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Wavelength Dependence of Intact Protein Extraction Using Femtosecond Laser Ablation

ABSTRACT: Raw files for the paper named: Wavelength Dependence of Intact Protein Extraction Using Femtosecond Laser Ablation

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Mastigocladopsis Repens Rhodopsin Equine Myoglobin Lysozyme From Chicken Egg White

SUBMITTER: R.J. Dwayne Miller

PROVIDER: MSV000097234 | MassIVE | Tue Mar 04 07:05:00 GMT 2025

REPOSITORIES: MassIVE

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Wavelength Dependence of Intact Protein Extraction Using Femtosecond Laser Ablation.

Wainwright Alexander A C AAC Madhoun Khaled K Su Pei P Janisse Samuel E SE Besaw Jessica E JE Grewal Harmanjot S HS Ernst Oliver P OP Kafader Jared O JO Kelleher Neil L NL Miller R J Dwayne RJD

The journal of physical chemistry letters 20250820 34

The extraction and analysis of intact proteins from complex biological samples via femtosecond laser ablation are influenced by wavelength-dependent multiphoton and avalanche ionization processes. To investigate these effects, an intact protein mixture with molecular weights ranging from 9 kDa to 68 kDa as well as individual proteins were sampled using laser wavelengths spanning from the ultraviolet to the near-infrared. Our results suggest that visible and infrared wavelengths enable intact pro ...[more]

PMID: 40833879

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Project description:Airway organoids derived from adult murine epithelial cells represent a complex 3D in vitro system mimicking the airway epithelial tissue’s native cell composition and physiological properties. In combination with a precise damage induction via femtosecond laser-based nanosurgery, this model allows for the examination of intra- and intercellular dynamics in the course of repair processes with a high spatio-temporal resolution, which can hardly be reached using in vivo approaches. For characterization of the organoids’ response to single or multiple-cell ablation, we first analyzed overall organoid survival and found that airway organoids were capable of efficiently repairing damage induced by femtosecond laser-based ablation of a single to ten cells within 24 h. An EdU staining assay further revealed a steady proliferative potential of airway organoid cells. Especially in the case of ablation of five cells, proliferation was enhanced within the first four hours upon damage induction, whereas ablation of ten cells was followed by a slight decrease in proliferation within this time frame. Analyzing individual trajectories of single cells within airway organoids, we found an increased migratory behavior in cells within close proximity to the ablation site following the ablation of ten, but not five cells. Bulk RNA sequencing and subsequent enrichment analysis revealed the differential expression of sets of genes involved in the regulation of epithelial repair, distinct signaling pathway activities such as Notch signaling, as well as cell migration after laser-based ablation. Together, our findings demonstrate that organoid repair upon ablation of ten cells involves the same key processes by which native airway epithelial wound healing is regulated. This marks the herein presented in vitro damage model suitable to study repair processes following acute airway injury, thereby posing a novel approach to gain insights into the mechanisms driving epithelial repair on a single-cell level.