Absolute quantitation of isoforms of post-translationally modified proteins in transgenic organism.
Mol Cell Proteomics
; 11(8): 272-85, 2012 Aug.
Article
en En
| MEDLINE
| ID: mdl-22442259
ABSTRACT
Post-translational modification isoforms of a protein are known to play versatile biological functions in diverse cellular processes. To measure the molar amount of each post-translational modification isoform (P(isf)) of a target protein present in the total protein extract using mass spectrometry, a quantitative proteomic protocol, absolute quantitation of isoforms of post-translationally modified proteins (AQUIP), was developed. A recombinant ERF110 gene overexpression transgenic Arabidopsis plant was used as the model organism for demonstration of the proof of concept. Both Ser-62-independent (14)N-coded synthetic peptide standards and (15)N-coded ERF110 protein standard isolated from the heavy nitrogen-labeled transgenic plants were employed simultaneously to determine the concentration of all isoforms (T(isf)) of ERF110 in the whole plant cell lysate, whereas a pair of Ser-62-dependent synthetic peptide standards were used to quantitate the Ser-62 phosphosite occupancy (R(aqu)). The P(isf) was finally determined by integrating the two empirically measured variables using the following equation P(isf) = T(isf) · R(aqu). The absolute amount of Ser-62-phosphorylated isoform of ERF110 determined using AQUIP was substantiated with a stable isotope labeling in Arabidopsis-based relative and accurate quantitative proteomic approach. The biological role of the Ser-62-phosphorylated isoform was demonstrated in transgenic plants.
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Procesamiento Proteico-Postraduccional
/
Plantas Modificadas Genéticamente
/
Arabidopsis
/
Proteínas de Arabidopsis
Idioma:
En
Revista:
Mol Cell Proteomics
Asunto de la revista:
BIOLOGIA MOLECULAR
/
BIOQUIMICA
Año:
2012
Tipo del documento:
Article
País de afiliación:
China