Aims: 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. Materials & methods: 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. Results: 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. Conclusion: Promega Rapid-Digestion Trypsin is the best-performing next-generation trypsin, surpassing an overnight tryptic digestion.
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