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.