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A proteomic method for the analysis of changes in protein concentrations in response to systemic perturbations using metabolic incorporation of stable isotopes and mass spectrometry

Gustavsson, Niklas LU ; Greber, B; Kreitler, T; Himmelbauer, H; Lehrach, H and Gobom, J (2005) In Proteomics 5(14). p.3563-3570
Abstract
While several techniques exist for assessing quantitative differences among proteomes representing different cell states, methods for assessing how these differences are mediated are largely missing. We present a method that allows one to differentiate between cellular processes, such as protein synthesis, degradation and PTMs which affect protein concentrations. An induced systemic perturbation of a cell culture was coupled to a replacement of the growth medium to one highly enriched in the stable isotope N-15. The relative abundance of the N-15- and N-14-enriched forms of proteins, isolated from cell cultures harvested at time points following the onset of the perturbation, were determined by MS. Alterations in protein synthesis and... (More)
While several techniques exist for assessing quantitative differences among proteomes representing different cell states, methods for assessing how these differences are mediated are largely missing. We present a method that allows one to differentiate between cellular processes, such as protein synthesis, degradation and PTMs which affect protein concentrations. An induced systemic perturbation of a cell culture was coupled to a replacement of the growth medium to one highly enriched in the stable isotope N-15. The relative abundance of the N-15- and N-14-enriched forms of proteins, isolated from cell cultures harvested at time points following the onset of the perturbation, were determined by MS. Alterations in protein synthesis and degradation were quantified by comparing proteins isolated from perturbed and unperturbed cultures, respectively. The method was evaluated by subjecting HeLa cells to heat stress. As expected, a number of known heat shock proteins (Hsp) increased in concentration during heat stress. For Hsp27, increased de novo synthesis accounted for the concentration increase, while for Hsp70, decreased degradation accounted for the increase. A protein that was detected only after prolonged heat stress, vimentin, was not primarily synthesized de novo, but appeared rather as a result of PTM. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Proteomics
volume
5
issue
14
pages
3563 - 3570
publisher
John Wiley & Sons
external identifiers
  • wos:000232247600001
  • scopus:25844518423
ISSN
1615-9861
DOI
10.1002/pmic.200401193
language
English
LU publication?
yes
id
5908d5f8-a61d-4dc5-9c98-0c2832deebdc (old id 152457)
date added to LUP
2007-07-05 10:34:41
date last changed
2017-08-06 03:31:56
@article{5908d5f8-a61d-4dc5-9c98-0c2832deebdc,
  abstract     = {While several techniques exist for assessing quantitative differences among proteomes representing different cell states, methods for assessing how these differences are mediated are largely missing. We present a method that allows one to differentiate between cellular processes, such as protein synthesis, degradation and PTMs which affect protein concentrations. An induced systemic perturbation of a cell culture was coupled to a replacement of the growth medium to one highly enriched in the stable isotope N-15. The relative abundance of the N-15- and N-14-enriched forms of proteins, isolated from cell cultures harvested at time points following the onset of the perturbation, were determined by MS. Alterations in protein synthesis and degradation were quantified by comparing proteins isolated from perturbed and unperturbed cultures, respectively. The method was evaluated by subjecting HeLa cells to heat stress. As expected, a number of known heat shock proteins (Hsp) increased in concentration during heat stress. For Hsp27, increased de novo synthesis accounted for the concentration increase, while for Hsp70, decreased degradation accounted for the increase. A protein that was detected only after prolonged heat stress, vimentin, was not primarily synthesized de novo, but appeared rather as a result of PTM.},
  author       = {Gustavsson, Niklas and Greber, B and Kreitler, T and Himmelbauer, H and Lehrach, H and Gobom, J},
  issn         = {1615-9861},
  language     = {eng},
  number       = {14},
  pages        = {3563--3570},
  publisher    = {John Wiley & Sons},
  series       = {Proteomics},
  title        = {A proteomic method for the analysis of changes in protein concentrations in response to systemic perturbations using metabolic incorporation of stable isotopes and mass spectrometry},
  url          = {http://dx.doi.org/10.1002/pmic.200401193},
  volume       = {5},
  year         = {2005},
}