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Direct analysis of biotransformations with mass spectrometry—DiBT-MS

Nature Protocols (2025)Cite this article

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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.

Key points

  • Engineering enzymes to perform specific chemical transformations is still an iterative process that involves expressing mutant proteins and screening them for reactivity under different conditions. The screening process can be time consuming if both the protein and the products need to be enriched before analysis.

  • With desorption electrospray ionization (DESI) mass spectrometry, it is possible to analyze the reaction mixture directly via the accurate mass of the products without extensive sample preparation or work-up.

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Fig. 1: Overview of the DiBT-MS workflow.
Fig. 2: Experiments to demonstrate the reproducibility of the DiBT-MS workflow with nylon membrane slides.
Fig. 3: Major physical components and comparison of DESI sources used for this protocol: Prosolia (top), Waters Desi XS (middle) and Waters AutoDesi Source (bottom).
Fig. 4: DESI stage default starting positions for Prosolia, DESI XS and AutoDESI source; see the left-hand side for labels.
Fig. 5: Solvent optimization visualization of 6,7-dimethoxymethyl-3,4-dihydroisoquinoline and 6,7-dimethoxymethyl-1,2,3,4-tetrahydroisoquinoline shown in HD Imaging.
Fig. 6: DiBT-MS of standards that are involved in CAR biotransformation reactions, namely the reactants and expected products in the absence of enzyme.
Fig. 7: Optimization of CAR biotransformation through DiBT-MS collaboration with normalized intensity for visualization.

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Data availability

Raw data files pertaining to Figs. 2, 5, 6 and 7 and the Excel file used to calculate reaction rates from DiBT data are available to download from Figshare https://doi.org/10.6084/m9.figshare.27931572.v1.

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Acknowledgements

The authors would like to acknowledge Waters Corporation, and in particular E. Jones, for their support of our MS development. We acknowledge the Engineering and Physical Sciences Research Council (EPSRC), the Biotechnology and Biological Sciences Research Council (BBSRC) and AstraZeneca plc for funding under the Prosperity Partnership EP/S005226/1. We acknowledge the support of EPSRC through the strategic equipment award EP/T019328/1 and BBSRC for funding the Centre for Synthetic Biology of Fine and Speciality Chemicals BB/M017702/1. R. Smith acknowledges Bristol Myers Squib and the Department of Chemistry for funding her PhD studentship.

Author information

Author notes

  1. These authors contributed equally: Ruth Knox, Rachel Smith.

Authors and Affiliations

  1. Michael Barber Centre for Collaborative Mass Spectrometry, Department of Chemistry, Manchester Institute of Biotechnology, University of Manchester, Manchester, UK

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

  2. Early Chemical Development, Pharmaceutical Sciences, R&D, AstraZeneca, Macclesfield, UK

    Emily E. Kempa

  3. Department of Chemistry, Durham University, Durham, UK

    Christian Schnepel

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Contributions

R.K. and R. Smith developed the sprayer head solvent optimization methods, acquired all spectra and drafted the manuscript. E.E.K. contributed to method development while studying at The University of Manchester and paper drafting while under employment by AstraZeneca plc. C.S. prepared CAR enzymes, and P.E.B., R. Smith, R. Spiess, C.S., S.L.F. and N.J.T. contributed to method development and paper revisions.

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Correspondence to Perdita E. Barran.

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E.E.K. is an employee of AstraZeneca and owns or has the option to own stocks in this company.

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Nature Protocols thanks Manfred Reetz, László Csaba Bencze and the other, anonymous, reviewer for their contribution to the peer review of this work.

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Key references

Yan, C. et al. J. Am. Chem. Soc. 139, 1408–1411 (2017): https://doi.org/10.1021/jacs.6b12165

Kempa, E. E. et al. JACS Au 1, 508–516 (2021): https://doi.org/10.1021/jacsau.1c00027

Extended data

Extended Data Fig. 1 Generating slide area and optimizing visualization parameters within HD Imaging.

a, Screenshots of the import and slide selection process (described in protocol Steps 14–17) for HD Imaging. A photo of the nylon slide can be imported to the acquisition computer by pen drive or email. 1. Select ‘DESI-MS’ as the experiment type and choose the slide position on the stage. 2. Align the DESI stage movements with the correct position of the slide. 3. Finalize the slide area and double-check boundaries. b, Screenshots of protocol Steps 18–22. The instrument parameters must be customized by mass range, analyzer mode and type of MS experiment (ion MS versus MS versus tandem MS). The area selected for recording (red rectangle) should include a margin of nylon membrane outside the target area, to ensure complete recording of the sample wells. Finally, the default scanning parameters (and associated time for recording the experiment) are displayed.

Extended Data Fig. 2 HD imaging data analysis window overview for 6,7-dimethoxymethyl-3,4-dihydroisoquinoline.

Top left square: List of imported acquired files (after protocol Steps 33–37). A preview of each file is shown as a small black image, with a species highlighted. The TIC over the complete slide and for the ions of interest is shown in Fig. S2 (Supplementary Data). The current file that is visualized in the rest of the HD Imaging window is highlighted in blue (bottom of this square). Bottom left square: Molecular ions associated with the selected imported data file, categorized by summed ion intensity (high to low), with the top 1,000 ions displayed. The highest ion selected corresponds to the reactant depicted in Fig. 2. Top right rectangle: Image selection tools to normalize visualization across image files. Additional options include blending results for multiple ions in the same image (data set blend) or producing a smoothed pixel image (image smoothing, linear interpolation). Bottom right rectangle: Mass spectra for the acquired file selected in the top right. The selected ion (highlighted in purple) at m/z 206.13 corresponds to the protonated starting material for the IRED biotransformation displayed in Fig. 2 (and Figs. S2 and S3) and is also the most abundant ion recorded. The corresponding selected ion chromatogram is shown in Fig. S4.

Supplementary information

Supplementary Information

Supplementary data in support of Extended Data Fig. 2 and Fig. 5; Supplementary notes on DESI stage control for the different DESI sources; Supplementary Figs. S1–S15

Reporting Summary

Supplementary Data 1

Supplementary data

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Knox, R., Smith, R., Kempa, E.E. et al. Direct analysis of biotransformations with mass spectrometry—DiBT-MS. Nat Protoc (2025). https://doi.org/10.1038/s41596-025-01161-9

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