Chemical and mass spectrometrical methods in protein analysis

Detta är en avhandling från Stockholm : Karolinska Institutet, Department of Medical Biochemistry and Biophysics

Sammanfattning: Sensitive analytical methods are essential in protein studies because of frequently encountered limited sample availability. There is a constant need for optimization of sample preparation and analytical procedures for structural characterization of biomedically relevant proteins. Amino acid sequences can be derived from genomic data. However, gene processing and post-translational modifications alter the expressed protein product which can be further processed in tissues to a functional form. Therefore, both protein quantity and structure must be evaluated per se. A method for chemical deblocking of N-terminally acetylated proteins useful for identification via sequence analysis by Edman degradation was developed and optimized. The protocol was applied to the characterization of native and recombinant proteins. Deacetylation yields were up to 70%. The method is efficient with both proteins and peptides, and has a fairly low yield of unspecific cleavage of peptide bonds. Mass spectrometry is a different method to analyze protein structure. The technique was applied to verify the removal of an acetyl group and also to analyze the structure of gel separated proteins from brain, prostate and archaea. Two-dimensional gel electrophoresis is efficient to separate complex protein mixtures. Protein characterization can then be carried through by mass spectrometry after in-gel digestion of polypeptides. These techniques were employed to analyze foetal human brain tissue from individuals with or without Down's syndrome. Forty proteins were identified. Protein patterns were largely similar, although actin and tubulin were observed not only at the expected molecular weights and isoelectric points, but also as negatively charged fragments. Proteins in benign and malignant prostate glands were also analyzed. The protein patterns indicated a high degree of heterogeneity. Several proteins were identified and prostatic acid phosphatase and tropomyosin I were found to be downregulated in prostatic cancer, whereas heat shock protein 70 was found to be upregulated in the neoplastic state. The results indicate that integration of analytical methods provides strategies for the characterization of proteins expressed under different conditions in goals toward determination of protein function.

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