Application of pressurized solvents for ultrafast trypsin hydrolysis in proteomics: proteomics on the fly

A new method for rapid proteolytic digestion of proteins under high pressure that uses pressure cycling technology in the range of 5-35 kpsi was demonstrated for proteomic analysis. Successful in-solution digestions of single proteins and complex protein mixtures were achieved in 60 s and then analyzed by reversed phase liquid chromatography-electrospray ionization ion trap-mass spectrometry. Method performance in terms of the number of Shewanella oneidensis peptides and proteins identified in a shotgun approach was evaluated relative to a traditional "overnight" sample preparation method. Advantages of the new method include greatly simplified sample processing, easy implementation, no cross contamination among samples, and cost effectiveness.