Alison Porter   Alison Porter began her college career at Big Sandy Community and Technical College in August of 2015, earning an Associate of Arts degree in May of 2017. She transferred to Morehead State University in August of 2017, and earned her Bachelor of Science in Veterinary Science in May of 2020. She accepted a position in University of Kentucky’s Master of Science in Veterinary Science program and will earn that degree in May of 2022. Through the master’s program, she worked as a graduate research assistant. This position exposed her to research in equine analytical chemistry which aided in furthering the research on the Equine Biological Passport. This abstract topic is one that comes directly from her thesis work over the last two years. She identified and validated four protein biomarkers that changed in response to bisphosphonate administration in horses, work which has not previously been done. She has been submerged in research using a mass spectrometer and has learned how to analyze data using programs such as Skyline. In the fall, she is planning on continuing her education and research efforts at the University of Kentucky College of Medicine.

Identifying and Validating Bisphosphonate Protein Biomarkers in Equine Sera Using Mass Spectrometry Methods

Bisphosphonates are a class of drugs used in humans and animals to treat resorptive diseases of the bone by inhibiting bone resorption. In equine, these anti-resorptive drugs are FDA-approved to treat clinical signs of navicular disease. Currently, there are two non-nitrogen containing bisphosphonate drugs approved for use in equine, tiludronate and clodronate. Bisphosphonates are heterogeneously distributed and tend to accumulate in areas of high bone turnover. These drugs do not undergo phase 1 or 2 metabolism, which prevents them from being readily detected using normal metabolite based drug testing techniques. Alternatively, we selected to monitor protein biomarkers, which demonstrated changes in response to bisphosphonate administration. This study developed a targeted acquisition method to validate four protein biomarkers that were identified using discovery analyses in response to bisphosphonate administration. Through untargeted LC-MS/MS methods, four key protein biomarkers—actin, carbonic anhydrase, fibrinogen, and fibronectin—were identified that exhibited changes across a time course and were found to have a role in bone remodeling. Following identification, a targeted parallel reaction monitoring (PRM) method was developed to validate these protein biomarkers. Read More
Samples were analyzed using an Orbitrap Exploris 480 mass spectrometer coupled with an UltiMate 3000 RSLCnano. In all serum samples, albumin was depleted using an organic solvent fractionation (isopropanol, trichloroacetic acid) protocol. Depleted samples were reduced, alkylated, and enzymatically digested using trypsin, followed by de-salting. Data were analyzed using Skyline (version 21) software, and were compared to pre-administration at day 0, as well as non-related equine sera that were used as a control. Using Skyline, optimal proteotypic peptides and transitions were selected for each protein biomarker using a data-driven approach. Initially, 103 candidate peptides were selected for evaluation. In Skyline, the 103 candidate peptides were refined down to 23 unique peptides. The refinement process was based on retention time, number of detectable transitions, and fragment ion ppm tolerance (±10 ppm). The 23 unique peptides were also chosen based on their peak area and proteotypic properties. Peptides with poor peak shape, low area counts, or that were non-distinguishable from the background matrix were removed. This iterative refinement process from initial peptide identification (103 candidate peptides) to optimized peptides (23 unique peptides) was all performed in Skyline software. Semi-quantitative analyses were performed using the top three most abundant peptides for each protein. These analyses led to determination of the average relative abundance of each time point, as well as percent difference of each time point from day 0 abundance. Each protein biomarker exhibited the same patterns that were established in the discovery phase with more intensity. Actin and carbonic anhydrase were both downregulated on day 18 and upregulated on days 7, 21, 28, 42, and 49 depending on the horse. Compared with day 0, these patterns of upregulation and downregulation remained true. Fibrinogen exhibited downregulation on day 18 and upregulation on day 7. Compared with day 0, fibrinogen exhibited downregulation on days 1, 3, 18, 34, 42, 49, 57, and 62. Fibronectin was initially identified as a protein that had an opposite abundance pattern to the other three proteins. This was also seen in the targeted phase. Fibronectin exhibited downregulation on day 7 and upregulation on day 18. When compared to day 0 abundance, it appeared that each time point was upregulated. However, even though day 7 was upregulated in comparison with day 0, it was downregulated when compared to day 18, keeping its initial established pattern. These patterns, while obvious in the discovery phase, were much clearer in the targeted phase. Skyline software was able to depict these patterns, as well as aid in refining the initial 103 peptides down to 23 unique peptides for all four proteins. These four protein biomarkers have relevance to bone remodeling and bisphosphonate administration. This study has developed a targeted LC-MS/MS method for detection of peptide biomarkers in equine sera, a technique which has not been previously established.