A mass spectrometry workflow for measuring protein turnover rates in vivo

AbstractPatient-derived organoids from induced pluripotent stem cells have emerged as a model for studying human diseases beyond conventional two-dimensional (2D) cell culture. Briefly, these three-dimensional organoids are highly complex, capable of self-organizing, recapitulate cellular architecture, and have the potential to model diseases in complex organs, such as the brain. For example, the hallmark of Parkinson’s disease - proteostatic dysfunction leading to the selective death of neurons in the substantia nigra - present a subtle distinction in cell type specificity that is simply lost in 2D cell culture models. As such, the development of robust methods to study global proteostasis and protein turnover in organoids will remain a critical need as organoid models evolve. To solve this problem, we have designed a workflow to extract proteins from organoids and measure global protein turnover using mass spectrometry and stable isotope labeling using amino acids in cell culture (SILAC). This allowed us to measure the turnover rates of 844 proteins and compare protein turnover to previously reported data in primary cell cultures and in vivo models. Taken together, this method will facilitate the study of proteostasis in organoid models of human disease and will provide an analytical and statistical framework to measure protein turnover in organoids of all cell types.

Download Full-text

Precise Estimation of In Vivo Protein Turnover Rates

10.1101/2020.11.10.377440 ◽

2020 ◽

Author(s):

Jonathon J. O’Brien ◽

Vikram Narayan ◽

Yao Wong ◽

Phillip Seitzer ◽

Celeste M. Sandoval ◽

...

Keyword(s):

Amino Acid ◽

Protein Turnover ◽

Search Algorithm ◽

Deuterium Oxide ◽

Isotopic Labeling ◽

Analytical Framework ◽

Turnover Rates ◽

Ion Distribution ◽

Common Technique

AbstractIsotopic labeling with deuterium oxide (D2O) is a common technique for estimating in vivo protein turnover, but its use has been limited by two long-standing problems: (1) identifying non-monoisotopic peptides; and (2) estimating protein turnover rates in the presence of dynamic amino acid enrichment. In this paper, we present a novel experimental and analytical framework for solving these two problems. Peptides with high probabilities of labeling in many amino acids present fragmentation spectra that frequently do not match the theoretical spectra used in standard identification algorithms. We resolve this difficulty using a modified search algorithm we call Conditional Ion Distribution Search (CIDS). Increased identifications from CIDS along with direct measurement of amino acid enrichment and statistical modeling that accounts for heterogeneous information across peptides, dramatically improves the accuracy and precision of half-life estimates. We benchmark the approach in cells, where near-complete labeling is possible, and conduct an in vivo experiment revealing, for the first time, differences in protein turnover between mice and naked mole-rats commensurate with their disparate longevity.

Download Full-text

Interrogation of Dystrophin and Dystroglycan Complex Protein Turnover After Exon Skipping Therapy

Journal of Neuromuscular Diseases ◽

10.3233/jnd-210696 ◽

2021 ◽

pp. 1-20

Author(s):

James S. Novak ◽

Rita Spathis ◽

Utkarsh J. Dang ◽

Alyson A. Fiorillo ◽

Ravi Hindupur ◽

...

Keyword(s):

Mass Spectrometry ◽

Protein Expression ◽

Protein Turnover ◽

Isotope Labeling ◽

Exon Skipping ◽

Mdx Mice ◽

Wild Type ◽

Gene Correction ◽

Dystrophin Protein

Recently, the Food and Drug Administration granted accelerated approvals for four exon skipping therapies –Eteplirsen, Golodirsen, Viltolarsen, and Casimersen –for Duchenne Muscular Dystrophy (DMD). However, these treatments have only demonstrated variable and largely sub-therapeutic levels of restored dystrophin protein in DMD patients, limiting their clinical impact. To better understand variable protein expression and the behavior of truncated dystrophin protein in vivo, we assessed turnover dynamics of restored dystrophin and dystroglycan complex (DGC) proteins in mdx mice after exon skipping therapy, compared to those dynamics in wild type mice, using a targeted, highly-reproducible and sensitive, in vivo stable isotope labeling mass spectrometry approach in multiple muscle tissues. Through statistical modeling, we found that restored dystrophin protein exhibited altered stability and slower turnover in treated mdx muscle compared with that in wild type muscle (∼44 d vs. ∼24 d, respectively). Assessment of mRNA transcript stability (quantitative real-time PCR, droplet digital PCR) and dystrophin protein expression (capillary gel electrophoresis, immunofluorescence) support our dystrophin protein turnover measurements and modeling. Further, we assessed pathology-induced muscle fiber turnover through bromodeoxyuridine (BrdU) labeling to model dystrophin and DGC protein turnover in the context persistent fiber degeneration. Our findings reveal sequestration of restored dystrophin protein after exon skipping therapy in mdx muscle leading to a significant extension of its half-life compared to the dynamics of full-length dystrophin in normal muscle. In contrast, DGC proteins show constant turnover attributable to myofiber degeneration and dysregulation of the extracellular matrix (ECM) in dystrophic muscle. Based on our results, we demonstrate the use of targeted mass spectrometry to evaluate the suitability and functionality of restored dystrophin isoforms in the context of disease and propose its use to optimize alternative gene correction strategies in development for DMD.

Download Full-text

Protein turnover measurement using selected reaction monitoring-mass spectrometry (SRM-MS)

Philosophical Transactions of The Royal Society A Mathematical Physical and Engineering Sciences ◽

10.1098/rsta.2015.0362 ◽

2016 ◽

Vol 374 (2079) ◽

pp. 20150362 ◽

Cited By ~ 1

Author(s):

Stephen W. Holman ◽

Dean E. Hammond ◽

Deborah M. Simpson ◽

John Waters ◽

Jane L. Hurst ◽

...

Keyword(s):

Mass Spectrometry ◽

Protein Turnover ◽

Tricarboxylic Acid Cycle ◽

High Mass ◽

Selected Reaction Monitoring ◽

Reaction Monitoring ◽

Turnover Rates ◽

Mass Spectrometers ◽

High Mass Resolution ◽

Sensitivity And Selectivity

Protein turnover represents an important mechanism in the functioning of cells, with deregulated synthesis and degradation of proteins implicated in many diseased states. Therefore, proteomics strategies to measure turnover rates with high confidence are of vital importance to understanding many biological processes. In this study, the more widely used approach of non-targeted precursor ion signal intensity (MS1) quantification is compared with selected reaction monitoring (SRM), a data acquisition strategy that records data for specific peptides, to determine if improved quantitative data would be obtained using a targeted quantification approach. Using mouse liver as a model system, turnover measurement of four tricarboxylic acid cycle proteins was performed using both MS1 and SRM quantification strategies. SRM outperformed MS1 in terms of sensitivity and selectivity of measurement, allowing more confident determination of protein turnover rates. SRM data are acquired using cheaper and more widely available tandem quadrupole mass spectrometers, making the approach accessible to a larger number of researchers than MS1 quantification, which is best performed on high mass resolution instruments. SRM acquisition is ideally suited to focused studies where the turnover of tens of proteins is measured, making it applicable in determining the dynamics of proteins complexes and complete metabolic pathways. This article is part of the themed issue ‘Quantitative mass spectrometry’.

Download Full-text

The number of catalytic cycles in an enzyme’s lifetime and why it matters to metabolic engineering

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2023348118 ◽

2021 ◽

Vol 118 (13) ◽

pp. e2023348118

Author(s):

Andrew D. Hanson ◽

Donald R. McCarty ◽

Christopher S. Henry ◽

Xiaochen Xian ◽

Jaya Joshi ◽

...

Keyword(s):

Metabolic Engineering ◽

Active Site ◽

Protein Turnover ◽

Metabolic Flux ◽

Failure Modes ◽

High Energy ◽

Enzyme Engineering ◽

Turnover Rates ◽

Catalytic Cycles

Metabolic engineering uses enzymes as parts to build biosystems for specified tasks. Although a part’s working life and failure modes are key engineering performance indicators, this is not yet so in metabolic engineering because it is not known how long enzymes remain functional in vivo or whether cumulative deterioration (wear-out), sudden random failure, or other causes drive replacement. Consequently, enzymes cannot be engineered to extend life and cut the high energy costs of replacement. Guided by catalyst engineering, we adopted catalytic cycles until replacement (CCR) as a metric for enzyme functional life span in vivo. CCR is the number of catalytic cycles that an enzyme mediates in vivo before failure or replacement, i.e., metabolic flux rate/protein turnover rate. We used estimated fluxes and measured protein turnover rates to calculate CCRs for ∼100–200 enzymes each from Lactococcus lactis, yeast, and Arabidopsis. CCRs in these organisms had similar ranges (<103 to >107) but different median values (3–4 × 104 in L. lactis and yeast versus 4 × 105 in Arabidopsis). In all organisms, enzymes whose substrates, products, or mechanisms can attack reactive amino acid residues had significantly lower median CCR values than other enzymes. Taken with literature on mechanism-based inactivation, the latter finding supports the proposal that 1) random active-site damage by reaction chemistry is an important cause of enzyme failure, and 2) reactive noncatalytic residues in the active-site region are likely contributors to damage susceptibility. Enzyme engineering to raise CCRs and lower replacement costs may thus be both beneficial and feasible.

Download Full-text

Long-lived metabolic enzymes in the crystalline lens identified by pulse-labeling of mice and mass spectrometry

eLife ◽

10.7554/elife.50170 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 4

Author(s):

Pan Liu ◽

Seby Louis Edassery ◽

Laith Ali ◽

Benjamin R Thomson ◽

Jeffrey N Savas ◽

...

Keyword(s):

Mass Spectrometry ◽

Protein Turnover ◽

Crystalline Lens ◽

Nuclear Proteins ◽

Turnover Rates ◽

Fiber Cells ◽

Oxidation Reduction ◽

Age Related ◽

Health And Disease ◽

Biochemical State

The lenticular fiber cells are comprised of extremely long-lived proteins while still maintaining an active biochemical state. Dysregulation of these activities has been implicated in diseases such as age-related cataracts. However, the lenticular protein dynamics underlying health and disease is unclear. We sought to measure the global protein turnover rates in the eye using nitrogen-15 labeling of mice and mass spectrometry. We measured the 14N/15N-peptide ratios of 248 lens proteins, including Crystallin, Aquaporin, Collagen and enzymes that catalyze glycolysis and oxidation/reduction reactions. Direct comparison of lens cortex versus nucleus revealed little or no 15N-protein contents in most nuclear proteins, while there were a broad range of 14N/15N ratios in cortex proteins. Unexpectedly, like Crystallins, many enzymes with relatively high abundance in nucleus were also exceedingly long-lived. The slow replacement of these enzymes in spite of young age of mice suggests their potential roles in age-related metabolic changes in the lens.

Download Full-text

Protein turnover and growth in the whole body, liver and kidney of the rat from the foetus to senility

Biochemical Journal ◽

10.1042/bj2170507 ◽

1984 ◽

Vol 217 (2) ◽

pp. 507-516 ◽

Cited By ~ 159

Author(s):

D F Goldspink ◽

F J Kelly

Keyword(s):

Protein Synthesis ◽

Protein Turnover ◽

Post Partum ◽

Protein Composition ◽

Whole Body ◽

Turnover Rates ◽

Tissue Mass ◽

Age Related ◽

Foetal Life

Changes in the growth and protein turnover (measured in vivo) of the rat liver, kidney and whole body were studied between 16 days of life in utero and 105 weeks post partum. Tissue and whole-body growth were related to changes in both cellular hyperplasia (i.e. changes in DNA) and hypertrophy (protein/DNA values) and to the protein composition within the enlarging tissue mass. The suitability of using a single large dose of phenylalanine for measuring the rates of protein synthesis during both pre- and post-natal life was established. The declining growth rates in the whole animal and the two visceral tissues were then explained by developmental changes in the fractional rates of protein synthesis and breakdown, turnover rates being age-for-age higher in the liver than in the kidney, which in turn were higher than those measured in the whole animal. The declining fractional rates of synthesis in both tissues and the whole body with increasing age were related to changes in the tissues' ribosomal capacity and activity. The fall in the hepatic rate between 18 and 20 days of foetal life (from 134 to 98% per day) corresponded to a decrease in both the ribosomal capacity and the rate of synthesis per ribosome. No significant changes in any of these parameters were, however, found in the liver between weaning (3 weeks) and senility (105 weeks). In contrast, the fractional synthetic (and degradative) rates progressively declined in the kidney (from 95 to 24% per day) and whole body (from 70 to 11% per day) throughout both pre- and post-natal life, mainly as a consequence of a progressive decline in the ribosomal capacity, but with some fall in the ribosomal activity also during foetal life. The age-related contributions of these visceral tissues to the total amount of protein synthesized per day by the whole animal were determined. The renal contribution remained fairly constant at 1.6-2.9%, whereas the hepatic contribution declined from 56 to 11%, with increasing age. Approximate-steady-state conditions were reached at, and between, 44 and 105 weeks post partum, the half-life values of mixed whole-body, kidney and liver proteins being 6.4, 3.0 and 1.5 days, respectively, at 105 weeks.

Download Full-text

An atlas of protein turnover rates in mouse tissues

Nature Communications ◽

10.1038/s41467-021-26842-3 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Zach Rolfs ◽

Brian L. Frey ◽

Xudong Shi ◽

Yoshitaka Kawai ◽

Lloyd M. Smith ◽

...

Keyword(s):

Protein Turnover ◽

Isotope Labeling ◽

Turnover Rates ◽

Visualization Software ◽

Degradation Rates ◽

Mouse Tissues ◽

Related Drug ◽

Synthesis And Degradation ◽

Mammalian Protein

AbstractProtein turnover is critical to cellular physiology as well as to the growth and maintenance of tissues. The unique synthesis and degradation rates of each protein help to define tissue phenotype, and knowledge of tissue- and protein-specific half-lives is directly relevant to protein-related drug development as well as the administration of medical therapies. Using stable isotope labeling and mass spectrometry, we determine the in vivo turnover rates of thousands of proteins—including those of the extracellular matrix—in a set of biologically important mouse tissues. We additionally develop a data visualization platform, named ApplE Turnover, that enables facile searching for any protein of interest in a tissue of interest and then displays its half-life, confidence interval, and supporting measurements. This extensive dataset and the corresponding visualization software provide a reference to guide future studies of mammalian protein turnover in response to physiologic perturbation, disease, or therapeutic intervention.

Download Full-text

Heterogeneity of proteome dynamics between connective tissue phases of adult tendon

10.1101/2020.01.27.921163 ◽

2020 ◽

Author(s):

Deborah Simpson ◽

Howard Choi ◽

Ding Wang ◽

Mark Prescott ◽

Andrew A. Pitsillides ◽

...

Keyword(s):

Mass Spectrometry ◽

Extracellular Matrix ◽

Connective Tissue ◽

Protein Turnover ◽

Tissue Homeostasis ◽

Turnover Rates ◽

Connective Tissues ◽

Isotope Labelling ◽

Tissue Integrity ◽

Tissue Proteome

AbstractMaintenance of connective tissue integrity is fundamental to sustain function, requiring protein turnover to repair damaged tissue. However, connective tissue proteome dynamics remain largely undefined, as do differences in turnover rates of individual proteins in the collagen and glycoprotein phases of connective tissue extracellular matrix (ECM). Here, we investigate proteome dynamics in the collagen and glycoprotein phases of connective tissues by exploiting the spatially distinct fascicular (collagen-rich) and interfascicular (glycoprotein-rich) ECM phases of tendon. Using isotope labelling, mass spectrometry and bioinformatics, we calculate turnover rates of individual proteins within rat Achilles tendon and its ECM phases. Our results demonstrate complex proteome dynamics in tendon, with ~1000-fold differences in protein turnover rates, and overall faster protein turnover within the glycoprotein-rich interfascicular matrix compared to the collagen-rich fascicular matrix. These data provide insights into the complexity of proteome dynamics in tendon, likely required to maintain tissue homeostasis.

Download Full-text

Protein turnover in the developing Triticum aestivum grain

10.1101/2021.06.15.448508 ◽

2021 ◽

Author(s):

Hui Cao ◽

Owen Duncan ◽

A. Harvey Millar

Keyword(s):

Protein Synthesis ◽

Protein Turnover ◽

Storage Proteins ◽

Spatiotemporal Pattern ◽

Turnover Rates ◽

Grain Development ◽

Wheat Grain ◽

Stable Isotope Labelling ◽

Atp Production

Protein abundance in cereal grains is determined by the relative rates of protein synthesis and protein degradation during grain development. Through combining in vivo stable isotope labelling and in-depth quantitative proteomics, we have measured the turnover of 1400 different types of proteins during wheat grain development. We demonstrate that there is a spatiotemporal pattern to protein turnover rates which explain part of the variation in protein abundances that is not attributable to differences in wheat gene expression. We show that approximately 20% of total grain ATP production is used for grain proteome biogenesis and maintenance, and nearly half of this budget is invested exclusively in storage protein synthesis. We calculate that 25% of newly synthesized storage proteins are turned over during grain development rather than stored. This approach to measure protein turnover rates at proteome scale reveals how different functional categories of grain proteins accumulate, calculates the costs of protein turnover during wheat grain development and identifies the most and the least stable proteins in the developing wheat grain.

Download Full-text