2D gel separation and sequence analysis of protein in micro amount : novel C-terminal sequencing methods.

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1) Novel C-terminal sequencing : Vapor from 90% perfluoroic acid aqueous solution on peptides at 90ﾟC for 4-24 hours results mixture of C-terminal truncate molecules. FAB-or ESI-mass spectrometry can alnalyze these C-terminal truncate degradated molecular ions. The mass differences of truncate ions directly give the C-terminal sequence. The reaction accompanies internal cleavage at C-terminal side of Asparatic acid and N-terminal side of serine. Vapor from 15-30% perfluoacyl anhydride on peptides-20ﾟC for 1 hour also results the mixture of C-terminal degraded molecules, analyzed by FAB-mass spectrometry as truncate molecular ions. Accompanying major degraded ions, -1, -18, -46 ions were observed and these ions were experimentally identified. The reaction mechanism was partly elucidated. Conditions of the reproducible experiments were extensively studied and the entire process to be in the glove box with N2 flow to avoid moisture at low temperature. Further more study is needed to big m … More olecule and automation of the method.2) N-terminal sequencing : Hetrogeneous reaction system is found to be favor for micro-sequencing chemical reaction. The intermediate ATZ compound (solid) in Edman degradation was amidated by gas and this reaction yields were found still 70% in femto mole range. According to this system, alternate amidation with two fluorescence-amines has been studied. A half of the native proteins waas found blocked at their N-termini. Anhydrous hydrazine vapor was found to be used for deblocking formyl-and pyroglutamyl residues but not deblock acetyl and acyl groups at N-termini, which is under progress.3) 2D-gel electrophoresis : A new concept of standardization was proposed with use of commercially available reference proteins. Three biological systems were used : rice, Arabidopsis and Fusarium (fungi). Numbers of separated protein spots were about 5,000,5,000 and 1,500 using the dimension, 20x20cm. The numbers of 5,000 may be maximum and may be one third or a half of these plant entire proteins. In Fusarium all proteins can be estimated to be 1,500. Partial N-terminal sequencings were carried out about 100-150 proteins for the respective system, and a half of them were sequenced and the other half were blocked at their N-termini. Altogether 70 proteins were carried out by our new deblocking method and more than one tenth were deblocked and sequenced. The approach with 2D may be complementary used for genome research. A novel homology research method was proposed. Less