Showing papers by "Shao En Ong published in 2006"

A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC).

Abstract: Stable isotope labeling by amino acids in cell culture (SILAC) is a simple, robust, yet powerful approach in mass spectrometry (MS)-based quantitative proteomics. SILAC labels cellular proteomes through normal metabolic processes, incorporating non-radioactive, stable isotope-containing amino acids in newly synthesized proteins. Growth medium is prepared where natural ("light") amino acids are replaced by "heavy" SILAC amino acids. Cells grown in this medium incorporate the heavy amino acids after five cell doublings and SILAC amino acids have no effect on cell morphology or growth rates. When light and heavy cell populations are mixed, they remain distinguishable by MS, and protein abundances are determined from the relative MS signal intensities. SILAC provides accurate relative quantification without any chemical derivatization or manipulation and enables development of elegant functional assays in proteomics. In this protocol, we describe how to apply SILAC and the use of nano-scale liquid chromatography coupled to electrospray ionization mass spectrometry for protein identification and quantification. This procedure can be completed in 8 days.

Identifying and quantifying sites of protein methylation by heavy methyl SILAC.

Abstract: A new appreciation of protein methylation comes with the recent discovery of demethylases, now placing methylation in the realm of a transient, reversible modification. Classical approaches to study methylation are laborious and involve radioactive, in vitro, enzyme-substrate labeling experiments with purified proteins. Mass spectrometry–based proteomics allows the unbiased analysis of complex protein mixtures and is increasingly applied to the study of post-translational modifications. However, it is particularly challenging to study methylation by proteomics because of the number of residues affected and the degree of methylation that can occur. Heavy methyl SILAC is a metabolic labeling strategy that harnesses the cell's machinery to convert a nonradioactive, stable isotope labeled version of methionine into the ‘heavy’ biological methyl donor S-adenosylmethionine. Cells incorporate this ‘heavy’ methyl group throughout their methylated substrates. This technique increases confidence in identifying and quantifying of sites of protein methylation. Keywords: SILAC; mass spectrometry; proteomics; metabolic labeling; methylation; quantification; post-translational modification

Chapter 54 – Stable Isotope Labeling by Amino Acids in Cell Culture for Quantitative Proteomics

Abstract: Publisher Summary Analysis by combined liquid chromatographic separation and mass spectrometry (LC-MS) is rapidly becoming the most popular and effective approach for large-scale protein identification. Quantitative methods in mass spectrometry (MS) rely largely on the principle of stable isotope labeling (SIL). The protein samples to be compared are derivatized separately with the two forms of ICAT, digested, and purified over an avidin column to enrich ICAT-labeled peptides for subsequent mass spectrometric analyses. Cells from both samples are mixed 1 to 1 and lyzed. Proteins extracted from the mixture are then analyzed by standard MS techniques. Many muscle-specific proteins, such as myosin, were found to be highly upregulated while other proteins, such as histones, were found at similar levels in both samples. SILAC-labeled cells are used to generate separate pools of proteins or protein complexes that are distinct to affinity bait. Cells may be differentially treated with a drug or growth factor; alternatively, cells may express the wild-type or mutant form of a component of a protein complex.