A high-throughput screening assay for mutant isocitrate dehydrogenase 1 using acoustic droplet ejection mass spectrometry

Speckmeier, Elisabeth; Pommereau, Antje; Grosser, Kay-Christoph; Mors, Hartmut; Maier, Thomas; Licher, Thomas; Bärenz, Felix

doi:10.1016/j.slasd.2022.04.002

Cited by 3 publications

(2 citation statements)

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“…Next, we conducted experiments to optimize the ADE-OPI-MS analysis protocol, focusing specifically on the carrier liquid and additional delay time, the two major factors that affect peak shape and sensitivity. 38 Three carrier fluids, 100% Methyl alcohol (MeOH), 70% Acetonitrile (ACN)/30% H 2 O (v/v) and 50% MeOH/50% ACN (v/v), were analyzed for their impact on CC peak shape and intensity (Table 1, Figure S2a,b). MeOH yielded burr-free peaks with minimal trailing edges while maintaining a superior signal-to-noise ratio (S/N), likely due to its advantages compared to the other two carrier liquids.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…Results indicated that NH 4 F performed better than the others. Based on prior findings, the sensitivity can be influenced by the concentration of ammonium fluoride. , So the optimal amount of NH 4 F was determined through testing three different concentrations (0.5 mM, 1 mM, and 2 mM) with varying carrier liquids (Figure S2e,f and Table S4). Our results revealed that the carrier liquids with changing NH 4 F concentrations did not yield a higher intensity.…”

Section: Resultsmentioning

confidence: 99%

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High-Throughput Screening for Oligonucleotide Detection by ADE-OPI-MS

Hu,

Zhang,

et al. 2024

Anal. Chem.

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Oligonucleotides represent a class of shorter DNA or RNA nucleic acid polymers extensively applied in the biomedical field. Despite progress in detecting and analyzing oligonucleotides, high-throughput analysis of the samples remains challenging. In this work, a high-throughput analysis method for oligonucleotide analysis was developed based on acoustic droplet ejection-open port interface-mass spectrometry (ADE-OPI-MS) technology. This approach was applied to determine the enzymatic activity of terminal deoxynucleotide transferase (TdT) for DNA synthesis, with a rate of 3 s/sample, which enhanced single-sample analysis efficiency approximately 60-fold over the previous gel analysis. After testing approximately 10,000 TdT mutants, we obtained three new variants with higher catalytic activities. Finally, by integrating these mutants, the catalytic activity of TdT was improved about 4 times compared to the starting mutant. Our results successfully established a high-throughput screening method for oligonucleotide analysis, which not only provides a foundation to engineer highly efficient TdT for ab initio synthesis of DNA but also paves the way for the potential application of oligonucleotide analysis in biomedical fields.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%