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The Investigation of Potential Salivary Protein Biomarkers of Acute Stress Using Proteomics and Mass Spectrometry

Marvin, Rachel K
http://rave.ohiolink.edu/etdc/view?acc_num=toledo1470409379

Abstract Details

, Doctor of Philosophy, University of Toledo, Chemistry.
The goal of the present research was to elucidate salivary biomarkers of acute stress using proteomic approaches. Acute stress is marked by an increased activity of the sympathetic branch of the autonomic nervous system. Prolonged exposure to acute stress may result in fatigue and chronic stress. Therefore, acute stress has potential social and economic implications in which the performance of workers in high stress occupations (e.g., health care and law enforcement professionals) may be negatively affected. To aide in the monitoring a stress an objective assessment using biomarkers is desirable. Saliva is an optimal body fluid for the discovery and investigation of biomarkers of this physiological state because its secretion is controlled by the autonomic nervous system, and its collection is easy and noninvasive. As people secrete 1-2 L of saliva per day, ample quantities of saliva are typically obtained for qualitative and quantitative analyses of potential biomarkers. In addition, saliva is primarily comprised of water, electrolytes, proteins and hormones whose abundance may be monitored. Consequently, the present analyses involved the examination of the human salivary proteomes at different time points corresponding to non-stressed and acutely stressed states to discover novel protein markers of acute stress. The first project utilized a model system employing the cold pressor test (CPT) to reproducibly induce stress. Participants immersed their non-dominant hand into cold water for a maximum of five minutes, which increases the activity of sympathetic nervous system for the majority of people. Whole saliva was collected before, immediately after and 20 minutes after the CPT. A variety of gel electrophoresis techniques (i.e., SDS-PAGE, 2D-PAGE and 2D-DIGE) was used to assess alterations in the salivary proteome at these three time points. When equal volumes of saliva were analyzed by SDS-PAGE, minor differences in the abundance of some salivary proteins were observed after the CPT. For instance, the amount of cystatins and alpha-amylase is increased immediately after and 20 minutes after the CPT in comparison to the sample obtained prior to the CPT. These differences were also noticed after HPLC was used to separate equal amounts of proteins from the whole saliva samples. Hypothesizing that protein phosphorylation may be influenced by the CPT, tryptic phosphopeptides were isolated from the salivary proteome using immobilized metal affinity chromatography (IMAC). After enrichment, the phosphopeptides were analyzed by nanoHPLC-MALDI-MS/MS, and some differences in the phosphorylation of salivary proteins were observed. Comparing the nanoHPLC chromatograms the peptides eluting at retention times of ~25 and 29.7 minutes had the highest absorbance for the samples collected immediately and 20 minutes after the CPT, indicating that these peptides are present in higher amounts after the stressor. In contrast, the peptide(s) at retention time of ~26.7 minutes had the highest absorbance, and the samples collected after the CPT stressor had a relatively low absorbance. The peptides at these retention times were identified as originating from salivary acidic proline-rich phosphopeptide 1/2. However, the changes in salivary protein abundances and phosphorylation require further characterization before they can be verified as biomarkers of acute stress induced by the CPT. The second project investigated proteins from saliva of medical residents who were placed into a stressed state by performing emergency medicine simulations. Whole saliva was collected from eight medical residents prior to performing the simulation, after the simulation, and the next morning upon waking. Salivary proteins were identified with SDS-PAGE and peptide mass fingerprinting, as well as by nanoHPLC-ESI-MS/MS. Relative quantification of the alpha-amylase, cystatin-type S family, a ~26 kDa band and a low-molecular weight (<10 kDa) band was investigated in relation to the stressful emergency medicine scenarios. Densitometry and statistical analyses of Coomassie stained SDS-PAGE gels indicated an increase in the relative amount of salivary alpha-amylase and cystatin type-S bands after simulated emergency interventions in comparison to the pre-simulation and waking time points. Some differences in the ~26 kda and the low-molecular weight bands at different time points were also observed. The results indicate that proteins, such as alpha-amylase, cystatins and histatin-3, may be considered as putative salivary biomarkers of acute stress, but they need further validation in a larger sample population.
Dragan Isailovic, PhD (Advisor)
Mark Mason, PhD (Committee Member)
John Bellizzi, PhD (Committee Member)
Kenneth Hensley, PhD (Committee Member)
Analytical Chemistry; Chemistry

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Citations

  • Marvin, R. K. (n.d.). The Investigation of Potential Salivary Protein Biomarkers of Acute Stress Using Proteomics and Mass Spectrometry [Doctoral dissertation, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1470409379

    APA Style (7th edition)

  • Marvin, Rachel. The Investigation of Potential Salivary Protein Biomarkers of Acute Stress Using Proteomics and Mass Spectrometry. University of Toledo, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=toledo1470409379.

    MLA Style (8th edition)

  • Marvin, Rachel. "The Investigation of Potential Salivary Protein Biomarkers of Acute Stress Using Proteomics and Mass Spectrometry." Doctoral dissertation, University of Toledo. Accessed APRIL 28, 2024. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1470409379

    Chicago Manual of Style (17th edition)

toledo1470409379
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