Screening of Cerebrospinal Fluid and Blood Sera of Multiple Sclerosis Patients for Oligoclonal Immunoglobulins by Capillary Electrophoresis

Summary Multiple sclerosis (MS) is an autoimmune disease characterized by the production of specific types of immunoglobulins into the central nervous system. These immunoglobulins appear as oligoclonal bands (OCBs) in agarose isoelectric focusing (IEF) of cerebrospinal fluid (CSF). Among the cases with clinically definite MS, up to 95% have oligoclonal IgG bands in their CSF. In this report, we describe a micellar electrokinetic capillary chromatography (MEKC) method for the separation of CSF and serum proteins. MEKC was performed using 25 mM borate buffer, pH 10, containing 25 mM SDS at 20 kV and normal polarity. High values of repeatability in migration times and of reproducibility in peak areas were obtained (R.S.D. values were less than 2%). Calibration graphs were linear up to 2000 mg L^–1. LOQ was 6.5 mg L^–1 and LOD determined as a signal to noise ration of 3:1 was 4.5 mg L^–1. Analysis of CSF and serum samples from patients with clinical definite MS and healthy individuals demonstrated the presence of two peaks migrating as -globulins in the CSF samples of patients. These peaks were absent from controls and the serum of the same patients. Correlation of the data obtained from IEF and MEKC analysis for 25 patients showed that the diagnostic sensitivity and specificity of MEKC were ca 89% and 92% respectively. The obtained results indicate that this MEKC method may be helpful for the diagnosis of multiple sclerosis. Capillary electrophoresis compared to flat bed IEF provides reproducible results, requires shorter analysis time, and allows direct quantitative determination.Presented at: International Symposium on Separation and Characterization of Natural and Synthetic Macromolecules, Amsterdam, TheNetherlands, February 5–7, 2003     

A rat brain protein expression map including cytosolic and enriched mitochondrial and microsomal fractions

Proteomics is a powerful tool to screen brain protein expression but the methodology is hampered by low abundance of proteins or compartmentalization or overload of high-abundance proteins. It was therefore the aim of the study to determine the expression of brain proteins by using enriched cellular subfractions and pre-electrophoretic chromatographical separation of brain homogenates. We used two-dimensional electrophoresis with subsequent matrix-assisted laser desorption/ionization (MALDI) detection and characterization of brain proteins. Subfractionation into cytosolic, mitochondrial and microsomal compartments was performed by ultracentrifugation. Pre-electrophoretic fractionation of the cytosolic fractions was carried out by ion exchange column chromatography. We detected and identified a large series of 437 proteins in rat brain and have shown proteins specific for the individual subcellular compartments. These proteins included housekeeping, signaling, cytoskeletal, intermediary metabolism, antioxidant proteins on the one and neuron and synaptosomal specific proteins on the other hand. Using fractionations of brain homogenates we were able to improve the power of the method on forming the basis for brain protein expressional studies and providing a reference map as a powerful tool for the neuroscientist.     

NMR spectroscopy techniques for screening and identifying ligand binding to protein receptors

Binding events of ligands to receptors are the key for an understanding of biological processes. Gaining insight into protein-protein and protein-ligand interactions in solution has recently become possible on an atomic level by new NMR spectroscopic techniques. These experiments identify binding events either by looking at the resonance signals of the ligand or the protein. Ideally, both techniques together deliver a complete picture of ligand binding to a receptor. The approaches discussed in this review allow screening of compound libraries as well as a detailed identification of the groups involved in the binding events. Also, characterization of the binding strength and kinetics is possible, competitive binding as well as allosteric effects can be identified, and it has even been possible to identify ligand binding to intact viruses and membrane-bound proteins.     

A meanfield approach to the thermodynamics of a protein-solvent system with application to the oligomerization of the tumor suppressor p53

The thermodynamic stability and oligomerization status of the tumor suppressor p53 tetramerization domain have been studied experimentally and theoretically. A series of hydrophilic mutations at Met-340 and Leu-344 of human p53 were designed to disrupt the hydrophobic dimer-dimer interface of the tetrameric oligomerization domain of p53. Meanfield calculations of the free energy of the solvated mutants as a function of interdimer distance were compared with experimental data on the thermal stability and oligomeric state [tetramer, dimer, or equilibrium mixture of both] of each mutant. The calculations predicted a decreasing stability and oligomeric state for the following amino acids at residue 340: Met [tetramer] > Ser Asp, His, Gin, > Glu, Lys [dimer], whereas the experimental results showed the following order: Met [tetramer] > Ser > Gln > His, Lys > Asp, Glu [dimers]. For residue 344, the calculated trend was Leu [tetramer] > Ala > Arg, Gln, Lys [dimer], and the experimental trend was Leu [tetramer] > Ala, Arg, Gln, Lys [dimer]. The discrepancy for the lysine side chain at residue 340 is attributed to the dual nature of lysine, both hydrophobic and charged. The incorrect prediction of stability of the mutant with Asp at residue 340 is attributed to the fact that within the meanfield approach, we use the wild-type backbone configuration for all mutants, but low melting temperatures suggest a softening of the α-helices at the dimer-dimer interface. This initial application of meanfield theory toward a protein-solvent system is encouraging for the application of the theoretical model to more complex systems.