Protein quantification through incorporation of stable isotopes has become a central technology in modern proteomics research. Quantification by iTRAQ (isobaric tags for relative and absolute quantification) is one of several techniques that our lab utilizes to monitor relative changes in protein and PTM abundance across perturbed biological systems1. Specifically, iTRAQ-based quantification facilitates the comparative analysis of peptide and proteins in a variety of settings including comparison of normal, disease or drug treated states.
Advantages of iTRAQ-based quantification include the following:
- iTRAQ is a chemical labeling method that can be implemented after cell or tissue lysis. Therefore, iTRAQ can be used to quantify proteins in any biological system and is not limited to only those systems that can accommodate incorporation of stable isotopes during cell culture.
- The iTRAQ method enables multiplexing (comparing) of up to four different samples in one mass spectrometry-based experiment.
- The nature of this technique results in concomitant increase in precursor ion intensity and reduction in sample complexity relative to multiplexed, precursor-based quantitative methods.
How does iTRAQ-based quantification work?
The iTRAQ technology utilizes isobaric reagents to label the primary amines of peptides and proteins. Our lab currently utilizes up to four iTRAQ reagents for quantification that each consist of an N-methyl piperazine reporter group with mass=114, 115, 116, or 117, a balance group with mass=31, 30, 29, or 28, and an N-hydroxy succinimide ester group that is reactive with the primary amines of peptides (Figure 1). The balance groups present in each of the iTRAQ reagents function to make the labeled peptides from each sample isobaric (same mass). Quantification is facilitated through analysis of reporter groups that are generated upon fragmentation in the mass spectrometer (discussed in more detail below).
Figure 1 iTRAQ reagent structure
Figure 2 illustrates the key steps in our iTRAQ workflow. 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. The sequence of a peptide is determined from the product ions that are generated from cleavage about peptide interresidue bonds. 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.
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. Abstract