Mass spectrometry-based identification and quantitation of endogenous peptides in complex biological matrices

mass spectrometry;peptides;neuropeptides;quantitative mass spectrometry

Dowd, Sarah E

关键词: mass spectrometry;    peptides;    neuropeptides;    quantitative mass spectrometry;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/89278/DOWD-DISSERTATION-2015.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

In the brain, chemical signaling molecules transfer information from cell-to-cell to modulate mood, behavior, and physiological functions like metabolism and circadian rhythm. The research presented in this dissertation is focused on characterizing a specific class of signaling molecules, endogenous signaling peptides. Despite being gene products, this class of signaling molecules is complex and diverse. Cell to cell signaling peptides are differentially processed, modified, and distributed depending on time and spatial location. The detection of the peptide complement in a biological sample is an analytical challenge due to the broad range of concentrations and diverse chemical properties of endogenous peptides. The presence of other classes of molecules, like salts and lipids, add to sample complexity and further complicates the characterization of signaling peptides. The ability to characterize endogenous peptides is dependent on the selection of appropriate, information-rich tools. In this dissertation, a set of mass spectrometry approaches coupled with optimized liquid chromatography separations and appropriate sample preparation were successfully utilized to measure chemical information for peptides in biological samples. Multiple MS platforms, each with distinct advantages and limitations, were utilized for identification and quantitation. To gain insight into the role of signaling peptides in a specific behavior, relative quantitation measurements with MS were performed in a non-targeted approach. Specifically, measurements were made for peptide level changes in the hippocampus and amygdala of mice to study the extinction of conditioned place preference (CPP) for cocaine in mice that have access to a running wheel after CPP training. MS measurements have yielded both known and novel candidate peptides that show relative abundance changes with exercise and context re-exposure, which yields insight into the biochemical mechanisms driving the observed extinguishment of CPP with running. Successful MS measurements of endogenous peptide measurements are dependent on appropriate and optimized sample preparation procedures. An optimized sampling procedure was developed and applied to detect the novel peptide alarin in the supernatant of a transfected cell line. While a synthetic version of the peptide spiked into the medium was detected by MS, the endogenous form of the peptide expressed by the cell line could not be unambiguously identified. Additionally, MS measurements were employed to evaluate the effects of different sample handling treatments on the detection of peptides in the mouse striatum and hypothalamus. The results of this preliminary study indicate for the sample treatments used, each has its limitations and the selection of an appropriate sample treatment in biological studies is dependent on the study goals and resources. This demonstrates the capabilities of mass spectrometric approaches to investigate peptide identities and dynamics, which can be utilized to enhance our understanding of the role of peptides in cell-to-cell signaling in the nervous system.