Direct analysis of biotransformations with mass spectrometry-DiBT-MS.

Knox, Ruth; Smith, Rachel; Kempa, Emily E.; Spiess, Reynard; Schnepel, Christian; Turner, Nicholas J.; Flitsch, Sabine L.; Barran, Perdita E.

Abstract

The development and analysis of engineered enzymes is greatly assisted by the use of high-throughput screening to quickly determine the efficacy of biotransformations under various conditions. Ambient ionization, particularly desorption electrospray ionization (DESI), coupled to high-resolution mass spectrometry has the advantages of minimal requirements for sample preparation before analysis, which renders it suitable for high-throughput screening, in which the accurate mass and potentially the tandem mass spectrometry (MS) fingerprint for any given product can be used for identification. We present a protocol that permits the application of this method in routine biotechnology and chemical biology laboratories that are using engineered enzymes (such as imine reductases and carboxylic acid reductases, mentioned herein) to produce target compounds from substrates (quinoline moieties and phenyl(piperazinyl) moieties, respectively). Through the use of DESI’s MS imaging capabilities, reaction monitoring can be easily visualized via imaging of selected substrate or product ions in a convenient, user-friendly workflow. We describe here how DESI-MS can be used to directly analyze the activity of biotransformations from crude cell lysate, which we term ‘DiBT-MS’. The DiBT-MS method presented here is 10–1,000 times as fast as liquid chromatography-MS, with the full procedure for 96 samples taking ~2 h and consuming far less solvent and sample. Also demonstrated in this protocol is the impact of solvent spray composition on ionization efficiency of the target analyte, the benefits of a nylon membrane slide and the reusability of sample slides in multiple experiments.

Citation

Knox, R., Smith, R., Kempa, E. E., Spiess, R., Schnepel, C., Turner, N. J., Flitsch, S. L., & Barran, P. E. (online). Direct analysis of biotransformations with mass spectrometry-DiBT-MS. Nature Protocols, https://doi.org/10.1038/s41596-025-01161-9

• Journal Article Type: Article
• Acceptance Date: Feb 17, 2025
• Online Publication Date: Apr 21, 2025
• Deposit Date: Apr 30, 2025
• Journal: Nature Protocols
• Print ISSN: 1754-2189
• Electronic ISSN: 1750-2799
• Publisher: Nature Research
• Peer Reviewed: Peer Reviewed
• DOI: https://doi.org/10.1038/s41596-025-01161-9
• Public URL: https://durham-repository.worktribe.com/output/3806919