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.