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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
Author(s) -
Gustavsson Niklas,
Greber Boris,
Kreitler Thomas,
Himmelbauer Heinz,
Lehrach Hans,
Gobom Johan
Publication year - 2005
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200401193
Subject(s) - proteome , protein biosynthesis , hsp70 , heat shock protein , proteomics , vimentin , mass spectrometry , protein turnover , chemistry , isotope , hsp27 , quantitative proteomics , isobaric labeling , protein degradation , stable isotope ratio , cell culture , biochemistry , stable isotope labeling by amino acids in cell culture , biology , chromatography , gene , genetics , immunology , physics , immunohistochemistry , quantum mechanics
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 15 N. The relative abundance of the 15 N‐ and 14 N‐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.