John C. Price

Analysis of proteome dynamics in the mouse brain

Advances in systems biology have allowed for global analyses of mRNA and protein expression, but large-scale studies of protein dynamics and turnover have not been conducted in vivo. Protein turnover is an important metabolic and regulatory mechanism in establishing proteome homeostasis, impacting many physiological and pathological processes. Here, we have used organism-wide isotopic labeling to measure the turnover rates of ~2,500 proteins in multiple mouse tissues, spanning four orders of magnitude. Through comparison of the brain with the liver and blood, we show that within the respective tissues, proteins performing similar functions often have similar turnover rates. Proteins in the brain have significantly slower turnover (average lifetime of 9.0 d) compared with those of the liver (3.0 d) and blood (3.5 d). Within some organelles (such as mitochondria), proteins have a narrow range of lifetimes, suggesting a synchronized turnover mechanism. Protein subunits within complexes of variable composition have a wide range of lifetimes, whereas those within well-defined complexes turn over in a coordinated manner. Together, the data represent the most comprehensive in vivo analysis of mammalian proteome turnover to date. The developed methodology can be adapted to assess in vivo proteome homeostasis in any model organism that will tolerate a labeled diet and may be particularly useful in the analysis of neurodegenerative diseases in vivo.

Measurement of human plasma proteome dynamics with 2H2O and liquid chromatography tandem mass spectrometry

Dysfunction of protein turnover is a feature of many human diseases, and proteins are substrates in important biological processes. Currently, no method exists for the measurement of global protein turnover (i.e., proteome dynamics) that can be applied in humans. Here we describe the use of metabolic labeling with deuterium ((2)H) from (2)H(2)O and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of mass isotopomer patterns to measure protein turnover. We show that the positions available for (2)H label incorporation in vivo can be calculated using peptide sequence. The isotopic incorporation values calculated by combinatorial analysis of mass isotopomer patterns in peptides correlate very closely with values established for individual amino acids. Inpatient and outpatient heavy water labeling protocols resulted in (2)H label incorporation sufficient for reproducible quantitation in humans. Replacement rates were similar for peptides deriving from the same protein. Using a kinetic model to account for the time course of each individuals (2)H(2)O enrichment curves, dynamics of approximately 100 proteins with half-lives ranging from 0.4 to 40 days were measured using 8 μl of plasma. The measured rates were consistent with literature values. This method can be used to measure in vivo proteome homeostasis in humans in disease and during therapeutic interventions.

A Data Processing Pipeline for Mammalian Proteome Dynamics Studies Using Stable Isotope Metabolic Labeling

In a recent study, in vivo metabolic labeling using 15N traced the rate of label incorporation among more than 1700 proteins simultaneously and enabled the determination of individual protein turnover rate constants over a dynamic range of three orders of magnitude (Price, J. C., Guan, S., Burlingame, A., Prusiner, S. B., and Ghaemmaghami, S. (2010) Analysis of proteome dynamics in the mouse brain. Proc. Natl. Acad. Sci. U. S. A. 107, 14508–14513). These studies of protein dynamics provide a deeper understanding of healthy development and well-being of complex organisms, as well as the possible causes and progression of disease. In addition to a fully labeled food source and appropriate mass spectrometry platform, an essential and enabling component of such large scale investigations is a robust data processing and analysis pipeline, which is capable of the reduction of large sets of liquid chromatography tandem MS raw data files into the desired protein turnover rate constants. The data processing pipeline described in this contribution is comprised of a suite of software modules required for the workflow that fulfills such requirements. This software platform includes established software tools such as a mass spectrometry database search engine together with several additional, novel data processing modules specifically developed for 15N metabolic labeling. These fulfill the following functions: (1) cross-extraction of 15N-containing ion intensities from raw data files at varying biosynthetic incorporation times, (2) computation of peptide 15N isotopic incorporation distributions, and (3) aggregation of relative isotope abundance curves for multiple peptides into single protein curves. In addition, processing parameter optimization and noise reduction procedures were found to be necessary in the processing modules in order to reduce propagation of errors in the long chain of the processing steps of the entire workflow.

The Effect of Long Term Calorie Restriction on in Vivo Hepatic Proteostatis: A Novel Combination of Dynamic and Quantitative Proteomics

Calorie restriction (CR) promotes longevity. A prevalent mechanistic hypothesis explaining this effect suggests that protein degradation, including mitochondrial autophagy, is increased with CR, removing damaged proteins and improving cellular fitness. At steady state, increased catabolism must be balanced by increasing mitochondrial biogenesis and protein synthesis, resulting in faster protein replacement rates. To test this hypothesis, we measured replacement kinetics and relative concentrations of hundreds of proteins in vivo in long-term CR and ad libitum-fed mice using metabolic 2H2O-labeling combined with the Stable Isotope Labeling in Mammals protocol and LC-MS/MS analysis of mass isotopomer abundances in tryptic peptides. CR reduced absolute synthesis and breakdown rates of almost all measured hepatic proteins and prolonged the half-lives of most (∼80%), particularly mitochondrial proteins (but not ribosomal subunits). Proteins with related functions exhibited coordinated changes in relative concentration and replacement rates. In silico expression pathway interrogation allowed the testing of potential regulators of altered network dynamics (e.g. peroxisome proliferator-activated receptor gamma coactivator 1-alpha). In summary, our combination of dynamic and quantitative proteomics suggests that long-term CR reduces mitochondrial biogenesis and mitophagy. Our findings contradict the theory that CR increases mitochondrial protein turnover and provide compelling evidence that cellular fitness is accompanied by reduced global protein synthetic burden.

Mass spectrometry imaging for in situ kinetic histochemistry

Tissues are composed of diverse cell subpopulations each with distinct metabolic characteristics that influence overall behavior. Unfortunately, traditional histopathology imaging techniques are ‘blind’ to the spatially ordered metabolic dynamics within tissue. While mass spectrometry imaging enables spatial mapping of molecular composition, resulting images are only a static snapshot in time of molecules involved in highly dynamic processes; kinetic information of flux through metabolic pathways is lacking. To address this limitation, we developed kinetic mass spectrometry imaging (kMSI), a novel technique integrating soft desorption/ionization mass spectrometry with clinically accepted in vivo metabolic labeling of tissue with deuterium to generate images of kinetic information of biological processes. Applied to a tumor, kMSI revealed heterogeneous spatial distributions of newly synthesized versus pre-existing lipids, with altered lipid synthesis patterns distinguishing region-specific intratumor subpopulations. Images also enabled identification and correlation of metabolic activity of specific lipids found in tumor regions of varying grade.

Circulating protein synthesis rates reveal skeletal muscle proteome dynamics

Here, we have described and validated a strategy for monitoring skeletal muscle protein synthesis rates in rodents and humans over days or weeks from blood samples. We based this approach on label incorporation into proteins that are synthesized specifically in skeletal muscle and escape into the circulation. Heavy water labeling combined with sensitive tandem mass spectrometric analysis allowed integrated synthesis rates of proteins in muscle tissue across the proteome to be measured over several weeks. Fractional synthesis rate (FSR) of plasma creatine kinase M-type (CK-M) and carbonic anhydrase 3 (CA-3) in the blood, more than 90% of which is derived from skeletal muscle, correlated closely with FSR of CK-M, CA-3, and other proteins of various ontologies in skeletal muscle tissue in both rodents and humans. Protein synthesis rates across the muscle proteome generally changed in a coordinate manner in response to a sprint interval exercise training regimen in humans and to denervation or clenbuterol treatment in rodents. FSR of plasma CK-M and CA-3 revealed changes and interindividual differences in muscle tissue proteome dynamics. In human subjects, sprint interval training primarily stimulated synthesis of structural and glycolytic proteins. Together, our results indicate that this approach provides a virtual biopsy, sensitively revealing individualized changes in proteome-wide synthesis rates in skeletal muscle without a muscle biopsy. Accordingly, this approach has potential applications for the diagnosis, management, and treatment of muscle disorders.

Mechanisms of in vivo ribosome maintenance change in response to nutrient signals

Control of protein homeostasis is fundamental to the health and longevity of all organisms. Because the rate of protein synthesis by ribosomes is a central control point in this process, regulation, and maintenance of ribosome function could have amplified importance in the overall regulatory circuit. Indeed, ribosomal defects are commonly associated with loss of protein homeostasis, aging, and disease (1–4), whereas improved protein homeostasis, implying optimal ribosomal function, is associated with disease resistance and increased lifespan (5–7). To maintain a high-quality ribosome population within the cell, dysfunctional ribosomes are targeted for autophagic degradation. It is not known if complete degradation is the only mechanism for eukaryotic ribosome maintenance or if they might also be repaired by replacement of defective components. We used stable-isotope feeding and protein mass spectrometry to measure the kinetics of turnover of ribosomal RNA (rRNA) and 71 ribosomal proteins (r-proteins) in mice. The results indicate that exchange of individual proteins and whole ribosome degradation both contribute to ribosome maintenance in vivo. In general, peripheral r-proteins and those with more direct roles in peptide-bond formation are replaced multiple times during the lifespan of the assembled structure, presumably by exchange with a free cytoplasmic pool, whereas the majority of r-proteins are stably incorporated for the lifetime of the ribosome. Dietary signals impact the rates of both new ribosome assembly and component exchange. Signal-specific modulation of ribosomal repair and degradation could provide a mechanistic link in the frequently observed associations among diminished rates of protein synthesis, increased autophagy, and greater longevity (5, 6, 8, 9).

Reduced in vivo hepatic proteome replacement rates but not cell proliferation rates predict maximum lifespan extension in mice.

Combating the social and economic consequences of a growing elderly population will require the identification of interventions that slow the development of age‐related diseases. Preserved cellular homeostasis and delayed aging have been previously linked to reduced cell proliferation and protein synthesis rates. To determine whether changes in these processes may contribute to or predict delayed aging in mammals, we measured cell proliferation rates and the synthesis and replacement rates (RRs) of over a hundred hepatic proteins in vivo in three different mouse models of extended maximum lifespan (maxLS): Snell Dwarf, calorie‐restricted (CR), and rapamycin (Rapa)‐treated mice. Cell proliferation rates were not consistently reduced across the models. In contrast, reduced hepatic protein RRs (longer half‐lives) were observed in all three models compared to controls. Intriguingly, the degree of mean hepatic protein RR reduction was significantly correlated with the degree of maxLS extension across the models and across different Rapa doses. Absolute rates of hepatic protein synthesis were reduced in Snell Dwarf and CR, but not Rapa‐treated mice. Hepatic chaperone levels were unchanged or reduced and glutathione S‐transferase synthesis was preserved or increased in all three models, suggesting a reduced demand for protein renewal, possibly due to reduced levels of unfolded or damaged proteins. These data demonstrate that maxLS extension in mammals is associated with improved hepatic proteome homeostasis, as reflected by a reduced demand for protein renewal, and that reduced hepatic protein RRs hold promise as an early biomarker and potential target for interventions that delay aging in mammals.

Compact Ultrahigh-Pressure Nanoflow Capillary Liquid Chromatograph

A compact ultrahigh-pressure nanoflow liquid chromatograph (LC) was developed with the purpose in mind of creating a portable system that could be easily moved to various testing locations or placed in close proximity to other instruments for optimal coupling, such as with mass spectrometry (MS). The system utilized innovative nanoflow pumps integrated with a very low volume stop-flow injector and mixing tee. The system weighed only 5.9 kg (13 lbs) or 4.5 kg (10 lbs) without a controller and could hold up to 1100 bar (16000 psi) of pressure. The total volume pump capacity was 60 μL. In this study, the sample injection volume was determined by either a 60 nL internal sample groove machined in a high-pressure valve rotor or by a 1 μL external sample loop, although other sample grooves or loops could be selected. The gradient dwell volume was approximately 640 nL, which allowed significant reduction in sample analysis time. Gradient performance was evaluated by determining the gradient step accuracy. A low RSD (0.6%, n = 4) was obtained for day-to-day experiments. Linear gradient reproducibility was evaluated by separating a three-component polycyclic aromatic hydrocarbon mixture on a commercial 150 μm inner diameter capillary column packed with 1.7 μm particles. Good retention-time reproducibility (RSD < 0.17%) demonstrated that the pumping system could successfully generate ultrahigh pressures for use in capillary LC. The system was successfully coupled to an LTQ Orbitrap MS in a simple and efficient way; LC-MS of a trypsin-digested bovine serum albumin (BSA) sample provided narrow peaks, short dwell time, and good peptide coverage.

Kinetics of Precursor Labeling in Stable Isotope Labeling in Cell Cultures (SILAC) Experiments

Recent advances in mass spectrometry have enabled proteome-wide analyses of cellular protein turnover. These studies have been greatly propelled by the development of stable isotope labeling in cell cultures (SILAC), a set of standardized protocols, reagents aimed at quantifying the incorporation of (15)N/(13)C labeled amino acids into proteins. In dynamic SILAC experiments, the degree of isotope incorporation in proteins is measured over time and used to determine turnover kinetics. However, the kinetics of isotope incorporation in proteins can potentially be influenced not only by their intracellular turnover but also by amino acid uptake, recycling and aminoacyl-tRNA synthesis. To assess the influence of these processes in dynamic SILAC experiments, we have measured the kinetics of isotopic enrichment within intracellular free amino acid and aminoacyl-tRNA precursor pools in dividing and division-arrested neuroblastoma cells following the introduction of extracellular (15)N labeled amino acids. We show that the total flux of extracellular amino acids into cells greatly exceeds that of intracellular amino acid recycling and synthesis. Furthermore, in comparison to internal sources, external amino acids are preferentially utilized as substrates for aminoacyl-tRNA precursors for protein synthesis. As a result, in dynamic SILAC experiments conducted in culture, the aminoacyl-tRNA precursor pool is near completely labeled in a few hours and protein turnover is the limiting factor in establishing the labeling kinetics of most proteins.