Quantitative mass spectrometry-based multiplexing compares the abundance of 5000 S. cerevisiae proteins across 10 carbon sources
Graphical abstract
Introduction
The budding yeast, Saccharomyces cerevisiae, is an exceptional model system for investigating biological functions and pathways by proteomic strategies. Its compact genome and low number of intron containing genes allow systematic coverage with comparative ease [1]. However, S. cerevisiae's value as a model is contingent on, and limited by, the number of uncharacterized ORFs (n = 677, 11.6% (omitting dubious ORFs)), most of which exist as homologs in other species, including humans (n = 388). As such, systematic characterization of these gene products and their co-expression profiles with known components is a valuable hypothesis-generating resource for the targeted characterization of gene function. However, uncharacterized ORFs are typically under-represented in systematic screens, many of which are assayed under standard metabolic conditions. Therefore, we aimed to measure protein abundance in highly diverse metabolic conditions to achieve an unprecedented depth of proteome coverage.
In industrial applications, S. cerevisiae has been used traditionally for brewing and baking, and more recently for the development of biofuels [2], [3]. A common mechanism of manipulating the metabolic processes governing S. cerevisiae is shifting carbon sources [4]. S. cerevisiae can survive on various carbon sources, both fermentable (e.g., glucose, maltose, fructose, sucrose, trehalose), and non-fermentable (e.g., pyruvate, acetate, lactate, oleate, glycerol) [5]. We expected that culturing yeast on a particular carbon source would result in pronounced proteomic changes associated with metabolic perturbation [6]. We investigate quantitatively the global proteomic alterations in wildtype S. cerevisiae following growth on minimal media supplemented with ten different carbon sources - maltose, trehalose, fructose, sucrose, glycerol, acetate, pyruvate, lactic acid, and oleate - using a multiplexed tandem mass tag (TMT) strategy.
The carbon sources used for this experiment consisted of monosaccharides, disaccharides, fatty acids, or building blocks of fatty acids. Glucose (dextrose) is a six carbon monosaccharide preferred as an energy source by many organisms, including S. cerevisiae, and is a component of standard YPD (yeast extract-peptone-dextrose) yeast media. Maltose and trehalose are disaccharides composed of two glucose units, with the former having an α,α-1,1-glucoside bond and the latter an α(1 → 4) bond between the glucose molecules [7]. Like glucose, fructose is a simple ketonic monosaccharide produced by many plants, and is the most water-soluble of all the sugars investigated [8]. The association of the monosaccharides glucose and fructose via a glycosidic linkage forms the disaccharide sucrose. Sucrose is highly abundant and is often used for ethanol-based fuel production [9]. While the aforementioned carbon sources are fermentable, we also examined several non-fermentable carbon sources - specifically glycerol, acetate, pyruvate, lactate, and oleate [4], [10], [11]. The backbone of glycerol is essential to all triglycerides, which are esters of glycerol with long-chain carboxylic acids [12]. Likewise, acetate is a building block for fatty acids, as the linking of the two of its carbon atoms forms a growing fatty acid [13]. Pyruvate is an alpha-keto acid with both a ketone functional group and a carboxylic acid. Pyruvate can be derived from glucose through glycolysis, converted back to carbohydrates (such as glucose) via gluconeogenesis, or to fatty acids via the acetyl-CoA pathway [14]. Like pyruvate, lactate (lactic acid) is an organic compound which is often a downstream product of pyruvate and glucose metabolism [15]. In addition, oleate (oleic acid) is an unsaturated fatty acid that occurs naturally in various animal and vegetable fats and oils. The processing of oleate by β-oxidative catabolism occurring in peroxisomes results in the formation of acetyl-CoA which can enter the TCA cycle [16]. These carbon sources have all been used previously for culturing S. cerevisiae, yet hitherto the alterations in the associated global proteome with respect to growth in glucose have not been investigated.
Previous work, upon which this study builds, has shown promise in elucidating the comprehensive proteome of S. cerevisiae via mass spectrometry-based techniques [17], [18], [19], [20], [21], [22]. We employed a TMT10-plex strategy to determine the relative protein abundance alterations resulting from a particular carbon source. The mass spectrometry measurements were performed on an Orbitrap Fusion Lumos mass spectrometer. This work represents the largest mass spectrometry-based analysis of the yeast S. cerevisiae to date. Our protocol can be used to direct future proteomic analyses, both global and targeted, to investigate the effects of altered carbon sources on the growth and metabolism of S. cerevisiae. Moreover, this dataset is a valuable resource, as it can be mined further to develop a better understanding of yeast metabolic pathways under different carbon sources and potentially manipulate these observed alterations for industrial applications.
Section snippets
Materials
Tandem mass tag (TMT) isobaric reagents were from Thermo Fisher Scientific (Waltham, MA). Water and organic solvents were from J.T. Baker (Center Valley, PA). Unless otherwise noted, all other chemicals were from Sigma (St. Louis, MO).
Media and growth
The yeast strain was BY4742, derived from S288c. The yeast minimal media was comprised of yeast nitrogenous base with amino acids, ammonium sulfate, and the appropriate carbon source. A starter culture grown in glucose-containing minimal media overnight from
Application of a TMT-based quantitative mass spectrometry strategy enabled the quantification of over 5000 proteins from yeast cultured with 10 different carbon sources
Based on our previously published protocol [31], [32], we analyzed the proteomes of S. cerevisiae grown in the presence of 10 carbon sources - glucose, maltose, oleate, fructose, sucrose, trehalose, lactate, acetate, pyruvate and glycerol. As outlined in Fig. 1, a starter culture was grown overnight in glucose and cultures of each of the 10 carbon sources were inoculated to OD600 0.05/mL. Cells were disrupted by bead beating, reduced and alkylated, and proteins were extracted by
Conclusions
We used a multiplexed isobaric tag-based quantitative mass spectrometry strategy to investigate comprehensively the proteomic alterations in S. cerevisiae resulting from growth in ten different carbon sources. Our analysis demonstrated the proteomic alterations resulting from the distinctive ability of S. cerevisiae to adapt to metabolic stress stemming from growth on sub-optimal carbon sources. Employing isobaric labeling [56], [57] permitted the quantification of protein samples from
Conflicts of interest
The authors acknowledge no conflict of interest.
Transparency document
Acknowledgements
We would like to thank members of the Gygi Lab at Harvard Medical School, particularly Dr. Ekaterina Stepanova for her insights into yeast growth and metabolism. This work was funded in part by an NIH/NIDDK grant K01 DK098285 (J.A.P.) and GM67945 (S.P.G).
References (60)
- et al.
Saccharomyces cerevisiae as anodic biocatalyst for power generation in biofuel cell: influence of redox condition and substrate load
Bioresour. Technol.
(2011) - et al.
Fructose and glucose differentially affect aging and carbonyl/oxidative stress parameters in Saccharomyces cerevisiae cells
Carbohydr. Res.
(2011) - et al.
A tissue-specific atlas of mouse protein phosphorylation and expression
Cell
(2010) - et al.
A new old yellow enzyme of Saccharomyces cerevisiae
J. Biol. Chem.
(1995) - et al.
The STL1 gene of Saccharomyces cerevisiae is predicted to encode a sugar transporter-like protein
Gene
(1994) - et al.
Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents
Mol. Cell. Proteomics
(2004) - et al.
Saccharomyces genome database: the genomics resource of budding yeast
Nucleic Acids Res.
(2012) - et al.
Comparative proteomics analysis of engineered Saccharomyces cerevisiae with enhanced biofuel precursor production
PLoS One
(2013) - et al.
Carbon source induces growth of stationary phase yeast cells, independent of carbon source metabolism
Yeast
(1993) - et al.
Transcriptional regulation of respiration in yeast metabolizing differently repressive carbon substrates
BMC Syst. Biol.
(2010)
Changes in the protein expression of yeast as a function of carbon source
J. Proteome Res.
Revisiting yeast trehalose metabolism
Curr. Genet.
Sucrose and Saccharomyces cerevisiae: a relationship most sweet
FEMS Yeast Res.
Transcriptional control of nonfermentative metabolism in the yeast Saccharomyces cerevisiae
Curr. Genet.
Carbon source dependent promoters in yeasts
Microb. Cell Factories
Increased expression and secretion of recombinant alpha-amylase in Saccharomyces cerevisiae by using glycerol as the carbon source
J. Protein Chem.
Ethanol and acetate acting as carbon/energy sources negatively affect yeast chronological aging
Oxidative Med. Cell. Longev.
Pyruvate metabolism in Saccharomyces cerevisiae
Yeast
Effects of acetic acid and lactic acid on the growth of Saccharomyces cerevisiae in a minimal medium
J. Ind. Microbiol. Biotechnol.
The sensitivity of yeast mutants to oleic acid implicates the peroxisome and other processes in membrane function
Genetics
A comprehensive proteomic and phosphoproteomic analysis of yeast deletion mutants of 14-3-3 orthologs and associated effects of rapamycin
Proteomics
The one hour yeast proteome
Mol. Cell. Proteomics
Modified MuDPIT separation identified 4488 proteins in a system-wide analysis of quiescence in yeast
J. Proteome Res.
A complete mass-spectrometric map of the yeast proteome applied to quantitative trait analysis
Nature
Analysis of the Saccharomyces cerevisiae proteome with PeptideAtlas
Genome Biol.
System-wide perturbation analysis with nearly complete coverage of the yeast proteome by single-shot ultra HPLC runs on a bench top Orbitrap
Mol. Cell. Proteomics
MultiNotch MS3 enables accurate, sensitive, and multiplexed detection of differential expression across cancer cell line proteomes
Anal. Chem.
A probability-based approach for high-throughput protein phosphorylation analysis and site localization
Nat. Biotechnol.
Target-decoy search strategy for mass spectrometry-based proteomics
Methods Mol. Biol.
Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry
Nat. Methods
Cited by (139)
Controlling circuitry underlies the growth optimization of Saccharomyces cerevisiae
2023, Metabolic EngineeringCombinatorial selective ER-phagy remodels the ER during neurogenesis
2024, Nature Cell Biology