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General Information info

Manuscript title Low oxygen levels as a trigger for enhancement of respiratory metabolism in Saccharomyces cerevisiae.
PubMed ID 19804647
Journal BMC Genomics
Year 2009
Authors Eija Rintala, Mervi Toivari, Juha-Pekka Pitkänen, Marilyn G Wiebe, Laura Ruohonen and Merja Penttilä
Affiliations VTT Technical Research Centre of Finland, Finland
Keywords Aerobic Condition, MAPK Signalling, Pathway Central Carbon Metabolism, Filamentous Growth, Ergosterol Biosynthesis
Full text article Downloadarticle Rintala_2009.pdf
Project name not specified

Experiment Description info

Organism Saccharomyces cerevisiae
Strain CEN.PK113-1A
Data type enzyme/protein concentrations
Data units
Execution date not specified

Experimental Details info

Temperature (0C) 30
pH 5
Carbon source glucose,
Culture mode chemostat
Process condition aerobic and anaerobic
Dilution rate (h-1) 0.1 ± 0.02
Working volume (L) 2.5
Biomass concentration (g/L) not specified
Medium composition

defined minimal medium, with 10 g glucose l-1 as carbon source, and supplemented with 10 mg ergosterol l-1 and 420 mg Tween 80 l-1.

General protocol information Measurement method: 2D gel-MALDI-TOF

Methods description - Notes

Cells for proteome analysis were collected in cold Na-phosphate buffer, pH 7 as described for the microarray analysis. 5-10 mg dry mass of cells was re-suspended in 150 μl of 10% (v/v) trichloro acetic acid (TCA, Merck) in 1.5 ml micro centrifuge tubes. 500 μl glass beads (0.5 mm diameter, Biospec Products) were added and the tube inserted into a MiniBeadbeater 8 (Biospec Products) and shaken at homogenisation speed, three times for 30 seconds. The tubes were cooled on ice between each homogenisation step. The suspensions were withdrawn and proteins were precipitated by adding 600 μl of -20°C acetone and incubating 30 min. on ice. Precipitated proteins were collected by centrifugation for 30 min., 13 000 rpm, at 4°C, rinsed once with 600 μl of -20°C acetone and re-suspended in 450 μl of 7 M urea (Promega, USA), 2 M thiourea (Fluka, USA), 4% (w/v) CHAPS (Fluka), 1% (w/v) Pharmalytes 3-10 (Pharmacia, Sweden) and 1% (w/v) DTT (Sigma) by gently shaking for 20 min. at room temperature. Supernatants were collected by centrifugation for 5 min. 13 000 rpm (Eppendorf bench centrifuge). The protein concentration of supernatants was determined by the Non-Interfering Protein Assay (Geno Technology, Inc.) and the samples were stored at -70°C before isoelectric focusing.

Isoelectric focusing and second dimension 11% (w/v) SDS-PAGE were carried out as described earlier [1]. After electrophoresis the gels were fixed for one and a half hours in 30% (v/v) ethanol and 0.5% (v/v) acetic acid and stained with Sypro Ruby (Molecular Probes), according to manufacturers' instructions. The stained gels were scanned with a resolution of 100 microns on a Typhoon instrument (GE Healthcare). The gel images were analysed using the Progenesis software (Nonlinear Dynamics). The gel patterns from different gels were automatically matched, with some additional manual editing, and the quantities of matching spots in different gels were compared. From each condition, samples from 2-4 independent cultivations were used and for each sample 4 gels were analysed. After background correction, the data was transferred to the R environment for normalisation and data analysis.

Background corrected proteome data of 500 spots was obtained from Progenesis software. For each condition, the spots that had zero values in more than 50% of the gels were treated as real zeroes and set to the lowest value of each gel [2]. The rest of the missing values were estimated using the k- nearest neighbour-method [3]. The data was log transformed and quantile normalised as described in [4]. The statistical analysis of differences was done using linear modelling [5].

Protein identifications were carried out in the Protein Chemistry Unit, Institute of Biomedicine, Anatomy, Biomedicum, Helsinki. For protein identification, excised gel spots were washed and dehydrated with acetonitrile (Rathburn, Scotland, HPLC grade S). Proteins were reduced with 20 mM DTT and incubated at 56°C for 30 min before alkylation with 55 mM Iodoacetamide (Sigma, USA)/100 mM ammonium hydrogen carbonate (NH4HCO3) in the dark at room temperature for 15 min. After washing with 100 mM NH4HCO3 and dehydration with acetonitrile the gel pieces were rehydrated in 10 to 15 μl sequencing grade trypsin (Promega, USA) in 100 mM NH4HCO3, to a final concentration of 0.01 μg/μl trypsin and incubated for trypsin digestion overnight at 37°C. Tryptic peptides were eluted from the gel pieces by incubating for 15 min at room temperature successively in 25 mM NH4HCO3 and then twice in 5% formic acid. The tryptic peptides were desalted using Zip Tip μC-18 reverse phase (Millipore, USA) and directly eluted with 50% v/v acetonitrile/0.1% v/v trifluoroacetic acid (TFA) onto a MALDI target plate. Then, a saturated matrix solution α-cyano-4-hydroxy cinnamic acid (CHCA) (Sigma, USA) in 33% ACN/0.1% TFA was added. MALDI-TOF analyses were carried out with an Autoflex (Bruker Daltonics, Bremen Germany) equipped with a nitrogen pulsed laser (337 nm) and operating in positive mode. Typically, mass spectra were acquired by accumulating spectra of 240 laser shots. External calibration was performed for molecular assignments using a peptide calibration standard (Bruker Daltonics GmbH, Leipzig, Germany). Trypsin autolytic peptide masses were used to check or correct the calibration.
-----------------References--------------------
[1] Salusjärvi L, Poutanen M, Pitkänen JP, Koivistoinen H, Aristidou A, Kalkkinen N, Ruohonen L, Penttilä M. Yeast. 2003, 20: 295-314. http://doi.org/bcmj9b
[2] Almeida JS, Stanislaus R, Krug E, Arthur JM. Proteomics. 2005, 5: 1242-1249. http://doi.org/c5whmc
[3] Gottardo R: EMV. R package version 1.3.1. 2006.
[4] Chang J, Van Remmen H, Ward WF, Regnier FE, Richardson A, Cornell J. J Proteome Res. 2004, 3: 1210-1218. http://doi.org/br5pr7 [5] Smyth GK. In:Bioinformatics and Computational Biology Solutions using R and Bioconductor. R package version 2.12.0. 2005

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Entered by Administrator KiMoSysFirst name: Administrator
Affiliation: INESC-ID/IST
Homepage: http://kdbio.inesc-id.pt/kimosys
Interests: mathematical modeling, accessible data, use of data

Created 2018-07-18 12:56:46 UTC

Updated 2018-07-18 13:01:13 UTC

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Status (reviewed) 2018-07-18 13:01:41 UTC




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