Analysis Of The Zebrafish Olfactory System Using Immunohistochemistry And Enhanced Techniques Of Desorption Electrospray Ionization Mass Spectrometry (Desi-Ms)
Date of Award
Doctor of Philosophy
Andre R. Venter, Ph.D.
Christine Byrd-Jacobs, Ph.D.
Steven Bertman, Ph.D.
John Spitsbergen, Ph.D.
Desorption electrospray ionization, lipid analysis, mass spectrometry, zebrafish, zebrafish olfactory system
Desorption electrospray ionization (DESI-MS) is an ambient ionization technique where the sample is analyzed directly from a surface with very minimal sample preparation under ambient conditions and follows ESI-like ionization mechanisms. DESI-MS has proven powerful in analyzing or imaging lipids and other small molecules directly from biological samples and even allows for subsequent histological staining and analyses. However, DESI-MS is less widely used for protein analysis due to a lack of sensitivity and the complex diversity of proteins in biological samples.
A major goal of this research has been to obtain new neurobiological knowledge by combining histology and mass spectrometry through a collaborative project. The work presented here focuses on (1) examining and comparing the different sensory neurons in the zebrafish olfactory system before and after chemical damage, (2) improving DESI-MS analysis of proteins and lipids through multiple approaches, and (3) utilizing signal improving techniques to analyze lipids and proteins present in zebrafish olfactory tissue.
Zebrafish olfactory organs were examined after chemical damage with Triton X- 100 detergent. Statistical analyses revealed that sensory neurons mediating food detection are more resistant to damage than the other neuronal subtypes. To investigate metabolic changes in different damage states, DESI-MS analyses revealed chemical disturbances in both the treatment and internal control rosettes of treated fish. Principal component analysis (PCA) was used to determine differences in spectral features displaying separation and clustering of samples between treatment groups. We discovered that internal control rosettes undergo lipid changes as well, even though they were not subjected to the chemical insult. Thus, great care should be taken when defining intra-animal controls in future studies.
Protein analysis improved with the addition of a pre-wetting solvent through a “wetting-quill” and through the addition of organic solvent vapors. The wetting-quill increased dissolution time, one of the major contributors to the loss of protein signal. However, results indicated a need to use the bare minimum amount of solvent additions. This minimum was best attained by using solvent vapors in the ionization region, rather than liquid application. Similar to previously reported ESI-MS results, protein signal intensity was improved when the DESI sprayer was exposed to polar vapors such as acetonitrile or ethyl acetate.
Finally, we successfully show that the novel DESI-improvement methods developed by our lab can be applied to the analysis of biological tissues. When analyzed with the addition of ethyl acetate and/or L-serine, both protein and lipid signal intensities and S/N ratios were improved. We also observed that some lipids, especially those carrying a phosphocholine headgroup, formed serine adducts making them detectable in the negative mode without the use of acidic modifiers which can disrupt histology results. This makes L-serine a potentially useful additive in experiments where histological analysis is required from the same tissue sample previously analyzed by DESI-MS.
Maser, Tara Lynn, "Analysis Of The Zebrafish Olfactory System Using Immunohistochemistry And Enhanced Techniques Of Desorption Electrospray Ionization Mass Spectrometry (Desi-Ms)" (2022). Dissertations. 3856.