Methods for the analysis of protein structures are being improved, applied and tested in independent and collaborative research projects in proteomics. Chromatographic systems, both on-line liquid chromatographic and gel-based are being tested. Collaborative studies on the identification of post-translational modifications or unique gene products are in progress. Methods are being devised for the selective isolation and characterization of low molecular weight proteins not usually detected by gel based methods. New methods for targeted functional proteomics have been devised and are being tested. Strategies for the quantitative detection of changes in phosphorylation of serine or threonine residues have been verified in model peptides. Beta-elimination of phosphoric acid from phosphoserine is followed by thioacetylation. After hydrolysis, the cysteine sulfur reacts with a biotinylated cleavable tag which facilitates separation of the family of peptides that previously contained phosphoserine. By reacting a protein mixture from one experimental group with deuterated biotinylated tag and proteins from a control group with non-deuterated reagent, it is possible to mass label and distinguish peptides from treated vs. control cells. Admixture of equal quantities of peptides allows a ratios to be measured mass spectrometrically and distinctions to be made between peptides from housekeeping proteins vs. those newly formed or modified with respect to cellular events. The affinity tagged peptides can be effectively separated from the total peptide mass and then analyzed by MALDI/TOF/MS or LC/MS. Liquid-chromatography-electrospray mass spectrometry methods have been applied for the quantitative analyses of 13C-labeled NAD as product of 13C-tryptophan metabolism. Improved methods of sample introduction and concentration have been devised for capillary zone electrophoresis. Magnetic beads that have been surface coated are washed into a column, magnetically trapped, and the sample applied. Peptides stick to the bead coating, permitting enrichment of dilute samples, consequently enhancing the applicability of capillary electrophoresis to dilute solutions.
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