Biological mass spectrometry. Development of methods for protein and peptide analysis applied in neuroscience

Abstract

This thesis describes the development and use of biological mass spectrometry for protein and peptide analysis, and its application in neuroscience. Emphasis is placed on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF-MS), which has been the technique primarily used in the studies. Aspects relevant to the mass spectrometric study of proteins and peptides in neuroscience include the ability to purify and perform enzymatic or chemical reactions on small amounts of analytes from complex biological matrices. This calls for the development of novel techniques. Various strategies have been explored in this work.A rapid and sensitive method for proteolytic peptide mapping and protein identification, based on immobilized enzyme was developed. A sensitive sample purification and preparation technique for MALDI-MS is presented, and demonstrated in the identification of proteins from human brain.A novel method for mass spectrometric peptide mapping was developed, based on vapor-phase partial acid hydrolysis. The optimization of the method is described, and its application for MALDI-MS peptide mapping and protein identification is demonstrated.The combination of antibody affinity-based purification and MALDI TOF-MS allows characterization of the molecular heterogeneity of immunoreactive species, and thus yields more specific information than traditional techniques such as radioimmunoassay (RIA) and Western blot. By developing a sensitive method for immuno-affinity isolation of neurotensin (NT)-like immunoreactive molecules, we were able to detect low femtomole amounts of NT and several processed forms thereof in human hypothalamus, starting from low mg of brain tissue.A new method was developed for mass spectrometric quantification of NT in biological samples, based on the use of a stable isotope-labeled synthetic NT as internal standard. By detecting the antigen at its specific molecular mass, artifacts due to antibody cross-reactivity and nonbiospecific retention of sample compounds are overcome. The method is demonstrated with hypothalamus samples and compared with results obtained with RIA.