The drive toward more sensitive LC-MS assays has resulted in long, complex methods. We assessed next-generation trypsins to identify a suitable candidate to integrate into protein LC-MS method development strategies, to simplify methods and increase throughput. The performance of commercially available next-generation trypsins was assessed based on the digestion of protein standards in buffer and complex matrix by LC-high-resolution MS. The performance of all next-generation trypsins assessed exceeded that of an overnight tryptic digest in a fraction of the time. Performing reduction and alkylation prior to digestion with heat-stable trypsins may be beneficial and should be investigated. Promega Rapid-Digestion Trypsin is the best-performing next-generation trypsin, surpassing an overnight tryptic digestion.
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