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General Information info

Manuscript title Absolute Quantification of Protein and mRNA Abundances Demonstrate Variability in Gene-Specific Translation Efficiency in Yeast.
PubMed ID 28365149
Journal Cell Systems
Year 2017
Authors Petri-Jaan Lahtvee, Benjamín J. Sánchez, Agata Smialowska, Sergo Kasvandik, Ibrahim E. Elsemman, Francesco Gatto, Jens Nielsen
Affiliations Department of Biology and Biological Engineering, Systems and Synthetic Biology, Chalmers University of Technology, Kemivägen 10, 412 96 Gothenburg, Sweden.
Keywords integrative data analysis, absolute proteome, absolute transcriptome, protein turnover, protein degradation rates, translation efficiency, genome-scale metabolic modeling, translational control
Full text article Downloadarticle Lahtvee_2017.pdf
Project name not specified

Experiment Description info

Organism Saccharomyces cerevisiae
Strain CEN.PK113-7D ΔLYS1
Data type enzyme/protein concentrations
Data units pgDW
Execution date not specified

Experimental Details info

Temperature (0C) 30
pH 5.5
Carbon source glucose,
Culture mode chemostat
Process condition aerobic
Dilution rate (h-1) 0.1
Working volume (L) 1
Biomass concentration (g/L) see original paper
Medium composition

Base medium used contained 10 g glucose, 5 g (NH4)2SO4, 3 g KH2PO4 and 0.5 g MgSO4 per litre, in addition to 1 mL of trace elements solution and 1 mL of vitamin solution. The trace element solution contained, per litre (pH=4): EDTA (sodium salt), 15.0 g; ZnSO4⋅7H2O, 4.5 g; MnCl2⋅2H2O, 0.84 g; CoCl2⋅6H2O, 0.3 g; CuSO4⋅5H2O, 0.3 g; Na2MoO4⋅2H2O, 0.4 g; CaCl2⋅2H2O, 4.5 g; FeSO4⋅7H2O, 3.0 g; H3BO3, 1.0 g; and KI, 0.10 g.). The vitamin solution contained, per liter (pH=6.5): biotin, 0.05 g; p-amino benzoic acid, 0.2 g; nicotinic acid, 1 g; Ca-pantothenate, 1 g; pyridoxine-HCl, 1 g; thiamine-HCl, 1 g and myo-inositol, 25 g.

General protocol information Measurement method: LC-MS/MS

Methods description - Notes

Quantitative Proteome Measurements: The peak intensity-based absolute quantification method iBAQ was chosen for protein quantification [1]. First, a lysine auxotrophic strain was created by deleting the LYS1 gene. Latter strain was cultivated with a labelled heavy 15N, 13C-lysine (Cambridge Isotope Laboratories), and fully labelled biomass was produced (in which 98% of proteogenic lysine was labelled, data not shown) and used as an internal standard in the measurements.
Nano-LC/MS/MS Analysis: Injected peptides (2 μg) were separated on an Ultimate 3000 RSLCnano system (Dionex, Sunnyvale, California, United States) using a C18 cartridge trap-column in a backflush configuration and an in-house packed (3 μm C18 particles, Dr Maisch) analytical 50 cm x 75 μm emitter-column (New Objective). The peptides were eluted at 200 nL min−1 with an 8%–40% B 240 min gradient (buffer B: 80% acetonitrile + 0.1% formic acid, buffer A: 0.1% formic acid) to a Q Exactive (Thermo Fisher Scientific) tandem mass spectrometer operating with a top-10 strategy and a cycle time of 0.9 seconds. Briefly, one 350-1 400 m/z MS scan at a resolution of R = 70,000 was followed by higher-energy collisional dissociation fragmentation (normalized collision energy of 25) of the 10 most-intense ions (charge states +2 to +6) at R = 17,500. The MS and MS/MS ion target values were 3x 106 and 5x 104, respectively. Dynamic exclusion was limited to 80 seconds.
------------------------------------References----------------------------------
[1] Schwanhäusser B., Busse D., Li N., Dittmar G., Schuchhardt J., Wolf J., Chen W., Selbach M.Nature. 2011; 473: 337-342. http://doi.org/bgjkh9

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Submission and curation info

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 2018-09-20 17:22:27 UTC

Updated 2018-09-20 17:22:27 UTC

Version 0

Status (reviewed) 2018-09-20 17:22:32 UTC




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