Ultrasonic-based protein quantitation by (18) O-labeling: optimization and comparison between different procedures

Herein we report results regarding the optimization and comparison between different ultrasonic‐based procedures for protein quantitation by the direct ¹⁸O‐labeling approach. The labeling procedure was evaluated using different proteins, different ultrasonic devices and different reaction times: from 30 s to 10 min with the ultrasonic probe and from 30 s to 30 min with the sonoreactor. Variables such as the enzyme‐to‐protein ratio and protein concentration were also assessed. The results show that it is possible to accelerate the labeling reaction from 12 h to only 15 min with the sonoreactor without compromising the labeling efficiency. A larger variation in the double labeling yield was obtained among the different peptides, but the values for the smaller peptides are similar to the ones achieved with the classic methodology. These findings were further confirmed by labeling a complex protein mixture from human plasma. It was also found that the labeling reaction is affected by the sample concentration, even when performed with the classic overnight procedure. Copyright © 2010 John Wiley & Sons, Ltd.