Micrococcin cysteine-to-thiazole conversion through transient interactions between the scaffolding protein TclI and the modification enzymes TclJ and TclN.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a broad group of compounds mediating microbial competition in nature. Azole/azoline heterocycle formation in the peptide backbone is a key step in the biosynthesis of many RiPPs. Heterocycle formation in RiPP precursors is often carried out by a scaffold protein, an ATP-dependent cyclodehydratase, and an FMN-dependent dehydrogenase. It has generally been assumed that the orchestration of these modifications is carried out by a stable complex including the scaffold, cyclodehydratase, and dehydrogenase. The antimicrobial RiPP micrococcin begins as a precursor peptide (TclE) with a 35-amino acid N-terminal leader and a 14-amino acid C-terminal core containing six Cys residues that are converted to thiazoles. The putative scaffold protein (TclI) presumably presents the TclE substrate to a cyclodehydratase (TclJ) and a dehydrogenase (TclN) to accomplish the two-step installation of the six thiazoles. In this study, we identify a minimal TclE leader region required for thiazole formation, demonstrate complex formation between TclI, TclJ, and TclN, and further define regions of these proteins required for complex formation. Our results point to a mechanism of thiazole installation in which TclI associates with the two enzymes in a mutually exclusive fashion, such that each enzyme competes for access to the peptide substrate in a dynamic equilibrium, thus ensuring complete modification of each Cys residue in the TclE core. IMPORTANCE: Thiopeptides are a family of antimicrobial peptides characterized for having sulfur-containing heterocycles and for being highly post-translationally modified. Numerous thiopeptides have been identified; almost all of which inhibit protein synthesis in gram-positive bacteria. These intrinsic antimicrobial properties make thiopeptides promising candidates for the development of new antibiotics. The thiopeptide micrococcin is synthesized by the ribosome and undergoes several post-translational modifications to acquire its bioactivity. In this study, we identify key interactions within the enzymatic complex that carries out cysteine to thiazole conversion in the biosynthesis of micrococcin.

Human tear film protein sampling using soft contact lenses.

BACKGROUND: Human tear protein biomarkers are useful for detecting ocular and systemic diseases. Unfortunately, existing tear film sampling methods (Schirmer strip; SS and microcapillary tube; MCT) have significant drawbacks, such as pain, risk of injury, sampling difficulty, and proteomic disparities between methods. Here, we present an alternative tear protein sampling method using soft contact lenses (SCLs). RESULTS: We optimized the SCL protein sampling in vitro and performed in vivo studies in 6 subjects. Using Etafilcon A SCLs and 4M guanidine-HCl for protein removal, we sampled an average of 60 ± 31 µg of protein per eye. We also performed objective and subjective assessments of all sampling methods. Signs of irritation post-sampling were observed with SS but not with MCT and SCLs. Proteomic analysis by mass spectrometry (MS) revealed that all sampling methods resulted in the detection of abundant tear proteins. However, smaller subsets of unique and shared proteins were identified, particularly for SS and MCT. Additionally, there was no significant intrasubject variation between MCT and SCL sampling. CONCLUSIONS: These experiments demonstrate that SCLs are an accessible tear-sampling method with the potential to surpass current methods in sampling basal tears.

CHalf: Folding Stability Made Simple.

The structure of a protein defines its function and integrity and correlates with the protein folding stability (PFS). Quantifying PFS allows researchers to assess differential stability of proteins in different disease or ligand binding states, providing insight into protein efficacy and potentially serving as a metric of protein quality. There are a number of mass spectrometry (MS)-based methods to assess PFS, such as Thermal Protein Profiling (TPP), Stability of Proteins from Rates of Oxidation (SPROX), and Iodination Protein Stability Assay (IPSA). Despite the critical value that PFS studies add to the understanding of mechanisms of disease and treatment development, proteomics research is still primarily dominated by concentration-based studies. We found that a major reason for the lack of PFS studies is the lack of a user-friendly data processing tool. Here we present the first user-friendly software, CHalf, with a graphical user interface for calculating PFS. Besides calculating site-specific PFS of a given protein from chemical denature folding stability assays, CHalf is also compatible with thermal denature folding stability assays. CHalf also includes a set of data visualization tools to help identify changes in PFS across protein sequences and in between different treatment conditions. We expect the introduction of CHalf to lower the barrier of entry for researchers to investigate PFS, promoting the usage of PFS in studies. In the long run, we expect this increase in PFS research to accelerate our understanding of the pathogenesis and pathophysiology of disease.

Quantifying In Situ Structural Stabilities of Human Blood Plasma Proteins Using a Novel Iodination Protein Stability Assay.

Many of the diseases that plague society today are driven by a loss of protein quality. One method to quantify protein quality is to measure the protein folding stability (PFS). Here, we present a novel mass spectrometry (MS)-based approach for PFS measurement, iodination protein stability assay (IPSA). IPSA quantifies the PFS by tracking the surface-accessibility differences of tyrosine, histidine, methionine, and cysteine under denaturing conditions. Relative to current methods, IPSA increases protein coverage and granularity to track the PFS changes of a protein along its sequence. To our knowledge, this study is the first time the PFS of human serum proteins has been measured in the context of the blood serum (in situ). We show that IPSA can quantify the PFS differences between different transferrin iron-binding states in near in vivo conditions. We also show that the direction of the denaturation curve reflects the in vivo surface accessibility of the amino acid residue and reproducibly reports a residue-specific PFS. Along with IPSA, we introduce an analysis tool Chalf that provides a simple workflow to calculate the residue-specific PFS. The introduction of IPSA increases the potential to use protein structural stability as a structural quality metric in understanding the etiology and progression of human disease. Data is openly available at Chorusproject.org (project ID 1771).

In-Depth Mass Spectrometry-Based Proteomics of Formalin-Fixed, Paraffin-Embedded Tissues with a Spatial Resolution of 50-200 µm.

Formalin-fixed, paraffin-embedded (FFPE) tissues are banked in large repositories to cost-effectively preserve valuable specimens for later study. With the rapid growth of spatial proteomics, FFPE tissues can serve as a more accessible alternative to more commonly used frozen tissues. However, extracting proteins from FFPE tissues is challenging due to cross-links formed between proteins and formaldehyde. Here, we have adapted the nanoPOTS sample processing workflow, which was previously applied to single cells and fresh-frozen tissues, to profile protein expression from FFPE tissues. Following the optimization of extraction solvents, times, and temperatures, we identified an average of 1312 and 3184 high-confidence master proteins from 10 µm thick FFPE-preserved mouse liver tissue squares having lateral dimensions of 50 and 200 µm, respectively. The observed proteome coverage for FFPE tissues was on average 88% of that achieved for similar fresh-frozen tissues. We also characterized the performance of our fully automated sample preparation and analysis workflow, termed autoPOTS, for FFPE spatial proteomics. This modified nanodroplet processing in one pot for trace samples (nanoPOTS) and fully automated processing in one pot for trace sample (autoPOTS) workflows provides the greatest coverage reported to date for high-resolution spatial proteomics applied to FFPE tissues. Data are available via ProteomeXchange with identifier PXD029729.

Utilizing Nonequilibrium Isotope Enrichments to Dramatically Increase Turnover Measurement Ranges in Single Biopsy Samples from Humans.

The synthesis of new proteins and the degradation of old proteins in vivo can be quantified in serial samples using metabolic isotope labeling to measure turnover. Because serial biopsies in humans are impractical, we set out to develop a method to calculate the turnover rates of proteins from single human biopsies. This method involved a new metabolic labeling approach and adjustments to the calculations used in previous work to calculate protein turnover. We demonstrate that using a nonequilibrium isotope enrichment strategy avoids the time dependent bias caused by variable lag in label delivery to different tissues observed in traditional metabolic labeling methods. Turnover rates are consistent for the same subject in biopsies from different labeling periods, and turnover rates calculated in this study are consistent with previously reported values. We also demonstrate that by measuring protein turnover we can determine where proteins are synthesized. In human subjects a significant difference in turnover rates differentiated proteins synthesized in the salivary glands versus those imported from the serum. We also provide a data analysis tool, DeuteRater-H, to calculate protein turnover using this nonequilibrium metabolic 2H2O method.

Improved Sensitivity for Protein Turnover Quantification by Monitoring Immonium Ion Isotopologue Abundance.

We describe an analytical strategy allowing for the direct quantification of stable isotope label incorporation in newly synthesized proteins following administration of the stable isotope tracer deuterium oxide. We present a demonstration of coupling high-resolution mass spectrometry, metabolic stable isotope labeling, and MS/MS-based isotopologue quantification for the measurement of protein turnover. Stable isotope labeling with deuterium oxide, followed by immonium ion isotopologue quantification, is a more sensitive strategy for determining protein fractional synthesis rates compared to peptide centric mass isotopomer distribution analysis approaches when labeling time and/or stable isotope tracer exposure is limited and, as such, offers a great advantage for human studies.

Analysis of thrombin-antithrombin complex formation using microchip electrophoresis and mass spectrometry.

Preterm birth (PTB) related health problems take over one million lives each year, and currently, no clinical analysis is available to determine if a fetus is at risk for PTB. Here, we describe the preparation of a key PTB risk biomarker, thrombin-antithrombin (TAT), and characterize it using dot blots, MS, and microchip electrophoresis (µCE). The pH for fluorescently labeling TAT was also optimized using spectrofluorometry and spectrophotometry. The LOD of TAT was measured in µCE. Lastly, TAT was combined with six other PTB risk biomarkers and separated in µCE. The ability to make and characterize TAT is an important step toward the development of an integrated microfluidic diagnostic for PTB risk.

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).

DeuteRater: a tool for quantifying peptide isotope precision and kinetic proteomics.

MOTIVATION: Using mass spectrometry to measure the concentration and turnover of the individual proteins in a proteome, enables the calculation of individual synthesis and degradation rates for each protein. Software to analyze concentration is readily available, but software to analyze turnover is lacking. Data analysis workflows typically don't access the full breadth of information about instrument precision and accuracy that is present in each peptide isotopic envelope measurement. This method utilizes both isotope distribution and changes in neutromer spacing, which benefits the analysis of both concentration and turnover. RESULTS: We have developed a data analysis tool, DeuteRater, to measure protein turnover from metabolic D 2 O labeling. DeuteRater uses theoretical predictions for label-dependent change in isotope abundance and inter-peak (neutromer) spacing within the isotope envelope to calculate protein turnover rate. We have also used these metrics to evaluate the accuracy and precision of peptide measurements and thereby determined the optimal data acquisition parameters of different instruments, as well as the effect of data processing steps. We show that these combined measurements can be used to remove noise and increase confidence in the protein turnover measurement for each protein. AVAILABILITY AND IMPLEMENTATION: Source code and ReadMe for Python 2 and 3 versions of DeuteRater are available at https://github.com/JC-Price/DeuteRater . Data is at https://chorusproject.org/pages/index.html project number 1147. Critical Intermediate calculation files provided as Tables S3 and S4. Software has only been tested on Windows machines. CONTACT: jcprice@chem.byu.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Imaging regiospecific lipid turnover in mouse brain with desorption electrospray ionization mass spectrometry.

Compartmentalization of metabolism into specific regions of the cell, tissue, and organ is critical to life for all organisms. Mass spectrometric imaging techniques have been valuable in identifying and quantifying concentrations of metabolites in specific locations of cells and tissues, but a true understanding of metabolism requires measurement of metabolite flux on a spatially resolved basis. Here, we utilize desorption ESI-MS (DESI-MS) to measure lipid turnover in the brains of mice. We show that anatomically distinct regions of the brain have distinct lipid turnover rates. These turnover measurements, in conjunction with relative concentration, will enable calculation of regiospecific synthesis rates for individual lipid species in vivo. Monitoring spatially dependent changes in metabolism has the potential to significantly facilitate research in many areas, such as brain development, cancer, and neurodegeneration.

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.

Whole blood and urine bioactive Hepcidin-25 determination using liquid chromatography mass spectrometry.

Hepcidin is a small cysteine-rich signaling peptide that regulates blood serum iron concentrations [1-4]. Patients with chronic inflammation are known to have elevated levels of hepcidin in their blood and urine and often suffer from anemia as a result [5-10]. Measuring and quantifying the amount of active hepcidin in blood and urine can help to determine the cause and severity of the anemia thereby helping physicians determine the correct course of treatment [11-16]. We have developed a simple technique to isolate, chemically modify, and concentrate hepcidin from blood and urine coupled to high-pressure liquid chromatography mass spectrometry that can accurately and reproducibly measure and quantify the active hormone.

Linear-response reflection coefficient of the recorder air-jet amplifier.

In a duct-flute such as the recorder, steady-state oscillations are controlled by two parameters, the blowing pressure and the frequency of the acoustic resonator. As in most feedback oscillators, the oscillation amplitude is determined by gain-saturation of the amplifier, and thus it cannot be controlled independently of blowing pressure and frequency unless the feedback loop is opened. In this work, the loop is opened by replacing the recorder body with a waveguide reflectometer: a section of transmission line with microphones, a signal source, and an absorbing termination. When the mean flow from the air-jet into the transmission line is not blocked, the air-jet amplifier is unstable to edge-tone oscillations through a feedback path that does not involve the acoustic resonator. When it is blocked, the air-jet is deflected somewhat outward and the system becomes stable. It is then possible to measure the reflection coefficient of the air-jet amplifier versus blowing pressure and acoustic frequency under linear response conditions, avoiding the complication of gain-saturation. The results provide a revealing test of flute drive models under the simplest conditions and with few unknown parameters. The strengths and weaknesses of flute drive models are discussed.

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.

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 (2)H(2)O-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.

Novel Approaches for the Accumulation of Oxygenated Intermediates to Multi-Millimolar Concentrations.

Metalloenzymes that utilize molecular oxygen as a co-substrate catalyze a wide variety of chemically difficult oxidation reactions. Significant insight into the reaction mechanisms of these enzymes can be obtained by the application of a combination of rapid kinetic and spectroscopic methods to the direct structural characterization of intermediate states. A key limitation of this approach is the low aqueous solubility (< 2 mM) of the co-substrate, O2, which undergoes further dilution (typically by one-third or one-half) upon initiation of reactions by rapid-mixing. This situation imposes a practical upper limit on [O2] (and therefore on the concentration of reactive intermediate(s) that can be rapidly accumulated) of ∼1-1.3 mM in such experiments as they are routinely carried out. However, many spectroscopic methods benefit from or require significantly greater concentrations of the species to be studied. To overcome this problem, we have recently developed two new approaches for the preparation of samples of oxygenated intermediates: (1) direct oxygenation of reduced metalloenzymes using gaseous O2 and (2) the in situ generation of O2 from chlorite catalyzed by the enzyme chlorite dismutase (Cld). Whereas the former method is applicable only to intermediates with half lives of several minutes, owing to the sluggishness of transport of O2 across the gas-liquid interface, the latter approach has been successfully applied to trap several intermediates at high concentration and purity by the freeze-quench method. The in situ approach permits generation of a pulse of at least 5 mM O2 within ∼ 1 ms and accumulation of O2 to effective concentrations of up to ∼ 11 mM (i.e. ∼ 10-fold greater than by the conventional approach). The use of these new techniques for studies of oxygenases and oxidases is discussed.

Inclusion compound based approach to arrays of artificial dipolar molecular rotors: bulk inclusions.

We examine the insertion of two dipolar molecular rotors as guests into a host, tris(o-phenylenedioxy)cyclotriphosphazine (TPP, 1), using differential scanning calorimetry, solid-state NMR, powder X-ray diffraction, and dielectric spectroscopy. The rotors are 1-(4'-n-pentylbiphenyl-4-yl)-12-(2,3-dichlorophenyl)-p-dicarba-closo-dodecaborane and 1,12-bis(2,3-dichlorophenyl)-p-dicarba-closo-dodecaborane. Both enter the bulk even though their nominal diameter exceeds the nominal channel diameter and although a closely related rotor, 1-n-hexadecyl-12-(2,3-dichlorophenyl)-p-dicarba-closo-dodecaborane, is known to produce a surface inclusion compound. Rotational barriers of 5.4-9.3 kcal/mol were found for the dichlorophenyl rotator contained within the TPP channel. Clearly, van der Waals diameters in themselves do not suffice to predict TPP channel entry. It is suggested that the efficacy of the p-carborane stopper is reduced by the presence of the two relatively bulky adjacent benzene rings, which help to stretch the channel, and by the axial direction of its axis, which prevents the attached rotator from contributing to the stopping action.

A data processing pipeline for mammalian proteome dynamics studies using stable isotope metabolic labeling.

In a recent study, in vivo metabolic labeling using (15)N 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 (15)N metabolic labeling. These fulfill the following functions: (1) cross-extraction of (15)N-containing ion intensities from raw data files at varying biosynthetic incorporation times, (2) computation of peptide (15)N 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.

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 approximately 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.