Amino acid residue specific stable isotope labeling for quantitative proteomics

Abstract

Various stable isotope labeling (SIL) techniques have recently emerged to improve the efficiency and accuracy of protein quantitation by mass spectrometry (MS). We have developed a mass-tagging strategy to incorporate stable isotope tagged amino acids into cellular proteins in a residue-specific manner during cell growth. In this study, we further extend this residue-specific SIL approach to the accurate quantitation of protein abundances in different cell populations. For proteins whose expression levels are the same in cells grown in the normal and labeled media, the relative areas of the normal (light) and labeled (heavy) isotopic peaks are linearly correlated with the cells mixing ratios. This approach was first used to determine the effect of the zinc-responsive transcription factor Zap1 on the yeast proteome. Ten protein spots from a PAGE gel were chosen randomly and their differential protein expression levels in wild-type and zap1delta cells were readily determined by the isotopic ratio. Methionine synthase (Met6) was identified to be up-regulated more than four times in the zap1delta mutant strain whereas the expression level of other nine proteins remained unchanged. Further, we applied this strategy to study the cellular response to radiation in human skin fibroblast cells. Analyzing one protein band randomly selected from SDS-PAGE, the expression level of a novel protein was found to increase two-fold in response to radiation whereas the expression level of a control protein remained unchanged. This strategy is generally applicable using any particular type of amino acid as the labeling precursors for accurate quantitation of protein relative abundances.

Analytical strategies for phosphoproteomics

“…Quantitation methods differ at the level in which the label is introduced into the sample. Some techniques introduce the label in viable cells in cell culture ( 14 N/ 15 N labeling [122] and stable isotope labeling by amino acids in cell culture, SILAC [123,124]). Others introduce the tag at the protein level (isotope-coded affinity tags, ICAT [125]) or peptide level (isobaric peptide tags for relative and absolute quantifi-cation, iTRAQ [126] or 18 O-labeling during tryptic digestion [127]), whereas yet others use a label-free strategy in which quantitative information is extracted from LC-MS (extracted ion current, XIC) or MS/MS data (protein abundance index, PAI) in sequential experiments [128][129][130].…”

Flightless I Homolog Negatively Modulates the TLR Pathway

“…We have previously shown that stable isotope-labeled amino acids can be naturally incorporated into cellular proteomes through cell culturing in a residue-specific manner (19). Subsequently, both our group and the group of Mann and colleagues (20,21) independently applied stable isotope labeling for quantitative protein profiling. In the current study, we combined stable isotope labeling and epitope tagging to allow the sensitive and selective identification of the MyD88-signaling complex formed in actual immune cells.…”

Differences in the secretome of cartilage explants and cultured chondrocytes unveiled by SILAC technology

“…Stable isotope labeling by amino acids in cell culture (SILAC) represents a simple and accurate method to explore expression proteomics in cell culture. [8][9][10][11] This methodology described by Ong et al 12 enables the identification of functional expression of proteins by administration to the culture medium of nonradioactive isotope labeled essential amino acids that are incorporated into all newly synthesized proteins. In our study, we have analyzed and compared the secreted proteome-profiling of cultured chondrocytes and cartilage explants by using a stable isotope labeling strategy in combination with 1D SDS-PAGE protein fractionation (shotgun), in-gel digestion, and LC/MS-MS analysis.…”