A goal of these studies is to better understand the molecular mechanisms underlying human nervous system development and function, as well as the pathogenesis of certain inherited brain disorders. Our studies have focused on the structural and active-site properties of proteins found in the nervous system including neurotrophic peptides/proteins, lysosomal hydrolases, and other proteins/peptides, which interact with excitable membranes, receptors and venom toxins. Proteins from human and animal tissues are purified by liquid chromatography (ion-exchange, gel permeation and affinity techniques), high performance liquid chromatography (HPLC) and electrophoretic separation. State-of-the-art micro sequencing analysis (gas-phase, liquid-phase and solid-phase), surface enhanced laser desorption ionization (SELDI) and matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) are performed to characterize proteins. The unambiguous identification of proteins/peptides is crucial for the definition of the proteome. Using ProteinChip Array technology (SELDI) we have developed experimental protocols and probed test conditions required for protein identification on ProteinChip Arrays. We were able to directly digest proteins/peptides on-chip surfaces by specific proteases, such as trypsin, Glu C and Asp N and obtain the peptide mass fingerprint of the sample under investigation by direct analysis on a simple laser desorption/ionization mass spectrometer. Furthermore, tandem mass spectrometry was performed on several of resulting tryptic peptides by using collision quadrupole time of flight (Qq-TOF) MS/MS via a ProteinChip interface, thus allowing the precise identification of the parent protein within the sample. In addition we were able to identify the C-terminal sequence of peptides after digestion with carboxypeptidase Y directly on ProteinChip surfaces coupled with SELDI-TOF mass spectral analysis, both under native and denaturing conditions. Utilizing the on chip digestion methodology we examined the structural differences between the disease and physiological forms of gross cystic disease fluid protein 15 (GCDFP-15), a breast tumor marker. The primary structure of both forms of the protein was identical by MS/MS analysis, however, an altered N-linked glycosylation pattern was observed in both forms by coupling enzymatic digestion and ProteinChip technology. Furthermore we examined the interaction of both forms of GCDFP-15 with fibronectin and CD4 (proteins known to interact with the physiological form). The biological characteristics of the pathological form are altered with regard to both CD4 and fibronectin binding. Utilizing the SELDI we have also examined the peptidic """"""""middle molecules"""""""" that accumulate in uremic patients' sera. In order to fully characterize these molecules we developed a new micro-preparative procedure (employing SDS electrophoresis and liquid chromatography) that provided separation of these peptides. Microsequencing and MS/MS analyses of the peptides showed that most of the identified middle molecules were fragments of fibrinogen. Combining the resources of the laboratory, we have clearly demonstrated our ability to analyze samples from a variety of patient disease states. We are presently examining CSF from Alzheimer disease patients to identify possible biomarkers for use in earlier diagnosis. By combining both SELDI analysis and on chip immunoassay we hope to be able to clearly identify those species that are different from patient to control as well as those that are post-translationally altered.
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