aTRAQ™ Kit for Amino Acid Analysis of Hydrolysates

For research use only. Not for use in diagnostic procedures.

The aTRAQ Reagent Kits for amino acid analysis of hydrolysates enables you to quantitate 20 amino acids from protein hydrolysate samples more than three-times faster than conventional amino acid analyzers. The kits are designed for use with LC/MS/MS systems.

Analysis is unaffected by chromatographic shifts due to column aging.
Reagents and derivatized samples are stable.
Internal standards for every amino acid provide confident identification and superior accuracy and precision.
High throughput with an analysis time of 18 minute per sample
Small sample requirement, 1 µg
Wide linear dynamic range of greater than four orders of magnitude with an LLOQ and ULOQ of 1 nmole, respectively on a 3200 QTRAP® System
Complete kit includes the required reagents, buffers, standards, controls, and column.
Extended ability to add custom amines, amino acids, and small peptides and create corresponding internal standards
Optional ability to automate pipetting steps

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Overview

Faster performance, better accuracy.

The aTRAQ Reagents provide a consistent mass tag to aid in 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 the molecular weight of the sample and standard of 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.

Hydrolysate labeling reaction is buffered at a basic pH. The samples are labeled with the aTRAQ Reagent Δ8, allowed to react for 30 mins, dried, and mixed with the internal standards pre-labeled with aTRAQ Reagent Δ0. The two aTRAQ Reagents have identical structures, but the number of isotopic sites vary. This mixture is then injected for LC/MS/MS analysis using either a triple quadrupole 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 (see 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, an amino acid differentially-labeled with separate tags cannot be distinguished by HPLC due to the identical retention times for differentially-tagged amino acids. 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 labeled amino acid masses and 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 (see Figure 3).

Figure 3: General scheme.

Warranty: Standard parts and labor warranty for one year starting from the completion of instrument commissioning.

Kits
Part Number	Kit Name/ Description
4442675	aTRAQ Starter Kit Hydrolysates 50 Assay Includes all the consumable materials except mobile phases (water and methanol) and the chromatography column (4374841) needed to run 50 aTRAQ reagent– labeled samples using the Amino Acid Analysis Hydrolysate Sample Protocol. Also includes the following: aTRAQ Reagent Kit Hydrolysates 50 Assay (4442677) Contains sufficient materials to label 50 hydrolysate samples with aTRAQ Reagent, the aTRAQ Internal Standard (4442688) with a Certificate of Analysis, and the Quick Reference Card. aTRAQ Unlabeled Standard (4442687) A mixture of unlabeled amino acids used for testing the aTRAQ Kit and protocol. Enough to do 10 tests. aTRAQTM Internal Standard (4442688) Amino acid standards labeled with aTRAQ™ reagent Δ0. This is added to each aTRAQ reagent Δ8-labeled sample to be used as the internal standard. Includes a Certificate of Analysis with the concentrations of each amino acid in the standard, enough internal standard for 50 samples.
4374841	Amino Acid Analyzer C18 Column Reverse Phase (5 µm, 4.6mm x 150mm) Column
4442677	aTRAQ Kit Hydrolysates 50 Assay Contains sufficient materials to label 50 hydrolysate samples with aTRAQ Reagent, the aTRAQ Internal Standard (4442688), a certificate of analysis, and a quick reference card.
4442678	aTRAQ Kit Hydrolysate 200 Assay Contains sufficient materials to label 200 hydrolysate samples with aTRAQ Reagent, the aTRAQ Internal Standard (4442688), a certificate of analysis, and a quick reference card.
4442679	aTRAQ Standards Set Hydrolysates Includes the following standards: aTRAQ Internal Standard (4442688) Amino acid standards labeled with aTRAQ Reagent Δ0. This is added to each sampled labeled with aTRAQ Reagent Δ8 to be used as the internal standard. Includes a certificate of analysis with the concentrations of each amino acid in the standard (enough for 50 samples). aTRAQ Unlabeled Standard (4442687) A mixture of unlabeled amino acids used for testing the aTRAQ Kit and protocol. Enough for 10 tests.

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