Faster performance, better accuracy: The aTRAQ Reagents provide a consistent mass tag to aid in the MS/MS fragmentation of amino acid samples and internal standards in a molecular weight region with reduced interference. The precision and accuracy of quantitation is improved through the use of an internal standard for each amino acid analyzed; an extra degree of confirmation is provided by comparing the retention time and molecular weight of the standard and sample for each amino acid. The mechanism of aTRAQ Reagent derivatization is shown below (Figure 1). Each aTRAQ Reagent tag consists of a reporter end and a reactive end that couples with the primary or secondary amine group of each amino acid. The reporter tag used for the sample has a molecular weight of 121 amu. The reporter tag used for the internal standard has a molecular weight of 113 amu.Figure 1: General labeling reaction.
The labeling protocol is compatible with a wide variety of physiological matrices including plasma, urine, cerebrospinal fluid, and tissue extracts. The sample (40 µL) is first mixed with a sulfosalicylic acid solution to precipitate proteins. The labeling reaction is buffered at a basic pH. The samples are labeled with aTRAQ Reagent Δ8 for 30 min, dried, and mixed with the internal standards pre-labeled with aTRAQ Reagent Δ0. The two aTRAQ Reagents have identical structures, except the number of isotopic sites vary. This mixture is analyzed by LC/MS/MS using either a triple quadrupole instrument or QTRAP System operating in mulitiple reaction monitoring (MRM) mode. HPLC separation is performed using a C18 column heated to 50 °C. The gradient, wash, and equilibration take a total of 18 min (Figure 2). The use of the Scheduled MRM Algorithm maximizes dwell time while monitoring of large numbers of MRM transitions, resulting in optimum data quality and reproducibility.
Figure 2: Representative chromatogram.
Using this method, the same amino acid differentially-labeled with separate tags cannot be distinguished by HPLC due to the identical retention times for the differentially-tagged amino acid. In the MS/MS mode, the tags fragment to yield reporter ions that have been encoded with isotopes to yield different masses. MRM transitions composed of the labeled amino acid masses and the reporter ion masses are used to differentiate the same amino acid from the sample and the internal standard; the ratio of these peak areas is used to calculate the concentration of the amino acid in the sample (Figure 3).
Figure 3: General scheme.