English
 
User Manual Privacy Policy Disclaimer Contact us
  Advanced SearchBrowse

Item

ITEM ACTIONSEXPORT

Released

Journal Article

A mass spectrometry workflow for measuring protein turnover rates in vivo.

MPS-Authors
/persons/resource/persons131071

Mandad,  S.
Research Group of Bioanalytical Mass Spectrometry, MPI for Biophysical Chemistry, Max Planck Society;

/persons/resource/persons15947

Urlaub,  H.
Research Group of Bioanalytical Mass Spectrometry, MPI for Biophysical Chemistry, Max Planck Society;

External Ressource
No external resources are shared
Fulltext (public)
There are no public fulltexts stored in PuRe
Supplementary Material (public)

3177397_Suppl_1.pdf
(Supplementary material), 162KB

3177397_Suppl_2.pdf
(Supplementary material), 71KB

3177397_Suppl_3.zip
(Supplementary material), 28KB

3177397_Suppl_4.xlsx
(Supplementary material), 287KB

3177397_Suppl_5.xlsx
(Supplementary material), 315KB

Citation

Alevra, M., Mandad, S., Ischebeck, T., Urlaub, H., Rizzoli, S. O., & Fornasiero, E. F. (2019). A mass spectrometry workflow for measuring protein turnover rates in vivo. Nature Protocols, 14, 3333-3365. doi:10.1038/s41596-019-0222-y.


Cite as: http://hdl.handle.net/21.11116/0000-0005-3A8E-B
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.