Preparation of heteroelement-incorporated and stable isotope-labeled protein standards for quantitative proteomics.
Methods Mol Biol
; 1156: 337-63, 2014.
Article
en En
| MEDLINE
| ID: mdl-24792000
ABSTRACT
A major obstacle for further development of quantitative proteomics is the lack of accurately quantified protein standards. The following protocol describes innovative methods for the production of stable isotope-labeled protein standards. Their production is achieved by cell-free protein synthesis, which enables simultaneous incorporation of selenomethionine and stable isotope-labeled amino acids. The selenium tag allows sensitive and accurate quantification by inductively coupled plasma mass spectrometry (ICP-MS). The stable isotope label allows internal standardization in mass spectrometry-based proteomics by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Both label types can be placed within a single protein RISQ standard (recombinant isotope-labeled and selenium quantified) or can be distributed over two types of related RSQ and RIQ standards for the same target protein (recombinant selenium quantified and recombinant isotope-labeled and quantified). The combination of cell-free synthesis as production method with ICP-MS and ESI-MS/MS as detection methods results in protein standards, which are quantified at an outstanding level of accuracy.
Texto completo:
1
Base de datos:
MEDLINE
Asunto principal:
Proteínas
/
Proteómica
/
Marcaje Isotópico
Idioma:
En
Revista:
Methods Mol Biol
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2014
Tipo del documento:
Article