Protein turnover: measurement of proteome dynamics by whole animal metabolic labelling with stable isotope labelled amino acids

The measurement of protein turnover in tissues of intact animals is obtained by whole animal dynamic labelling studies, requiring dietary administration of precursor label. It is difficult to obtain full labelling of precursor amino acids in the diet and if partial labelling is used, calculation of the rate of turnover of each protein requires knowledge of the precursor relative isotope abundance (RIA). We describe an approach to dynamic labelling of proteins in the mouse with a commercial diet supplemented with a pure, deuterated essential amino acid. The pattern of isotopomer labelling can be used to recover the precursor RIA, and sampling of urinary secreted proteins can monitor the development of liver precursor RIA non-invasively. Time-series analysis of the labelling trajectories for individual proteins allows accurate determination of the first order rate constant for degradation. The acquisition of this parameter over multiple proteins permits turnover profiling of cellular proteins and comparisons of different tissues. The median rate of degradation of muscle protein is considerably lower than liver or kidney, with heart occupying an intermediate position.     

Harmonizing Labeling and Analytical Strategies to Obtain Protein Turnover Rates in Intact Adult Animals

Changes in the abundance of individual proteins in the proteome can be elicited by modulation of protein synthesis (the rate of input of newly synthesized proteins into the protein pool) or degradation (the rate of removal of protein molecules from the pool). A full understanding of proteome changes therefore requires a definition of the roles of these two processes in proteostasis, collectively known as protein turnover. Because protein turnover occurs even in the absence of overt changes in pool abundance, turnover measurements necessitate monitoring the flux of stable isotope–labeled precursors through the protein pool such as labeled amino acids or metabolic precursors such as ammonium chloride or heavy water. In cells in culture, the ability to manipulate precursor pools by rapid medium changes is simple, but for more complex systems such as intact animals, the approach becomes more convoluted. Individual methods bring specific complications, and the suitability of different methods has not been comprehensively explored. In this study, we compare the turnover rates of proteins across four mouse tissues, obtained from the same inbred mouse strain maintained under identical husbandry conditions, measured using either [¹³C₆]lysine or [²H₂]O as the labeling precursor. We show that for long-lived proteins, the two approaches yield essentially identical measures of the first-order rate constant for degradation. For short-lived proteins, there is a need to compensate for the slower equilibration of lysine through the precursor pools. We evaluate different approaches to provide that compensation. We conclude that both labels are suitable, but careful determination of precursor enrichment kinetics in amino acid labeling is critical and has a considerable influence on the numerical values of the derived protein turnover rates.     

Turnover of the human proteome: determination of protein intracellular stability by dynamic SILAC

The proteome of any system is a dynamic entity, such that the intracellular concentration of a protein is dictated by the relative rates of synthesis and degradation. In this work, we have analyzed time-dependent changes in the incorporation of a stable amino acid resolved precursor, a protocol we refer to as "dynamic SILAC", using 1-D gel separation followed by in-gel digestion and LC-MS/MS analyses to profile the intracellular stability of almost 600 proteins from human A549 adenocarcinoma cells, requiring multiple measures of the extent of labeling with stable isotope labeled amino acids in a classic label-chase experiment. As turnover rates are acquired, a profile can be built up that allows exploration of the 'dynamic proteome' and of putative features that predispose a protein to a high or a low rate of turnover. Moreover, measurement of the turnover rate of individual components of supramolecular complexes provides a unique insight in processes of protein complex assembly and turnover.     

Determining synthesis rates of individual proteins in zebrafish (Danio rerio) with low levels of a stable isotope labelled amino acid

The zebrafish is a powerful model organism for the analysis of human cardiovascular development and disease. Understanding these processes at the protein level not only requires changes in protein concentration to be determined but also the rate at which these changes occur on a protein‐by‐protein basis. The ability to measure protein synthesis and degradation rates on a proteome‐wide scale, using stable isotope labelling in conjunction with mass spectrometry is now a well‐established experimental approach. With the advent of more selective and sensitive mass spectrometers, it is possible to accurately measure lower levels of stable isotope incorporation, even when sample is limited. In order to challenge the sensitivity of this approach, we successfully determined the synthesis rates of over 600 proteins from the cardiac muscle of the zebrafish using a diet where either 30% or 50% of the L‐leucine was replaced with a stable isotope labelled analogue ([²H₇]L‐leucine]. It was possible to extract sufficient protein from individual zebrafish hearts to determine the incorporation rate of the label into hundreds of proteins simultaneously, with the two labelling regimens showing a good correlation of synthesis rates.     

A quantitative spatial proteomics analysis of proteome turnover in human cells

Measuring the properties of endogenous cell proteins, such as expression level, subcellular localization, and turnover rates, on a whole proteome level remains a major challenge in the postgenome era. Quantitative methods for measuring mRNA expression do not reliably predict corresponding protein levels and provide little or no information on other protein properties. Here we describe a combined pulse-labeling, spatial proteomics and data analysis strategy to characterize the expression, localization, synthesis, degradation, and turnover rates of endogenously expressed, untagged human proteins in different subcellular compartments. Using quantitative mass spectrometry and stable isotope labeling with amino acids in cell culture, a total of 80,098 peptides from 8,041 HeLa proteins were quantified, and their spatial distribution between the cytoplasm, nucleus and nucleolus determined and visualized using specialized software tools developed in PepTracker. Using information from ion intensities and rates of change in isotope ratios, protein abundance levels and protein synthesis, degradation and turnover rates were calculated for the whole cell and for the respective cytoplasmic, nuclear, and nucleolar compartments. Expression levels of endogenous HeLa proteins varied by up to seven orders of magnitude. The average turnover rate for HeLa proteins was ~20 h. Turnover rate did not correlate with either molecular weight or net charge, but did correlate with abundance, with highly abundant proteins showing longer than average half-lives. Fast turnover proteins had overall a higher frequency of PEST motifs than slow turnover proteins but no general correlation was observed between amino or carboxyl terminal amino acid identities and turnover rates. A subset of proteins was identified that exist in pools with different turnover rates depending on their subcellular localization. This strongly correlated with subunits of large, multiprotein complexes, suggesting a general mechanism whereby their assembly is controlled in a different subcellular location to their main site of function.     

Global analysis of cellular protein translation by pulsed SILAC

Current methods for system‐wide gene expression analysis detect changes in mRNA abundance, but neglect regulation at the level of translation. Pulse labeling with stable isotopes has been used to measure protein turnover rates, but this does not directly provide information about translation rates. Here, we developed pulsed stable isotope labeling by amino acids in cell culture (pSILAC) with two heavy isotope labels to directly quantify protein translation on a proteome‐wide scale. We applied the method to cellular iron homeostasis as a model system and demonstrate that it can confidently identify proteins that are translationally regulated by iron availability.     

The subunit structure and dynamics of the 20S proteasome in chicken skeletal muscle

We have succeeded in purifying the 20S core proteasome particle from less than 1 g of skeletal muscle in a rapid process involving two chromatographic steps. The individual subunits were readily resolved by two-dimensional PAGE, and the identities of each of the 14 subunits were assigned by a combination of peptide mass fingerprinting and MS/MS/de novo sequencing. To assess the dynamics of proteasome biogenesis, chicks were fed a diet containing stable isotope-labeled valine, and the rate of incorporation of label into valine-containing peptides derived from each subunit was assessed by mass spectrometric analysis after two-dimensional separation. Peptides containing multiple valine residues from the 20S proteasome and other soluble muscle proteins were analyzed to yield the relative isotope abundance of the precursor pool, a piece of information that is essential for calculation of turnover parameters. The rates of synthesis of each subunit are rather similar, although there is evidence for high turnover subunits in both the alpha (nonproteolytic) and beta (proteolytic) rings. The variability in synthesis rate for the different subunits is consistent with a model in which some subunits are produced in excess, whereas others may be the rate-limiting factor in the concentration of 20S subunits in the cell. The ability to measure turnover rates of proteins on a proteome-wide scale in protein assemblies and in a complex organism provides a new dimension to the understanding of the dynamic proteome.     

Brain Protein Synthesis in the Conscious Rat Using L-[35S]Methionine: Relationship of Methionine Specific Activity Between Plasma and Precursor Compartment and Evaluation of Methionine Metabolic Pathways

The method previously developed for the measurement of rates of methionine incorporation into brain proteins assumed that methionine derived from protein degradation did not recycle into the precursor pool for protein synthesis and that the metabolism of methionine via the transmethylation pathway was negligible. To evaluate the degree of recycling, we have compared, under steady-state conditions, the specific activity of L-[35S] methionine in the tRNA-bound pool to that of plasma. The relative contribution of methionine from protein degradation to the precursor pool was 26%. Under the same conditions, the relative rate of methionine flux into the transmethylation cycle was estimated to be 10% of the rate of methionine incorporation into brain proteins. These results indicate the following: (a) there is significant recycling of unlabeled methionine derived from protein degradation in brain; and (b) the metabolism of methionine is directed mainly towards protein synthesis. At normal plasma amino acid levels, methionine is the amino acid which, to date, presents the lowest degree of dilution in the precursor pool for protein synthesis. L-[35S]-Methionine, therefore, presents radiobiochemical properties required to measure, with minimal underestimation, rates of brain protein synthesis in vivo.     

Effect of Loading Doses of l-Valine on Relative Contributions of Valine Derived from Protein Degradation and Plasma to the Precursor Pool for Protein Synthesis in Rat Brain

"Flooding" amino acid pools with high doses of labeled amino acids of low specific activity has been proposed to minimize the effects of recycling of amino acids derived from protein degradation on the specific activity of the amino acid precursor pool for protein synthesis. We have examined the influence of recycling on the precursor pool for protein synthesis under conditions in which plasma valine concentrations were normal (0.19 mM) and "flooded" (10-28 mM) by comparing the steady-state specific activity of the tRNA-bound valine with that of the plasma valine. Under normal and "flooding" conditions, the relative contributions of valine from protein degradation to the precursor pool were 63 and 26%, respectively; "flooding" with a plasma level of 28 mM raised the brain acid-soluble pool level to 3.1 mM but was no more effective in decreasing the relative contribution of valine from protein degradation to the precursor pool than "flooding" with a plasma level of 17 mM valine, which raised the brain acid-soluble level only to 2.3 mM. The results of these studies show that "flooding" amino acid pools does indeed reduce the effect of recycling on the precursor amino acid pool for protein synthesis, but it does not totally eliminate it.     

Topograph,a Software Platform for Precursor Enrichment Corrected Global Protein Turnover Measurements

Defects in protein turnover have been implicated in a broad range of diseases, but current proteomics methods of measuring protein turnover are limited by the software tools available. Conventional methods require indirect approaches to differentiate newly synthesized protein when synthesized from partially labeled precursor pools. To address this, we have developed Topograph, a software platform which calculates the fraction of peptides that are from newly synthesized proteins and their turnover rates. A unique feature of Topograph is the ability to calculate amino acid precursor pool enrichment levels which allows for accurate calculations when the precursor pool is not fully labeled, and the approach used by Topograph is applicable regardless of the stable isotope label used. We validate the Topograph algorithms using data acquired from a mouse labeling experiment and demonstrate the influence that precursor pool corrections can have on protein turnover measurements.Methods of measuring protein synthesis and degradation using stable or radioactive isotope labels have existed for decades. The isotope label is introduced in the form of a labeled amino acid or amino acid precursor, and the incorporation or removal of that label from protein is used to estimate average protein turnover rates (1, 2). Historically, the amount of stable isotope label incorporated into a protein is measured by enriching for the protein (e.g. affinity chromatography, gel electrophoresis, and other biochemical methods), hydrolyzing the protein to amino acids, derivatizing the amino acids, and measuring the labeled amino acid by gas chromatography-mass spectrometry or gas chromatography-combustion-isotope ratio mass spectrometry (3, 4). More recently, proteomics methods have been developed that measure the labeled amino acid on the peptide level, eliminating the need for a protein enrichment step and enabling the monitoring of many proteins in a single experiment (5).Proteomics approaches to measuring protein turnover rates in mice have been accomplished by the introduction of a ¹⁵N stable isotope label. The labeled diets were created by supplementing a protein-free diet with a ¹⁵N enriched protein source. Price et al. (6) generated ¹⁵N-labeled protein from the alga, Spirulina platensis and Zhang et al. (7) introduced ¹⁵N-label in the form of lysate from the bacterium, Ralstonia eutropha. An advantage of using complete ¹⁵N labeling is the rapid incorporation of ¹⁵N and separation of isotope distributions between labeled and natural isotope abundance peptides, which reduces the need to deconvolute the two distributions. However, current methods require that the dietary protein content be derived from bacterial or alga lysate, a diet that is not normally fed to laboratory mice. As a result, measurements of protein turnover may not reflect conventional mouse model systems because of effects of diet on protein and amino acid metabolism. A more recent work by Claydon et al. (8) demonstrated a stable isotope labeling method by supplementing labeled valine into a standard mouse diet.The complex data generated from these analyses creates a data processing and analysis challenge; exemplified by recent software platforms that have been developed. Guan et al. (9) and Hoopmann et al. (10) demonstrated data analysis pipelines for ¹⁵N labeled SILAM and SILAC experiments. Here we describe the software platform, Topograph, we have developed for the analysis of liquid chromatography-tandem MS (LC-MS/MS) data from samples with isotopic labels. Topograph is able to deconvolute the complex spectra that may result from overlapping isotope distributions, regardless of the isotope label used. More uniquely, Topograph is able to calculate the relative isotope abundance (RIA)¹ of the amino acid precursor pool, which is necessary to correctly determine the amount of newly synthesized peptide and to subsequently calculate peptide and protein turnover rates.     

A proteomics strategy for determining the synthesis and degradation rates of individual proteins in fish

In order to study the protein dynamics in the tissues of fish we have developed a proteomics-based strategy to determine the rates of synthesis and degradation of individual proteins. We have demonstrated the feasibility of this approach by measuring the turnover of multiple isoforms of parvalbumin (β1-7) in the skeletal muscle of common carp (Cyprinus carpio). A stable isotope-labelled amino acid ([(2)H(7)] l-leucine) was administered to the carp via the diet and its incorporation into the isoforms of parvalbumin in muscle over time was monitored by LC-MS analysis of signature peptides. The relative isotope abundance was calculated and used to deconvolute the data. The β7 parvalbumin isoform had a rate of synthesis that was greater than the rate of degradation. In contrast the rate of degradation of the β5 isoform exceeded its rate of synthesis, whilst the analysis revealed that the other parvalbumin β-isoforms (β1, β2, β3, β4 and β6) had a rate of synthesis that was equal to the rate of degradation. This work has addressed a number of technical challenges and represents the first study to use proteomic approaches to measure the turnover of individual proteins in fish.     

Recent advancements in mass spectrometry–based tools to investigate newly synthesized proteins

Tight regulation of protein translation drives the proteome to undergo changes under influence of extracellular or intracellular signals. Despite mass spectrometry–based proteomics being an excellent method to study differences in protein abundance in complex proteomes, analyzing minute or rapid changes in protein synthesis and abundance remains challenging. Therefore, several dedicated techniques to directly detect and quantify newly synthesized proteins have been developed, notably puromycin-based, bio-orthogonal noncanonical amino acid tagging–based, and stable isotope labeling by amino acids in cell culture–based methods, combined with mass spectrometry. These techniques have enabled the investigation of perturbations, stress, or stimuli on protein synthesis. Improvements of these methods are still necessary to overcome various remaining limitations. Recent improvements include enhanced enrichment approaches and combinations with various stable isotope labeling techniques, which allow for more accurate analysis and comparison between conditions on shorter timeframes and in more challenging systems. Here, we aim to review the current state in this field.     

METABOLISM OF GLUTAMATE AND RELATED AMINO ACIDS IN THE 10-DAY-OLD MOUSE BRAIN: EXPERIMENTS WITH LABELLED ACETATE AND β-HYDROXYBUTYRATE

Abstract– The pattern of incorporation of [³H, 1-¹⁴C]- and [³H. 2-¹⁴C]acetate into glutamate and related amino acids was studied in the brain of 10-day-old mice. A comparison of these patterns with those obtained for the adult brain led to the suggestion that the glutamate pool labelled directly by acetate is a much larger fraction of the total glutamate pool in the 10-day-old brain than it is in the adult brain.
Some data on the pattern of labelling of brain amino acids by 3-hydroxybutyrate. glucose and acetate support the hypothesis that direct carboxylation of pyruvate is somewhat more active in the immature than in the mature brain.
Differences in the labelling patterns of free and protein-bound brain amino acids by acetate, do indicate that the free amino acid pool labelled by acetate is not the precursor pool for protein synthesis.     

A Novel Pulse-Chase SILAC Strategy Measures Changes in Protein Decay and Synthesis Rates Induced by Perturbation of Proteostasis with an Hsp90 Inhibitor

Standard proteomics methods allow the relative quantitation of levels of thousands of proteins in two or more samples. While such methods are invaluable for defining the variations in protein concentrations which follow the perturbation of a biological system, they do not offer information on the mechanisms underlying such changes. Expanding on previous work [1], we developed a pulse-chase (pc) variant of SILAC (stable isotope labeling by amino acids in cell culture). pcSILAC can quantitate in one experiment and for two conditions the relative levels of proteins newly synthesized in a given time as well as the relative levels of remaining preexisting proteins. We validated the method studying the drug-mediated inhibition of the Hsp90 molecular chaperone, which is known to lead to increased synthesis of stress response proteins as well as the increased decay of Hsp90 “clients”. We showed that pcSILAC can give information on changes in global cellular proteostasis induced by treatment with the inhibitor, which are normally not captured by standard relative quantitation techniques. Furthermore, we have developed a mathematical model and computational framework that uses pcSILAC data to determine degradation constants k_d and synthesis rates V_s for proteins in both control and drug-treated cells. The results show that Hsp90 inhibition induced a generalized slowdown of protein synthesis and an increase in protein decay. Treatment with the inhibitor also resulted in widespread protein-specific changes in relative synthesis rates, together with variations in protein decay rates. The latter were more restricted to individual proteins or protein families than the variations in synthesis. Our results establish pcSILAC as a viable workflow for the mechanistic dissection of changes in the proteome which follow perturbations. Data are available via ProteomeXchange with identifier PXD000538.     

Application of Mass Spectrometry in Proteomics

Mass spectrometry has arguably become the core technology in proteomics. The application of mass spectrometry based techniques for the qualitative and quantitative analysis of global proteome samples derived from complex mixtures has had a big impact in the understanding of cellular function. Here, we give a brief introduction to principles of mass spectrometry and instrumentation currently used in proteomics experiments. In addition, recent developments in the application of mass spectrometry in proteomics are summarised. Strategies allowing high-throughput identification of proteins from highly complex mixtures include accurate mass measurement of peptides derived from total proteome digests and multidimensional peptide separations coupled with mass spectrometry. Mass spectrometric analysis of intact proteins permits the characterisation of protein isoforms. Recent developments in stable isotope labelling techniques and chemical tagging allow the mass spectrometry based differential display and quantitation of proteins, and newly established affinity procedures enable the targeted characterisation of post-translationally modified proteins. Finally, advances in mass spectrometric imaging allow the gathering of specific information on the local molecular composition, relative abundance and spatial distribution of peptides and proteins in thin tissue sections.     

A novel strategy for quantitative proteomics using isotope-coded protein labels

Stable isotope labelling in combination with mass spectrometry has emerged as a powerful tool to identify and relatively quantify thousands of proteins within complex protein mixtures. Here we describe a novel method, termed isotope-coded protein label (ICPL), which is capable of high-throughput quantitative proteome profiling on a global scale. Since ICPL is based on stable isotope tagging at the frequent free amino groups of isolated intact proteins, it is applicable to any protein sample, including extracts from tissues or body fluids, and compatible to all separation methods currently employed in proteome studies. The method showed highly accurate and reproducible quantification of proteins and yielded high sequence coverage, indispensable for the detection of post-translational modifications and protein isoforms. The efficiency (e.g. accuracy, dynamic range, sensitivity, speed) of the approach is demonstrated by comparative analysis of two differentially spiked proteomes.     

On the application of compartmental models to radioactive tracer kinetic studies of in vivo protein turnover in animals

A mathematical framework is presented for unifying and extending the various compartmental models and formulae used to calculate fractional protein synthesis and degradation rates in animals from data obtained by infusing labelled amino acids. It is shown how the various schemes can be derived as special cases of the product-precursor model or some three-pool variant. Three-compartment representations, which circumvent the need to measure the specific radioactivity of the precursor pool, are proposed. The mathematical solutions are generally presented in a form that is amenable to parameter estimation by non-linear least squares. The problems of measuring the true precursor pool for protein synthesis are addressed, and theoretical consideration is given to assaying aminoacyl-tRNA.     

Comparison of the turnover patterns of total and individual muscle proteins in normal mice and those with hereditary muscular dystrophy

1. The incorporation of amino acids into hindleg muscle proteins of normal and dystrophic mice was measured (1/2)h to 16 days after administration of the radioactive pulse. 2. Dystrophic animals showed a faster initial rate of incorporation into total and soluble proteins in the first few hours after injection, but the extent of incorporation relative to the size of the amino acid pool was similar in both. There was little difference between the overall degradation rates although this started later in the dystrophic proteins. An initial fast phase of degradation reached a plateau after 3 days whereupon the residual label in the protein remained constant up to 16 days after injection. 3. Analyses of individual radioactive proteins fractionated by polyacrylamide-gel electrophoresis showed that the distribution of label was similar in all the soluble proteins from normal and dystrophic muscle. Time-course experiments revealed that in dystrophic mice the two major soluble proteins of the muscle, creatine kinase and adenylate kinase, initially incorporated 2-3 times more label relative to the initial size of the precursor pool. This label was then lost equally rapidly and the final plateau value was much less than that in normal mice. This initial peak of activity was not observed in normal mice. 4. A group of dehydrogenases showed similar initial turnover patterns in both dystrophic and normal mice but the final plateau value was much higher in the former. 5. The results provide support for the hypothesis that there is no obvious defect in the protein synthetic machinery of dystrophic muscle. However, certain proteins do show anomalous turnover patterns relative to those in normal animals. A single structural gene mutation giving rise to one particularly unstable and readily degradable muscle protein is excluded as the cause of the dystrophy.