iTRAQ

“Untargeted Biomarker Discovery using iTRAQ Relative Quantitation"
Our iTRAQ protein quantification service is ideally 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.

 

Applied Biosystems iTRAQ isobaric affinity labels allow for multiplexing up to 4 or 8 samples (4-Plex or 8-Plex) per experiment. After tryptic digestion, multiple peptides are labeled, including peptides with post translational modifications. Quantitation is achieved by comparison of the peak areas and resultant peak ratios for either four MS/MS reporter ions, which range from 114 to 117 Da or eight MS/MS reporter ions, which range from 113-119 and 121 Da.

 

Our iTRAQ protein quantification workflow includes strong cation exchange prior to LC-MS/MS analysis on our Thermo Fisher LTQ Orbitrap Velos mass spectrometer. Please refer to the Applied Biosystems website for further information on iTRAQ.

 

Advantages of our iTRAQ technology include:

• Ideal for comparative studies: up to 4 or 8 labels can be used for multiplexing experiments
• Non-targeted approach for discovery studies
• Large dynamic range, high and low abundant proteins are identified
• Thousands of protein peptides can be identified and quantified in one analysis
• Post translational modifications can be detected
• Relative quantitation
• SOP-driven work flow
• Established Quality Control procedures

 

The key steps in our iTRAQ workflow are illustrated below. 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 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 on our Thermo Fisher LTQ Orbitrap Velos mass spectrometer for both identification and quantification. The relative quantity of a peptide among the treated samples is determined by comparing the intensities of reporter ion signals also present in the MS/MS scan.

iTraq

1. Ross PL, Huang YN, et al. (2004) "Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents". Mol. Cell. Proteomics 3: 1154–69.

MRM Proteomics – Protein Quantification and Biomarker Discovery by Multiple Reaction Monitoring