Label-free protein quantification after ultrafast digestion of complex proteomes using ultrasonic energy and immobilized-trypsin magnetic nanoparticles.

The ultimate high-throughput, high robustness and easy-to-handling sample treatment method for label-free shotgun proteomics is presented in this work. It is based on joining the effectiveness of immobilized trypsin at the nanoscale level with the latest technology to deliver ultrasonic energy. The new method can be used to reduce sample preparation time comprising the steps of reduction, alkylation and digestion time to just 15 min without compromising shotgun label-free protein quantification. It is demonstrated that trypsin immobilized at the nano-scale performs better than the commercially available counterpart macroparticles. Considering the current advances in (i) ultrasonic energy delivery that allows 96 samples to be treated at once in 30 min, and (ii) chromatography and mass spectrometry for shotgun proteomics, that allow to analyze complex proteomes in 5 min, we envision this methodology as the universal one to digest complex proteomes as it allows to profile quantitatively more than 200 samples per day.