samples were short-term stored at -80°C. For all experimental conditions, four independent replicates (50 animals each) were analyzed. This GEO record is for the contrasts between animals conditioned to 10 degrees Celsius (condition 1) versus 20 degrees Celsius (condition 2), 10 degrees Celsius (condition 1) versus 24 degrees Celsius (condition 3), and 20 degrees Celsius (condition 2) versus 24 degrees Celsius (condition 3)

Project description:1ml of WT, delta-pka, delta-arcA and delta-pka delta-arcA culture broth was harvested at u=0.4 h-1, centrifuged (14000 x g for 1 min at 4 degrees C), pellet washed once with PBS, flash frozen with liquid N2 and kept at -80 degrees C until further processing. Frozen pellets were melted on ice after which 100 ug of sample biomass was mixed with 100 ug of SILAC-labeled E. coli biomass (internal standard) and frozen at -80 degrees C. Cell pellets were suspended in 100 uL SDS lysis buffer (4% SDS/100 mM TRIS-HCl pH 8/100 mM DTT) and heated at 95 degrees C for 15 min. Cell lysates were sonicated with ultrasound for a few pulses and pelleted by centrifugation. Cell lysates were digested with trypsin according to Filter Aided Sample Preparation protocol (FASP) (Wiśniewski et al. 2009) and purified with C-18 StageTips (Rappsilber et al. 2007). LC-MS/MS analysis was performed using an Agilent 1200 series nanoflow system (Agilent Technologies) connected to a LTQ Orbitrap mass-spectrometer (Thermo Electron, San Jose, CA, USA) equipped with a nanoelectrospray ion source (Proxeon, Odense, Denmark). Peptides were separated with a 240-minute gradient from 2 to 40% B (A: 0.5% acetic acid, B: 0.5% acetic acid/80% acetonitrile) using a flow-rate of 200 nl/min. Data analysis of raw MS-files was performed by the MaxQuant software package version 1.3.0.5 (Cox and Mann 2008). Peak lists were searched using the Andromeda search engine (built into MaxQuant) against an E. coli database (downloaded 09.04.2013 from http://www.uniprot.org/) which was supplemented with common contaminants (e.g human keratin, trypsin). Full tryptic specificity, a maximum of two missed cleavages and a mass tolerance of 0.5 Da for fragment ions was specified in the MaxQuant search. Carbamidomethylation of cysteine was set as a fixed modification, and methionine oxidation and protein N-terminal acetylation were set as variable modifications. The required false discovery rate was set to 1% for both peptide and protein levels and the minimum required peptide length was set to six amino acids. “Match between runs” option with a time window of 1.5 min was allowed.

Project description:This study measured the differential expression of genes from a single clonal Daphnia pulex isolate obtained from an eutrophic flooded opencast mine near Gräfenhain (Saxony, Germany) and has been kept in the laboratory since 2002. The daphniids (50 adult animals per batch) were raised in 3-L glass beakers under a 16 h:8 h L:D photoperiod. Three-quarter of the culture medium (M4) was renewed once a week, and the animals were equally fed ad libitum with algae (Desmodesmus subspicatus; SAG 53.80, Göttingen, Germany). Long-term-acclimated animals were kept at three different temperatures (i.e., 10 ± 0.5°C, 20 ± 0.3°C, and 24 ± 0.3°C) under normoxia (Po2 ≥ 20 kPa) for at least twelve weeks. Time-resolved experiments (acute heat stress) were carried out with long-term 20°C acclimated animals, which were exposed for three different time intervals (2, 4, and 8 h) to either 30 ± 0.2°C (test conditions) or 20 ± 0.3°C (control conditions). For this, 50 animals each were rapidly transferred with a sieve (mesh size: 1.2 mm) from 20°C-medium to a set of beakers containing 30°C-medium. As control, 50 animals each were transferred from 20°C-medium to another set of beakers with 20°C-medium. Only adult female animals with a body length of 2−2.5 mm (between the base of the apical spine and the anterior part of the head) and carrying parthenogenetic eggs and embryos were used for experiments. Animals were not fed during short-term exposures. During experiments, all animals were in good physical condition. After thermal acclimation or heat exposure, animals were transferred into 1.5-ml microcentrifuge tubes and shock-frozen in liquid nitrogen after removing adhering water with a tissue paper. Samples were short-term stored at -80°C. For all experimental conditions, four independent replicates (50 animals each) were analyzed. This GEO record is for the acute heat stress temperature contrasts between animals conditioned to 20 degrees Celsius (condition 1) versus heat shock to 30 degrees Celsius (condition 2) exposed for 2 hours (treatment 1), 4 hours (treatment 2), and 8 hours (treatment 3).

Dataset Information

Quantitative proteomic analyses of Chlorella vulgaris NJ-7 and UTEX259

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