Dynamics of Protein Turnover, a Missing Dimension in Proteomics

Functional genomic experiments frequently involve a comparison of the levels of gene expression between two or more genetic, developmental, or physiological states. Such comparisons can be carried out at either the RNA (transcriptome) or protein (proteome) level, but there is often a lack of congruence between parallel analyses using these two approaches. To fully interpret protein abundance data from proteomic experiments, it is necessary to understand the contributions made by the opposing processes of synthesis and degradation to the transition between the states compared. Thus, there is a need for reliable methods to determine the rates of turnover of individual proteins at amounts comparable to those obtained in proteomic experiments. Here, we show that stable isotope-labeled amino acids can be used to define the rate of breakdown of individual proteins by inspection of mass shifts in tryptic fragments. The approach has been applied to an analysis of abundant proteins in glucose-limited yeast cells grown in aerobic chemostat culture at steady state. The average rate of degradation of 50 proteins was 2.2%/h, although some proteins were turned over at imperceptible rates, and others had degradation rates of almost 10%/h. This range of values suggests that protein turnover is a significant missing dimension in proteomic experiments and needs to be considered when assessing protein abundance data and comparing it to the relative abundance of cognate mRNA species. Functional genomic experiments frequently involve a comparison of the levels of gene expression between two or more genetic, developmental, or physiological states. Such comparisons can be carried out at either the RNA (transcriptome) or protein (proteome) level, but there is often a lack of congruence between parallel analyses using these two approaches. To fully interpret protein abundance data from proteomic experiments, it is necessary to understand the contributions made by the opposing processes of synthesis and degradation to the transition between the states compared. Thus, there is a need for reliable methods to determine the rates of turnover of individual proteins at amounts comparable to those obtained in proteomic experiments. Here, we show that stable isotope-labeled amino acids can be used to define the rate of breakdown of individual proteins by inspection of mass shifts in tryptic fragments. The approach has been applied to an analysis of abundant proteins in glucose-limited yeast cells grown in aerobic chemostat culture at steady state. The average rate of degradation of 50 proteins was 2.2%/h, although some proteins were turned over at imperceptible rates, and others had degradation rates of almost 10%/h. This range of values suggests that protein turnover is a significant missing dimension in proteomic experiments and needs to be considered when assessing protein abundance data and comparing it to the relative abundance of cognate mRNA species. Four levels of analysis are commonly exploited in functional genomics: genome, transcriptome, proteome, and metabolome. The last three levels are all context-dependent; the complement of mRNA molecules, protein molecules, and metabolites all change with the physiological, developmental, or pathological state of living cells. A change in the proteome is probably the most important of these three for the analysis of gene action and interaction, but it is also the most difficult to study in a truly comprehensive manner (1.Miklos G.L. Maleszka R. Protein functions and biological contexts.Proteomics. 2001; 1: 169-178Google Scholar). “Classical” proteomics only compares amounts of proteins in cells in two different states or conditions; it does not address the dynamics of the proteome in the different biological states that are being compared nor does it provide information about the mechanisms whereby the system changes from one state to the other. The acquisition of a new steady-state level of any protein will be the outcome of the change in its rate of synthesis as compared with the change in its rate of degradation (2.Gottesman S. Maurizi M.R. Regulation by proteolysis: energy-dependent proteases and their targets.Microbiol. Rev. 1992; 56: 592-621Google Scholar, 3.Hochstrasser M. Johnson P.R. Arendt C.S. Amerik A. Swaminathan S. Swanson R. Li S.J. Laney J. Pals-Rylaarsdam R. Nowak J. Connerly P.L. The Saccharomyces cerevisiae ubiquitin-proteasome system.Philos. Trans. R. Soc. Lond. B Biol. Sci. 1999; 354: 1513-1522Google Scholar). At the steady state, it is the balance between these two opposing processes that determines the concentration of any protein (4.Benaroudj N. Tarcsa E. Cascio P. Goldberg A.L. The unfolding of substrates and ubiquitin-independent protein degradation by proteasomes.Biochimie (Paris). 2001; 83: 311-318Google Scholar). To illustrate, an increase in the level of expression of a protein could be achieved by an enhanced rate of synthesis or a diminished rate of degradation. Despite its evident importance, the role of protein turnover has not previously been considered in analyses of the proteome. Yet the determination of the half-life of a large number of proteome components might do much to explain the marked disparity that is sometimes seen between transcriptome and proteome data (5.Gygi S.P. Rochon Y. Franza B.R. Aebersold R. Correlation between protein and mRNA abundance in yeast.Mol. Cell. Biol. 1999; 19: 1720-1730Google Scholar, 6.Ideker T. Thorsson V. Ranish J.A. Christmas R. Buhler J. Eng J.K. Bumgarner R. Goodlett D.R. Aebersold R. Hood L. Integrated genomic and proteomic analyses of a systematically perturbed metabolic network.Science. 2001; 292: 929-934Google Scholar, 7.Griffin T.J. Gygi S.P. Ideker T. Rist B. Eng J. Hood L. Aebersold R. Complementary profiling of gene expression at the transcriptome and proteome levels in Saccharomyces cerevisiae..Mol. Cell. Proteomics. 2002; 1: 323-333Google Scholar, 8.Chen G. Gharib T.G. Huang C.C. Taylor J.M. Misek D.E. Kardia S.L. Giordano T.J. Iannettoni M.D. Orringer M.B. Hanash S.M. Beer D.G. Discordant protein and mRNA expression in lung adenocarcinomas.Mol. Cell. Proteomics. 2002; 1: 304-313Google Scholar). In addition, the requirements of one of the most energy-demanding processes in the cell, the aggregate process of protein synthesis and degradation, protein turnover, can be quantified on a protein-by-protein basis. In this article, we define an experimental strategy to analyze the dynamics of protein turnover, a missing dimension of proteomics. The diploid yeast strain BY4743 (EUROSCARF accession number Y23935, www.uni-frankfurt.de/fb15/mikro/euroscarf/index.html) (9.Brachmann C.B. Davies A. Cost G.J. Caputo E. Li J. Hieter P. Boeke J.D. Designer deletion strains derived from Saccharomyces cerevisiae S288c: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications.Yeast. 1998; 14: 115-132Google Scholar), a leucine auxotroph, was used throughout. Yeast were grown in glucose-limited chemostat culture as described previously (10.Baganz F. Hayes A. Farquhar R. Butler P.R. Gardner D.C. Oliver S.G. Quantitative analysis of yeast gene function using competition experiments in continuous culture.Yeast. 1998; 14: 1417-1427Google Scholar) in a medium (Table I) containing 100 mg/liter dl-2H10leucine (98.5 atom % excess) at a dilution rate of 0.1 h−1. After a minimum of seven doubling times, sufficient to ensure that cells were fully labeled, unlabeled l-leucine (1 g in 50 ml) was added, and the incoming medium was changed to one containing unlabeled l-leucine at 50 mg/liter. Sampling was at 0, 0.167, 0.667, 1, 2, 4, 6, 8, 10, 12, 24.5, and 51 h into the chase. This sampling frequency served to reduce the true dilution rate in the chase phase from a nominal 0.1 h−1 to an actual 0.086 h−1. At each time point, cells were collected directly into ice-cold tubes containing cycloheximide (final concentration 100 μg/ml). Cells (40 ml at an A600 of ∼1.6) were harvested and centrifuged at 5000 rpm for 5 min at 4 °C. The pellet was resuspended in 1 ml of ice-cold double distilled H2O and transferred to a 1.5-ml microcentrifuge tube. Cells were repelleted by centrifugation at 10,000 rpm, the supernatant was discarded, and the yeast pellet was frozen in dry ice and stored at −80 °C. Cell pellets were thawed briefly on ice and resuspended in 300 μl of 20 mm HEPES, pH 7.5 containing one EDTA-free protease inhibitor mixture tablet/10 ml (Roche Diagnostics) and lysed by vortexing with glass beads (6 × 45 s with 45 s of cooling). DNase (6 μl of l mg/ml, Sigma) and RNase (2 μl of l mg/ml, Sigma) were added, and the lysate was held at 4 °C for 1 h. The lysate was centrifuged at 4000 rpm for 10 min at 4 °C, and the supernatant was assayed for protein (Coomassie plus protein assay, Pierce).Table ICarbon-limited minimal medium for chemostat cultureComponentFinal concentrationg/literKH2PO42MgSO4·7H2O0.55NaCl0.1CaCl2·2H2O0.09Glucose2.5NH4SO43.13Uracil0.02Histidine0.02dl-Leucineadl-2H10Leucine was used in labeled media.0.1Trace elementsbTrace elements were made as two 10,000× stock solutions: solution 1, 10,000× FeCl3·6H2O; solution 2, 10,000× ZnSO4·7H2O, CuSO4·5H2O, H3BO, KI. ZnSO4·7H2OcVitamins were made as a 600× stock solution and stored at −20 °C.0.00007 CuSO4·5H2O0.00001 H3BO30.00001 KI0.00001 FeCl3·6H2O0.00005Vitamins Inositol0.062 Thiamine/HCl0.014 Pyridoxine0.004 Calcium pantothenate0.004 Biotin0.0003a dl-2H10Leucine was used in labeled media.b Trace elements were made as two 10,000× stock solutions: solution 1, 10,000× FeCl3·6H2O; solution 2, 10,000× ZnSO4·7H2O, CuSO4·5H2O, H3BO, KI.c Vitamins were made as a 600× stock solution and stored at −20 °C. Open table in a new tab Proteins (150 μg of soluble protein) from each of the 12 time points were then solubilized in 8 m urea, 2% (w/v) CHAPS, 20 mm dithiothreitol, and 0.5% 3–10 IPG Buffer (AP Biotech) for 1 h at 37 °C before centrifugation at 10,000 rpm for 10 min at 4 °C and application to 13-cm Immobiline pH 3–10 dry strips (AP Biotech) for in-gel rehydration (180 V-h at 30 V, 360 V-h at 60 V) and isoelectric focusing (500 V-h at 500 V, l000 V-h at 1000 V, and 16,000 V-h at 8000 V) using an IPGphor isoelectric focusing system (AP Biotech). Second-dimension analysis was by 12% (w/v) linear SDS-PAGE followed by Coomassie Blue staining. Gels were visually inspected, the same spot was excised from each gel, and peptides were obtained by in-gel reduction, alkylation with iodoacetamide, tryptic digestion, and extraction using a MassPrep™ digestion robot (Micromass, Manchester, UK). Peptides were analyzed using a MALDI-TOF mass spectrometer (email protected™, Micromass, Manchester UK) covering the m/z range of 1000–4000 Th. Spectra were stacked above each other, and the peak differences between 0 h (heavy, fully labeled) and 51 h (light, fully unlabeled) were identified. Intermediate time points showed the gradual disappearance of peptides carrying heavy leucine and the gradual appearance of peptides carrying unlabeled leucine. Depending on the number of leucine residues in the peptide, the “heavy” and “light” peptides differed in mass by 9n Da, where n was the number of leucine residues in the peptide. The protein was identified by recording the masses of peptides in the 51-h spectrum (fully unlabeled) and including the leucine composition of each peptide (derived from comparison of the 0- and 51-h spectra) in a manual search of the yeast data base using MASCOT (www.matrixscience.co.uk), which allows inclusion of composition data in its search (11.Pratt J.M. Robertson D.H. Gaskell S.J. Riba-Garcia I. Hubbard S.J. Sidhu Oliver S.G. Butler P. Hayes A. J. in as an to protein in peptide mass 2002; Scholar). The peak of the heavy and tryptic peptides and were obtained and were used to the relative abundance at each used relative CHAPS, of as the The of changes over time as the with heavy are by those labeled with leucine. This is a of two of cells from the chemostat and protein The of the the at any to the values for at 0 and in 51 to the values of three and was for peptides derived from a of different proteins and a of n The in this was that it was as the in the the for was set to 51 to seven doubling times, over of the heavy labeled cells in the at 0 h been from the these we also some of the in determination of the at the and of the where either the heavy or the peak was relative to the other and the data were sometimes by in the mass The The of this was to the data using to the in the and the for the In time experiments, was from the of at a to the the true rate of degradation was by of the dilution rate from of a does not the of the of the rate of turnover of proteins is with and strategy was to turnover rates approach the of of proteins with stable isotope-labeled amino acids and mass to determine the of those labeled amino acids in tryptic In this it from other that proteins with stable isotope-labeled amino acids either to expression levels B. I. A. M. by amino acids in as a and approach to expression Cell. Proteomics. 2002; 1: Scholar, J. Quantitative analysis of the yeast proteome by of labeled 2002; 1: Scholar), to determine the of amino acids to in protein by peptide mass (11.Pratt J.M. Robertson D.H. Gaskell S.J. Riba-Garcia I. Hubbard S.J. Sidhu Oliver S.G. Butler P. Hayes A. J. in as an to protein in peptide mass 2002; Scholar, R. N. Y. J. Y. I. peptide of and between leucine and residues by mass using Soc. 1998; Scholar, mass with stable in proteins for and protein Scholar, L. V. mass with stable peptides for of protein 2001; Scholar, proteins for in 2002; Scholar), or to determine S. mass for the of protein by mass 2002; Scholar). the rate of turnover of Saccharomyces cerevisiae proteins in glucose-limited aerobic continuous culture at the steady state. In continuous the cells are in a metabolic state, and the in is by of cells as the is by incoming In this continuous culture is to in culture in which are number medium pH can and the rate of The yeast cells were grown at a dilution and proteins were labeled with a amino in the incoming a large of unlabeled amino was to the culture and at the same the medium was to one containing the unlabeled amino the cells are the of an of the unlabeled amino does not the but the labeled proteins are into the cells. The was labeled at all other the and and leucine is in the of tryptic peptides derived from the yeast proteome (11.Pratt J.M. Robertson D.H. Gaskell S.J. Riba-Garcia I. Hubbard S.J. Sidhu Oliver S.G. Butler P. Hayes A. J. in as an to protein in peptide mass 2002; Scholar). of a leucine of S. cerevisiae that dilution of the by leucine be we that the atom to the atom is and the relative of or leucine a into the metabolic of the important information in the of the strategy (11.Pratt J.M. Robertson D.H. Gaskell S.J. Riba-Garcia I. Hubbard S.J. Sidhu Oliver S.G. Butler P. Hayes A. J. in as an to protein in peptide mass 2002; Scholar, R. N. Y. J. Y. I. peptide of and between leucine and residues by mass using Soc. 1998; Scholar). Proteins were with heavy leucine for 50 more seven doubling at a dilution rate of 0.1 h−1. Thus, over of the leucine in the cells be the stable isotope-labeled of the leucine in this had on rate or on the of proteins in a not The chemostat was then with a large of unlabeled l-leucine to reduce the abundance of the The was to unlabeled and of cells were from the culture over the 51 h. The unlabeled leucine to the had on that leucine was not being used as an there were differences in from cells before or the chase with unlabeled leucine not the chase all proteins only unlabeled leucine. The rate of of labeled protein is a the of dilution of the cells or degradation At each sampling time before and the chase cells were and proteins from the lysate were by The of on the was this is not a of the and we could the same protein from each gel, which was then to in-gel tryptic digestion followed by MALDI-TOF mass A. F. M. P. M. and proteome by mass of yeast proteins from two Sci. S. A. Scholar). The for individual in the peptide mass the of the labeled protein by the unlabeled protein as the cells to in A set of MALDI-TOF data over 51 to the of individual peptides with one or leucine that the transition from fully labeled to fully unlabeled peptides was Proteins were identified by peptide mass by the data on the leucine composition of each peptide derived from the between the heavy and the had not been peptides of be protein synthesis by mass analysis 1999; Scholar). for peptides containing more one leucine the lack of of mass values between the fully labeled and fully unlabeled is that the relative abundance of the had been to a of this the on of these peptides of mass is by the in the the lack of any above the is for an chase. The abundance of labeled peptides also that all of the in the medium was to in The of the of the heavy and tryptic were for each sampling time in the chase The transition in between the fully labeled and the fully unlabeled peptides is most by a that the rate for of the from the protein To determine this rate a was to the set of data for each tryptic peptide. peptides in a peptide mass be to each peptide an of the rate of turnover of the each the rate was derived from peptides with one or more one leucine The in the rate were of the and the between the degradation rates, by peptides derived from a was all we the data from different peptides to a on of at each time (Table of degradation of yeast of analysis determination protein protein or protein protein rate 8 rate all peptides protein of of A A or A to and of most to in that for of with and from or Open table in a new tab of the degradation rate of a protein is of in the of that when applied to a of proteins in a proteome, determination of degradation rate be A more to proteomic might be derived by analysis of a time the chase each analysis of peptides a of at one of which is to an of by the for proteins from 4 and almost all proteins in this we were to data from peptides containing between one and three leucine protein the values of were by sampling the cells at two time points and 8 the degradation rates were and data from peptides were to a of the of the rate the degradation rate had previously been by and for a of these was using data for peptides but at a time The between the values of by the two methods was n The whereby turnover rate is from peptides from the same can as reliable an as the more To the of the we the determination of turnover from for a number of proteins the between the in each was between and for the of by was the obtained from analysis using peptides in the MALDI-TOF spectrum but at a time of 8 h. to the of the for a of the proteins we the determination of turnover from using the the is on the the cells are in true steady state in the the rate of of by of cells from the system at a true rate of 0.086 h−1 for Thus, the degradation rates of the proteins be for this by of the dilution rate the proteins analyzed in this are at rates h−1. of these and and are not at all and are only by dilution into cells. At the other one protein was at a rate of over the that we abundance the range of degradation rates is over in cells that are in on the more abundant proteins to the approach and to the dynamics of proteins in cells. abundant proteins might be to be the degradation rates are which important of of degradation of this of a process of any on degradation rates, and the of mechanisms A study of yeast proteins analyzed that were not in B. P. C.S. A sampling of the yeast Cell. Biol. 1999; 19: Scholar), but the time were which to the of the in turnover the approach used previously B. P. C.S. A sampling of the yeast Cell. Biol. 1999; 19: Scholar, B. G. J. analysis of the Sci. S. A. 2001; Scholar) be with the and of the protein spot that the enhanced from peptides is The to with a of and the rate of degradation of individual proteins an dimension in proteomics. The a reliable and a of and be to of relative rates of protein degradation. more analyses of the and rates of degradation of individual sampling and time determination of abundance a data set that is to The strategy used when cells are in steady-state but there will be other that are more to or for be to the rate of synthesis of each protein by to the although this of the and the of of the stable has to be for reliable of the abundance of mass The in with all other methods to protein turnover rates, is in the to define the rate of turnover turnover proteins be by a of heavy and the rate of degradation was not the protein be as the same the proteins for which the rate of of is to dilution rate can be as and two proteins been identified in this The whereby a protein the of the can as much on the and of the process as can the study of turnover it is most to of turnover rate from the peptides derived from an excised there is this could not be applied to peptides by A but this be as a in the of the be obtained either from the of time to which the approach could be or by of a to turnover rates from peptides from one In this the of leucine is important as it is the most abundant amino in the proteome (11.Pratt J.M. Robertson D.H. Gaskell S.J. Riba-Garcia I. Hubbard S.J. Sidhu Oliver S.G. Butler P. Hayes A. J. in as an to protein in peptide mass 2002; Scholar). to this analysis to the study of proteins commonly in the and of which on function and In this study we the turnover rate of a protein that to a these protein could been of different functional with other and the turnover rate could be an average of a number of rates that at the level of a that to analysis proteins that are in into different analysis of turnover could the same protein to a different turnover rate on the function of the from which it Despite the of protein degradation in of the proteome, the process The of the and has identified the and mechanisms that are for degradation of proteins A. The Sci. S. A. Scholar, The a protease by Biol. 1999; Scholar). The that is is that of the of the process whereby different proteins are to degradation at different The determination of degradation rate is an in the study of the and of protein turnover, for in the of the peptide approach can provide a of the range and of protein degradation for large of of the proteome. the of the dynamics of the proteome need be considered and the between transcriptome and proteome be are to and for