Determination of Dynorphins And Tnf- A By Lc-ms/ms In Biological Samples: Applicable To Studying Inflammatory Mechanisms
Date of Award
Doctor of Philosophy In Clinical-bioanalytical Chemistry
Dynorphins are endogenous opioid peptides that have been implicated as initiators ofimmune and inflammatory response through upregulation ofinflammatory cytokine and chemokine production, as well having a role in glutamate-induced neuro-inflammation and neurotoxicity. Being extremely potent peptides, the physiologic concentrations of dynorphins are very low ranging from 0.16 pg/mL during the absence of a stimulus to 23.5 pg/mL when stimulated in disease condition. Previously published HPLC-mass spectrometry techniques have insufficient detection capabilities for quantification and detection ofdynorphins. As a result, immunoassay quantification has been the most utilized technique for analysis of dynorphins in physiologic samples. Although being sensitive, immunoassays have some inherent drawbacks of being complex multi-step process taking a long time to complete, challenge with reproducibility due to the non-specific binding interactions and the requirement of high sample volume. My dissertation focused on developing a sensitive LC-MS/MS technique to overcome such challenges in the analysis. A sensitive and robust LC-MS/MS assay has been developed and validated in the present work which can quantify the dynorphins below their low physiologic concentrations in mouse serum. To achieve this level of sensitivity, the intact peptide was digested using a novel metalloendopeptidase called Lys-N. This digestion process produced fragments which are extremely sensitive to detection by mass spectrometer and very specific to vi dynorphin B. Sensitivity achieved by this method is 800 times more than previously published HPLC-mass spectrometry techniques.
Chandu, Karthik, "Determination of Dynorphins And Tnf- A By Lc-ms/ms In Biological Samples: Applicable To Studying Inflammatory Mechanisms" (2020). ETD Archive. 1262.