Creative Proteomics offers iTRAQ protein quantification service suited for unbiased untargeted biomarker discovery. Relative quantification of proteins for biomarker discovery in complex mixtures by mass spectrometry can easily and quickly be achieved using iTRAQ technology. iTRAQ is ideally suited for comparing normal, diseased, and drug-treated samples, time course studies, biological replicates and provides relative quantitation.
iTRAQ technology utilizes isobaric reagents to label the primary amines of peptides and proteins. The iTRAQ reagents usually consist of an N-methyl piperazine reporter group, a balance group, and an N-hydroxy succinimide ester group that is reactive with the primary amines of peptides. The balance groups present in each of the iTRAQ reagents function to make the labeled peptides from each sample isobaric and the quantification is facilitated through analysis of reporter groups that are generated upon fragmentation in the mass spectrometer. There are currently two mainly used reagents: 4-plex and 8-plex, which can be used to label all peptides from different samples/treatments. These samples are then pooled and usually fractionated by nano liquid chromatography and analyzed by tandem mass spectrometry (MS/MS).
iTRAQ workflow (4-plex) is shown above. Samples to be quantified are prepared under various treatment conditions followed by cell lysis to extract proteins. After using a standard protein assay to estimate the protein concentration of each sample, proteins are digested using an enzyme, such as trypsin, to generate proteolytic peptides. Each peptide digest is labeled with a different iTRAQ reagent and then the labeled digests are combined into one sample mixture. The combined peptide mixture is analyzed by LC-MS/MS for both identification and quantification.
A database search is then performed using the fragmentation data to identify the labeled peptides and hence the corresponding proteins. The fragmentation of the attached tag generates a low molecular mass reporter ion that can be used to relatively quantify the peptides and the proteins from which they originated.
Advantage of our iTRAQ technique:
In 2D LC mass spectrometry with iTRAQ all proteins are analysed, identified and quantified in one experiment, whereas in gels every spot must be imaged, its intensity measured, and then the spot cut out and identified individually.
Note: iTRAQ analysis cannot be performed on unknown genome.
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