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Fluorescence-Guided Matrix-assisted Laser Desorption/Ionization with Laser-Induced Postionization Mass Spectrometry of Individual Rat Neural Cells

Single-cell measurements are critical to understanding the rich spatiochemical heterogeneity of the brain. Matrix-assisted laser/desorption ionization ...

University of Illinois Urbana-Champaign

Recent multigenomic analysis of adrenocortical carcinomas (ACCs) identified SLC7A11/xCT as a novel biomarker. The Food and Drug Administration-approved anti-inflammatory drug, sulfasalazine (SAS), induces ferroptosis by blocking SLC7A11 expression. We hypothesize that SAS could be repurposed to target ACC cells.

Novel repurposing of sulfasalazine for the treatment of adrenocortical carcinomas, probably through the SLC7A11/xCT-hsa-miR-92a-3p-OIP5-AS1 network pathway.

Plant bioengineering is a time-consuming and labor-intensive process with no guarantee of achieving desired traits. Here, we present a fast, automated, scalable, high-throughput pipeline for plant bioengineering (FAST-PB) in maize (Zea mays) and Nicotiana benthamiana. FAST-PB enables genome editing and product characterization by integrating automated biofoundry engineering of callus and protoplast cells with single-cell matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). We first demonstrated that FAST-PB could streamline Golden Gate cloning, with the capacity to construct 96 vectors in parallel. Using FAST-PB in protoplasts, we found that PEG2050 increased transfection efficiency by over 45%. For proof-of-concept, we established a reporter-gene-free method for CRISPR editing and phenotyping via mutation of high chlorophyll fluorescence 136. We show that diverse lipids were enhanced up to 6-fold using CRISPR activation of lipid controlling genes. In callus cells, an automated transformation platform was employed to regenerate plants with enhanced lipid traits through introducing multigene cassettes. Lastly, FAST-PB enabled high-throughput single-cell lipid profiling by integrating MALDI-MS with the biofoundry, protoplast, and callus cells, differentiating engineered and unengineered cells using single-cell lipidomics. These innovations massively increase the throughput of synthetic biology, genome editing, and metabolic engineering and change what is possible using single-cell metabolomics in plants.

Enhancing lipid production in plant cells through automated high-throughput genome engineering and phenotyping.

Matrix-assisted laser desorption/ionization mass spectrometry with laser postionization (MALDI-2 MS) has become an important technique for the analysis of a wide range of biomolecules. It has traditionally been limited to custom lab-built setups until the recent introduction of a commercial timsTOF fleX MALDI-2 system. A comprehensive optimization of the timsTOF fleX system for lipid analysis was performed using a factorial design of experiments (DOE). By examining 13 instrumental parameters across three full factorial DOEs, we performed over 1500 individual runs to assess the impact and cross interactions of these parameters on the lipid signal intensity. We found optimal values for both ion transmission and MALDI-2 parameters to maximize the signals within the lipid region. These results show that laser shot frequency, collision RF, and pre pulse storage were essential for enhancing lipid ion transmission, resulting in a nearly 5-fold increase in signal intensity compared to default parameters. For MALDI-2 optimization, positive and negative modes showed similar optimized values, with TIMS In pressure and laser power being crucial. Overall, optimization of ion optics and MALDI-2 resulted in signal enhancements of nearly 2 orders of magnitude for certain lipid species.

Article	Year
Novel repurposing of sulfasalazine for the treatment of adrenocortical carcinomas, probably through the SLC7A11/xCT-hsa-miR-92a-3p-OIP5-AS1 network pathway. Surgery\| PubMed ID: 39424480	2025
Enhancing lipid production in plant cells through automated high-throughput genome engineering and phenotyping. The Plant cell\| PubMed ID: 39899469	2025
Factorial-Design-Based Optimization of a Commercial MALDI-2 timsTOF Mass Spectrometer for Lipid Analysis. Journal of the American Society for Mass Spectrometry\| PubMed ID: 40155311	2025

Seth W. Croslow

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