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Relative Quan SILAC

Discovery-based relative quantification is an analytical approach that allows the scientist to determine relative protein abundance changes across a set of samples simultaneously and without the requirement for prior knowledge of the proteins involved.

To understand the functions of individual proteins and their place in complex biological systems, it is often necessary to measure changes in protein abundance relative to changes in the state of the system. These measurements have traditionally been performed using Western blot analyses. More recently, modern proteomics has evolved to include a variety of technologies for the routine quantitative analyses of both known and unknown targets. Discovery-based relative quantification is an analytical approach that allows the scientist to determine relative protein abundance changes across a set of samples simultaneously and without the requirement for prior knowledge of the proteins involved. Here we describe three commonly used techniques for relative quantitation of unknown protein/peptide targets using mass spectrometry.

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Workflow Overview for SILAC Quantitation


SILAC-based quantitation is a powerful and widely used technique for identifying and quantifying relative changes in complex protein samples. It can be applied to complex biomarker discovery and systems biology studies as well as to isolated proteins and protein complexes. As its name implies, SILAC involves labeling protein samples in vivo with a heavy-isotope-labeled form of an amino acid. Inclusion of the labeled amino acid in cell or tissue culture media results in replacement of the natural light amino acid with the heavy form in newly synthesized proteins. Cells grown under differing experimental conditions (and in heavy or light media) can be mixed and all subsequent processing steps can be performed on the combined sample. This serves to greatly reduce sample handling variability, resulting in more accurate quantitation. Additionally, the labeled peptides do not require any specific fragmentation modality, meaning the samples can be analyzed by CID, ETD, and/or HCD fragmentation, which leads to the identification of more peptides.











Literature Highlights


Original SILAC paper:

Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics

Ong SE, Blagoev B, et al.
Mol Cell Proteomics. 2002 May;1(5):376-86.


Super-SILAC allows classification of diffuse large B-cell lymphoma subtypes by their protein expression profiles

Deeb SJ, D'Souza R, et al.
Mol Cell Proteomics. 2012 Mar 21.


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