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.