A Novel Combined Microstrip Resonator/Nanospray Ionization Source for Microwave-Assisted Trypsin Digestion of Proteins

Enzymatic digestion of proteins is a critical step in bottom-up and middle-down proteomics. Here, we demonstrate a method for decreasing the time required for proteolytic digestion of proteins from multiple hours to minutes by using an in-line microstrip cavity for programmed microwave heating. When a nanospray emitter tip, containing a digestion sample, is exposed to a region of highly focused microwave field, the rate of proteolytic digestion is enhanced and the time required for digestion greatly decreased. The design is advantageous for mass spectrometry because the solution-based digestion can then be directly sprayed from a nanoelectrospray tip emitter, decreasing sample transfer loss and allowing the system to be used in a flow-through proteolytic workflow. Microwave-assisted digestion using this method is evaluated against standard overnight digestion protocols using a variety of proteins, evaluating sequence coverage and observed peptide location, digestion rate, and overall efficacy. The influence of applied microwave power is investigated, and enzymatic kinetic parameters are evaluated to estimate temperature within the microreactor. Finally, the modulation of the proteolytic digestion of proteins based upon the modulation of applied microwave power is demonstrated on a time scale of seconds in a flow-through system.