TY - JOUR
T1 - Use of stable isotope labeling by amino acids in cell culture as a spike-in standard in quantitative proteomics
AU - Geiger, Tamar
AU - Wisniewski, Jacek R.
AU - Cox, Juergen
AU - Zanivan, Sara
AU - Kruger, Marcus
AU - Ishihama, Yasushi
AU - Mann, Matthias
N1 - Funding Information:
acknowleDGMents We thank P. Roerth for suggesting the term ‘spike-in SILAC’. T.G. is supported by the Humboldt Foundation. This project was supported by the European Commission’s 7th Framework Program PROteomics SPECification in Time and Space (PROSPECTS, HEALTH-F4-2008-021,648).
PY - 2011/2
Y1 - 2011/2
N2 - Mass spectrometry (MS)-based proteomics is increasingly applied in a quantitative format, often based on labeling of samples with stable isotopes that are introduced chemically or metabolically. In the stable isotope labeling by amino acids in cell culture (SILAC) method, two cell populations are cultured in the presence of heavy or light amino acids (typically lysine and/or arginine), one of them is subjected to a perturbation, and then both are combined and processed together. In this study, we describe a different approach-the use of SILAC as an internal or 'spike-in' standard-wherein SILAC is only used to produce heavy labeled reference proteins or proteomes. These are added to the proteomes under investigation after cell lysis and before protein digestion. The actual experiment is therefore completely decoupled from the labeling procedure. Spike-in SILAC is very economical, robust and in principle applicable to all cell- or tissue-based proteomic analyses. Applications range from absolute quantification of single proteins to the quantification of whole proteomes. Spike-in SILAC is especially advantageous when analyzing the proteomes of whole tissues or organisms. The protocol describes the quantitative analysis of a tissue sample relative to super-SILAC spike-in, a mixture of five SILAC-labeled cell lines that accurately represents the tissue. It includes the selection and preparation of the spike-in SILAC standard, the sample preparation procedure, and analysis and evaluation of the results.
AB - Mass spectrometry (MS)-based proteomics is increasingly applied in a quantitative format, often based on labeling of samples with stable isotopes that are introduced chemically or metabolically. In the stable isotope labeling by amino acids in cell culture (SILAC) method, two cell populations are cultured in the presence of heavy or light amino acids (typically lysine and/or arginine), one of them is subjected to a perturbation, and then both are combined and processed together. In this study, we describe a different approach-the use of SILAC as an internal or 'spike-in' standard-wherein SILAC is only used to produce heavy labeled reference proteins or proteomes. These are added to the proteomes under investigation after cell lysis and before protein digestion. The actual experiment is therefore completely decoupled from the labeling procedure. Spike-in SILAC is very economical, robust and in principle applicable to all cell- or tissue-based proteomic analyses. Applications range from absolute quantification of single proteins to the quantification of whole proteomes. Spike-in SILAC is especially advantageous when analyzing the proteomes of whole tissues or organisms. The protocol describes the quantitative analysis of a tissue sample relative to super-SILAC spike-in, a mixture of five SILAC-labeled cell lines that accurately represents the tissue. It includes the selection and preparation of the spike-in SILAC standard, the sample preparation procedure, and analysis and evaluation of the results.
UR - http://www.scopus.com/inward/record.url?scp=79551663189&partnerID=8YFLogxK
U2 - 10.1038/nprot.2010.192
DO - 10.1038/nprot.2010.192
M3 - מאמר
AN - SCOPUS:79551663189
VL - 6
SP - 147
EP - 157
JO - Nature Protocols
JF - Nature Protocols
SN - 1754-2189
IS - 2
ER -