Evaluation of a high-intensity focused ultrasound-immobilized trypsin digestion and 18O-labeling method for...

A new method that uses immobilized trypsin concomitant with ultrasonic irradiation results in ultrarapid digestion and more thorough (18)O labeling for quantitative protein comparisons. The method was reproducible and provided effective digestions within <1 min with lower amounts of enzyme, compared to traditional methods. This method was demonstrated for digestion of both simple and complex protein mixtures, including bovine serum albumin, a global proteome extract from the bacteria Shewanella oneidensis, and mouse plasma, as well as (18)O labeling of complex protein mixtures, validating this method for differential proteomic measurements. This approach is simple, reproducible, cost-effective, rapid, and well suited for automation.