Quantitative proteomics using stable isotope labeling with amino acids in cell culture

H. C. Harsha, Henrik Molina, Akhilesh Pandey

Research output: Contribution to journalArticle

144 Citations (Scopus)

Abstract

Stable isotope labeling with amino acids in cell culture (SILAC) is a simple in vivo labeling strategy for mass spectrometry-based quantitative proteomics. It relies on the metabolic incorporation of nonradioactive heavy isotopic forms of amino acids into cellular proteins, which can be readily distinguished in a mass spectrometer. As the samples are mixed before processing in the SILAC methodology, the sample handling errors are also minimized. Here we present protocols for using SILAC in the following types of experiments: (i) studying inducible protein complexes, (ii) identification of Tyr kinase substrates, (iii) differential membrane proteomics and (iv) studying temporal dynamics using SILAC 5-plexing. Although the overall time is largely dependent on the rate of cell growth and various sample processing steps employed, a typical SILAC experiment from start to finish, including data analysis, should take anywhere between 20 and 25 d.

Original languageEnglish (US)
Pages (from-to)505-516
Number of pages12
JournalNature Protocols
Volume3
Issue number3
DOIs
StatePublished - Mar 1 2008
Externally publishedYes

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Isotope Labeling
Cell culture
Isotopes
Proteomics
Labeling
Cell Culture Techniques
Amino Acids
Cell growth
Mass spectrometers
Processing
Mass spectrometry
Mass Spectrometry
Proteins
Phosphotransferases
Experiments
Membranes
Substrates
Growth

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Quantitative proteomics using stable isotope labeling with amino acids in cell culture. / Harsha, H. C.; Molina, Henrik; Pandey, Akhilesh.

In: Nature Protocols, Vol. 3, No. 3, 01.03.2008, p. 505-516.

Research output: Contribution to journalArticle

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