Project PXD005795

PRIDE Assigned Tags:
Biological Dataset



Feasibility of protein turnover studies in prototroph yeast (auxotroph data)


uantitative proteomics studies of yeast that use metabolic labeling with amino acids rely on auxotrophic mutations of one or more genes on the amino acid biosynthesis pathways. These mutations affect yeast metabolism, and preclude the study of some biological processes. Overcoming this limitation, it has recently been described that proteins in a yeast prototrophic strain can also be metabolically labeled with heavy amino acids. However, the temporal profiles of label incorporation under the different phases of the prototroph’s growth have not been examined. Labeling trajectories are important in the study of protein turnover and dynamics, in which label incorporation into proteins is monitored across many timepoints. Here we monitored protein labeling trajectories for 48 h after a pulse with heavy lysine in a yeast prototrophic strain and compared them with those of a lysine auxotrophic yeast. Labeling was successful in prototroph yeast during exponential growth phase but not in stationary phase. Furthermore, we were able to determine the half lives of more than 1,700 proteins during exponential phase of growth with high accuracy and reproducibility. We found a median half life of 2 h in both strains which corresponds with the cellular doubling time. Nucleolar and ribosomal proteins showed short half-lives whereas mitochondrial proteins and other energy production enzymes presented longer half-lives. Except for some proteins involved in lysine biosynthesis, we observed a high correlation in protein half lives between prototroph and auxotroph strains. Overall, our results demonstrate the feasibility of using prototrophs for proteomic turnover studies and provide a reliable dataset of protein half lives in exponentially growing yeast.

Sample Processing Protocol

All experiments were performed in duplicate with diploid S. cerevisiae strains from the FY background, a wild type prototrophic strain (parental strains FY4H and FY3G) and a lysine auxotrophic counterpart (parental strains FY4 and FY1856) generated by the insertion of a Ty retrotransposon into LYS2 gene in both alleles (lys2-128Δ/lys2-128Δ), resulting in a lysine auxotrophy due to transcriptional block. Both strains were grown in synthetic complete medium containing 6.7 g/l yeast nitrogen base, 2 g/l of drop-out mix (Bufferad, Lake Bluff, IL) containing all amino acids except lysine, and 2% glucose. Light lysine, containing natural abundance isotopes, (Sigma-Aldrich, St. Louis, MO) or heavy labeled (13C6/15N2) lysine (Cambridge Isotope Labs, Andover, MA) was added to a final concentration of 0.436 mM. Cells were pre-cultured in 5 ml medium containing light lysine overnight at 30 °C. Then 400 ml of heavy medium were inoculated from the pre-cultures to OD600 = 0.02, and cells were grown for 48 h, collecting samples at 0.5, 1, 1.5, 2, 4, 6, 8, 10, 12, 24, and 48 hours into the labeling. At each time point OD600 was measured and cells were collected directly into ice-cold tubes. Cells were harvested and centrifuged at 10,000g for 5 min at 4 °C. The pellet was washed twice with ice-cold ultra pure water, the supernatant was discarded and the yeast pellet was frozen in liquid nitrogen and stored at -80 °C. Cell pellets were thawed on ice and resuspended in lysis buffer composed of 8 M urea, 300 mM NaCl, 50 mM Tris, pH 8.2, 5 mM dithiothreitol (DTT), phosphatase inhibitors (50 mM NaF, 50 mM sodium β-glycerophosphate, 10 mM sodium pyrophosphate, 1 mM sodium orthovanadate) and protease inhibitors (EDTA free, Roche, Nutley, NJ). Sample processing from cell lysis through elution of purified peptides was performed following the in-StageTip (ST) method described previously10 with some modifications. Briefly, 100 μl of cell suspension containing approximately 20 μg protein (assuming 3 pg protein per cell) were loaded directly onto bottom sealed STs containing 14-gauge four-layered Empore C18 material (3M, St. Paul, MN) plugs already conditioned with methanol and 80% acetonitrile, 0.1% acetic acid, and equilibrated with 0.1% acetic acid. The enclosed ST was filled with ~100 μl of 0.5 mm diameter zirconia/silica beads (BioSpec, Bartlesville, OK) and placed inside the bead-milling adaptor. Cells were lysed by four repetitions of bead beating (1 min beating, 1.5 min rest). Lysate protein concentration was measured by Bradford assay (Biorad, Hercules, CA). Already reduced protein was alkylated of free thiols by addition of 15 mM iodoacetamide in the dark for 30 min. The alkylation reaction was quenched with 5 mM DTT. Urea concentration was diluted two fold with 50 mM Tris pH 8.9 and 2 mM calcium chloride. Proteolytic digestion was performed by addition of lysyl endopeptidase (LysC, Wako Chemicals, Richmond, VA), at 1:50 enzyme to protein ratio, and incubation at room temperature overnight. The digestion was quenched by addition of 10% TFA to pH < 2. Bottom seals from ST were removed and samples were centrifuged at 2000g. Peptides were desalted by 3 continuous washes with 0.1% acetic acid and directly eluted into a 96-well plate with 60 μl of 80% acetonitrile, 0.1% acetic acid. Samples were dried down by vacuum centrifugation and stored at -20 °C until LC-MS/MS analysis. Peptides were resuspended in 4% formic acid, 3% acetonitrile and loaded onto a 100-μm ID × 3-cm pre-column packed with Maccel C18 3-μm diameter, 200-Å pore size reverse-phase material (The Nest Group, Southborough, MA). Peptides were separated on a 75-μm ID × 40-cm analytical column packed with the same material and heated to 50 °C. The gradient was 3 to 32% acetonitrile in 0.125% formic acid. Eluted peptides were online analyzed in a hybrid quadrupole-Orbitrap (QExactive) mass spectrometer (Thermo Fisher, Bremen, Germany) using data dependent acquisition in which the 20 most abundant ions on an MS scan where selected for fragmentation by beam-type collision-activated dissociation (HCD), and fragmented ions were excluded from further selection during 40 s. Full MS scans were acquired in centroid mode from 300 to 1500 m/z at 70,000 FWHM resolution with a maximum injection time of 100 ms and fill target of 3e6 ions. MS/MS fragmentation spectra were collected at 17,500 FWHM with maximum injection time of 50 ms, using a 2.0 m/z precursor isolation window and fill target of 5e4 ions. Acquisition time for each fraction was 90 min, and included column wash and equilibration.

Data Processing Protocol

MS/MS spectra were searched with MaxQuant (version 11 against the SGD yeast protein sequence database (downloaded January 2011, 6,717 entries) with common contaminants added. The precursor mass tolerance was set to 4.5 ppm, and the fragment ion tolerance was set to 20 ppm. All searches used a fixed modification of cysteine carbamidomethylation (+57.0215 Da) and variable modifications of methionine oxidation (+15.9949 Da) and protein N-terminal acetylation (+42.0106 Da). LysC was the specified enzyme allowing for up to two missed cleavages. The false discovery rate (FDR) was estimated by searching against a concatenated forward-reverse database12. The maximum FDR was 0.01 on both the peptide and the protein level. The minimum required peptide length was seven residues. Proteins with at least two peptides (one of them unique to the protein) were considered identified. The “match between runs” option was enabled with a time window of 1 min to match identifications between replicates. The “requant option” of MaxQuant was disabled. Peptide intensities were grouped into proteins according to MaxQuant standard procedures. Protein quantification of heavy to light intensity ratios from the MaxQuant protein groups output table were used for the calculation of heavy label incorporation.


Miguel Martin Perez, University of Washington
Judit Villen, Department of Genome Sciences, University of Washington, Seattle, WA, USA ( lab head )

Submission Date


Publication Date


Corresponding dataset(s) in other omics resources

PXD001823 (ProteomeXchange)


Not available

Cell Type

diploid cell


Q Exactive


Not available



Experiment Type

Shotgun proteomics


    Martin-Perez M, Villén J. Feasibility of protein turnover studies in prototroph Saccharomyces cerevisiae strains. Anal Chem. 2015 Apr 7;87(7):4008-14 PubMed: 25767917