Analysis of tissues that reflect central nervous system disease by mass spectrometry

Abstract

Five different model systems exist for physical studies of disorders that afflict the human central nervous system: 1) brain tissue, 2) cerebrospinal fluid (CSF), 3) laboratory animals, 4) genetics, and 5) peripheral blood cells. Post-mortem brain samples, CSF, and peripheral blood cells harbor a great number of neuropeptides and proteins of diagnostic importance. Studies of these molecules have traditionally relied on antibody-based methods such as radioimmunoassay (RIA) and Western blotting. While sensitive, these types of assays are limited by the fact that antibodies recognize only an epitope on the molecule of interest. Thus, there is an omnipresent risk of the detecting antibodyís cross-reacting with other molecules containing this epitope. Likewise, truncated or otherwise modified forms of the peptide or protein may escape recognition. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS), a technique first described in 1988, has been utilized to provide molecular mass and sequence information for peptides, including neuropeptides, and proteins isolated from tissues that reflect disease states of the central nervous system. By combining affinity chromatography with HPLC separation, RIA, MALDI TOF MS, and post-source decay analysis, we were able to characterize the substance P-like immunoreactivity measured in porcine cortex and cortical nuclei. By studying the processing of four neuropeptides, neuropeptide Y 1-36, neuropeptide Y 18-36, somatostatin 1-28, and somatostatin 15-28 in neat CSF by MALDI TOF MS, and comparing our results to the HPLC-RIA profiles obtained from CSF, we were able to characterize the neuropeptide Y- and somatostatin-like immunoreactivity measured in these assays. Fundamental information about how neuropeptides are processed in CSF, depending on size and/or secondary structure, was also obtained in this study. The processing of neuropeptide Y 1-36, neuropeptide Y 18-36, somatostatin 1-28, and galanin was studied using MALDI TOF MS alone, in CSF samples taken from patients suffering from Alzheimerís disease, frontotemporal dementia, and neurologically normal controls. Differences in individual rates of neuropeptide processing and patterns of processing could be observed using this technique. Many proteins in CSF are of diagnostic importance. We have demonstrated that at least three of these can be monitored through MALDI TOF MS analysis of neat CSF. Using MALDI TOF MS analysis of neat CSF, it may be possible to diagnose certain inherited diseases directly, at the level of protein expression. In the last investigation included in this thesis, proteins in a human lymphocyte nuclear extract were identified using peptide mapping, post-source decay analysis, and database searching. Our results illustrate the usefulness of MALDI TOF MS-based techniques in the characterization of peptides and proteins from biological tissues used for the diagnosis of central nervous system disease; however, new uses for these techniques are rapidly becoming established in all areas of biomedicine.