Abstract：
:Proteins are continually produced and degraded, to avoid the accumulation of old or damaged molecules and to maintain the efficiency of physiological processes. Despite its importance, protein turnover has been difficult to measure in vivo. Previous approaches to evaluating turnover in vivo have required custom labeling approaches, involved complex mass spectrometry (MS) analyses, or used comparative strategies that do not allow direct quantitative measurements. Here, we describe a robust protocol for quantitative proteome turnover analysis in mice that is based on a commercially available diet for stable isotope labeling of amino acids in mammals (SILAM). We start by discussing fundamental concepts of protein turnover, including different methodological approaches. We then cover in detail the practical aspects of metabolic labeling and explain both the experimental and computational steps that must be taken to obtain accurate in vivo results. Finally, we present a simple experimental workflow that enables measurement of precise turnover rates in a time frame of ~4-5 weeks, including the labeling time. We also provide all the scripts needed for the interpretation of the MS results and for comparing turnover across different conditions. Overall, the workflow presented here comprises several improvements in the determination of protein lifetimes with respect to other available methods, including a minimally invasive labeling strategy and a robust interpretation of MS results, thus enhancing reproducibility across laboratories.
journal_name
Nat Protocjournal_title
Nature protocolsauthors
Alevra M,Mandad S,Ischebeck T,Urlaub H,Rizzoli SO,Fornasiero EFdoi
10.1038/s41596-019-0222-ysubject
Has Abstractpub_date
2019-12-01 00:00:00pages
3333-3365issue
12eissn
1754-2189issn
1750-2799pii
10.1038/s41596-019-0222-yjournal_volume
14pub_type
杂志文章相关文献
Nature Protocols文献大全abstract：:Interactions of proteins with DNA mediate many critical nuclear functions. Chromatin immunoprecipitation (ChIP) is a robust technique for studying protein-DNA interactions. Current ChIP assays, however, either require large cell numbers, which prevent their application to rare cell samples or small-tissue biopsies, or...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2008.68
更新日期：2008-01-01 00:00:00
abstract：:Live single-cell mass spectrometry (live MS) provides a mass spectrum that shows thousands of metabolite peaks from a single live plant cell within minutes. By using an optical microscope, a cell is chosen for analysis and a metal-coated nanospray microcapillary tip is used to remove the cell's contents. After adding ...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2015.084
更新日期：2015-09-01 00:00:00
abstract：:The membrane-permeant fluorogenic biarsenicals FlAsH-EDT(2) and ReAsH-EDT(2) can be prepared in good yields by a straightforward two-step procedure from the inexpensive precursor dyes fluorescein and resorufin, respectively. Handling of toxic reagents such as arsenic trichloride is minimized so the synthesis can be ca...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2008.144
更新日期：2008-01-01 00:00:00
abstract：:The radial arm water maze (RAWM) contains six swim paths (arms) extending out of an open central area, with an escape platform located at the end of one arm (the goal arm). The goal arm location remains constant for a given mouse. On day 1, mice are trained for 15 trials (spaced over 3 h), with trials alternating betw...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2006.275
更新日期：2006-01-01 00:00:00
abstract：:Cell proliferation may be measured in vivo by quantifying DNA synthesis with isotopically labeled deoxyribonucleotide precursors. Deuterium-labeled glucose is one such precursor which, because it achieves high levels of enrichment for a short period, is well suited to the study of rapidly dividing cells, in contrast t...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2009.117
更新日期：2009-01-01 00:00:00
abstract：:The Golgi apparatus undergoes extensive disassembly during mitosis and reassembly in post-mitotic daughter cells. This process has been mimicked in vitro by treating Golgi membranes with mitotic and interphase cytosol. To determine the minimal machinery that controls the morphological change, we have developed a defin...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2010.38
更新日期：2010-04-01 00:00:00
abstract：:The plant cell wall is a chemically complex structure composed mostly of polysaccharides. Detailed analyses of these cell wall polysaccharides are essential for our understanding of plant development and for our use of plant biomass (largely wall material) in the food, agriculture, fabric, timber, biofuel and biocompo...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2012.081
更新日期：2012-09-01 00:00:00
abstract：:This protocol describes a method for direct labeling and detection of small RNAs present in total RNA by splinted ligation. The assay uses a small RNA-specific bridge oligonucleotide to form base pairs with the small RNA and a 5'-end-radiolabeled ligation oligonucleotide. The captured small RNA is directly labeled by ...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2007.530
更新日期：2008-01-01 00:00:00
abstract：:In this report, we describe a two-step protocol for labeling of an affinity-purified antibody to biotin with horseradish peroxidase (HRP) using cyanuric chloride (CC) as a bridge. The enzyme was first modified with CC, and following chromatography on a PD-10 column, the activated HRP was incubated with the antibody to...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2009.6
更新日期：2009-01-01 00:00:00
abstract：:Gram-negative and Gram-positive bacteria release a variety of membrane vesicles through different formation routes. Knowledge of the structure, molecular cargo and function of bacterial extracellular vesicles (BEVs) is primarily obtained from bacteria cultured in laboratory conditions. BEVs in human body fluids have b...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/s41596-019-0236-5
更新日期：2020-01-01 00:00:00
abstract：:MicroRNAs (miRNAs) are short, about 21 nucleotides in length, noncoding, regulatory RNA molecules representing a new layer in post-transcriptional regulation of gene expression. Intensive miRNA research has necessitated the development of effective miRNA detection methods such as northern analyses, quantitative real-t...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2007.528
更新日期：2008-01-01 00:00:00
abstract：:The combination of ChIP-seq and transcriptome analysis is a compelling approach to unravel the regulation of gene expression. Several recently published methods combine transcription factor (TF) binding and gene expression for target prediction, but few of them provide an efficient software package for the community. ...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2013.150
更新日期：2013-12-01 00:00:00
abstract：:Small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS) are techniques used to extract structural parameters and determine the overall structures and shapes of biological macromolecules, complexes and assemblies in solution. The scattering intensities measured from a sample contain contributions ...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2016.113
更新日期：2016-11-01 00:00:00
abstract：:The lung epithelium is derived from the endodermal germ layer, which undergoes a complex series of endoderm-mesoderm-mediated signaling events to generate the final arborized network of conducting airways (bronchi, bronchioles) and gas-exchanging units (alveoli). These stages include endoderm induction, anterior-poste...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/s41596-018-0104-8
更新日期：2019-02-01 00:00:00
abstract：:The elucidation of protein-protein interaction networks is a crucial task in the postgenomic era. In this protocol, we describe our approach to discover protein-protein interactions using the surface plasmon resonance technique coupled to mass spectrometry (MS). A peptide or a protein is immobilized on a sensor chip a...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2009.84
更新日期：2009-01-01 00:00:00
abstract：:The scototaxis (dark/light preference) protocol is a behavioral model for fish that is being validated to assess the antianxiety effects of pharmacological agents and the behavioral effects of toxic substances, and to investigate the (epi)genetic bases of anxiety-related behavior. Briefly, a fish is placed in a centra...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2009.225
更新日期：2010-02-01 00:00:00
abstract：:As proteins within cells are spatially organized according to their role, knowledge about protein localization gives insight into protein function. Here, we describe the LOPIT technique (localization of organelle proteins by isotope tagging) developed for the simultaneous and confident determination of the steady-stat...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2006.254
更新日期：2006-01-01 00:00:00
abstract：:This protocol describes a method combining phase-contrast and fluorescence microscopy, Raman spectroscopy and optical tweezers to characterize the germination of single bacterial spores. The characterization consists of the following steps: (i) loading heat-activated dormant spores into a temperature-controlled micros...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2011.307
更新日期：2011-05-01 00:00:00
abstract：:Mass spectrometry-based phosphoproteomic analysis is a powerful method for gaining a global, unbiased understanding of cellular signaling. Its accuracy and comprehensiveness stands or falls with the quality and choice of the applied phosphopeptide prefractionation strategy. This protocol covers a powerful but simple a...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2015.134
更新日期：2016-01-01 00:00:00
abstract：:Metabolic labeling of glycans with a bioorthogonal chemical reporter such as the azide enables their visualization in cells and organisms as well as the enrichment of specific glycoprotein types for proteomic analysis. This process involves two steps. Azido sugars are fed to cells or organisms and integrated by the gl...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2007.422
更新日期：2007-01-01 00:00:00
abstract：:Freestanding plasmonic nanoparticle (NP) superlattice sheets are novel 2D nanomaterials with tailorable properties that enable their use for broad applications in sensing, anticounterfeit measures, ionic gating, nanophotonics and flat lenses. We recently developed a robust, yet general, two-step drying-mediated approa...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/s41596-019-0200-4
更新日期：2019-09-01 00:00:00
abstract：:The ability to rapidly generate large panels of antigen-specific human antibodies in a rodent would enable the efficient discovery of novel therapeutically useful antibodies. We have developed a system wherein human antigen-specific antibody-secreting plasmablasts can be enriched in vivo, in a severe combined immunode...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2014.104
更新日期：2014-07-01 00:00:00
abstract：:Hypochlorous acid (HOCl) is a critical member of the reactive oxygen species (ROS) produced by immune cells to fight infections. On the other hand, HOCl in homeostasis causes oxidative damage to biomolecules and is linked to many diseases, including inflammatory, neurodegenerative, and cardiovascular diseases. Herein,...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/s41596-018-0041-6
更新日期：2018-10-01 00:00:00
abstract：:The goal of many current studies of neovascularization is to define the phenotype of vascular cell populations of different origins and to determine how such cells promote assembly of vascular channel. Here, we describe a protocol to immunophenotype vascular cells by high-resolution imaging and by fluorescence-activat...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2007.537
更新日期：2008-01-01 00:00:00
abstract：:The mouse embryo hindbrain is a robust and adaptable model for studying sprouting angiogenesis. It permits the spatiotemporal analysis of organ vascularization in normal mice and in mouse strains with genetic mutations that result in late embryonic or perinatal lethality. Unlike postnatal models such as retinal angiog...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2013.015
更新日期：2013-02-01 00:00:00
abstract：:In this report we describe the development of a standardized three-dimensional (3D) system of the human oral mucosa based on an immortalized human oral keratinocyte cell line (OKF6/TERT-2). The procedure takes approximately 2-3 weeks to complete and includes three main stages: preparation of collagen-embedded fibrobla...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2006.323
更新日期：2006-01-01 00:00:00
abstract：:The immune system operates at the scale of the whole organism in mammals. We currently lack experimental approaches to systematically track and study organism-wide molecular processes in mice. Here we describe an integrated toolkit for measuring gene expression in whole tissues, 3-prime mRNA extension sequencing, that...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/s41596-019-0291-y
更新日期：2020-04-01 00:00:00
abstract：:The analysis of large microbiome data sets holds great promise for the delineation of the biological and metabolic functioning of living organisms and their role in the environment. In the midst of this genomic puzzle, viruses, especially those that infect microbial communities, represent a major reservoir of genetic ...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2017.063
更新日期：2017-08-01 00:00:00
abstract：:Targeted nucleases, including zinc-finger nucleases (ZFNs), transcription activator-like (TAL) effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9), have provided researchers with the ability to manipulate nearly any genomic sequence in hu...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2015.117
更新日期：2015-11-01 00:00:00
abstract：:Cell migration is a key feature of virtually every biological process, and it can be studied in a variety of ways. Here we outline a protocol for the in vitro study of cell migration using a ring barrier-based assay. A 'barrier' is inserted in the culture chamber, which prevents cells from entering a defined area. Cel...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2015.056
更新日期：2015-06-01 00:00:00
