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An optimized automated synthesizer is presented for assembling oligonucleotides, thiooligonucleotides and 5′-modified oligonucleotides including: chemical phosphorylation, multihydroxyl derivatization with a non-nucleosidic phosphoramidite. The incorporation of biotin, fluorescein and rhodamine phosphoramidites is described. The purification and structure determination of oligo-nucleotides was confirmed using high performance liquid chromatography (HPLC), capillary electrophoresis (CE) and laser desorption mass spectrometry (LDMS). Several applications and confirming data will be presented for gene synthesis and polymerase chain reaction (PCR) experiments.

As demonstrated by selected examples from our laboratories, CE is a unique methodology for purity control of synthetic as well as natural tissue-isolated biopolymers, a prerequisite before reliable biotestings should be performed. A combination of rapid matrix-assisted laser desorption ionization mass and CE electrophoretic mobility determinations facilitates primary sequence determinations of enzymatic peptide digest mixtures often making costly Edman degradations unnecessary. The enormous separation efficiency and large variety of different possible separation modes in CE, allow detection of single components in complex mixtures which is demonstrated by the apolipoprotein A-I determination in human blood serum in this communication.

A combination of high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) spiking, Edman degradation, amino acid analysis and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) was applied for the analysis of the primary structure of beta-thymosins. CE has been used to resolve peaks which coelute on HPLC, as well as to help establish the identity of tryptic fragments in the peptide mapping experiments of the two highly homologous beta-thymosins.

A rapid and efficient primary structure elucidation of prothymosin (ProT alpha) and its enzymatic fragments was performed by a combination of matrix-assisted laser desorption ionization mass spectrometry (MALDI), automatic N-terminal Edman degradation, and the available theoretical predictions of electrophoretic mobility in capillary electrophoresis (CE) as a basis suggesting active sites of ProT alpha in the different bioassays.

A method for the determination of apolipoprotein A-I, the major protein compartment of HDL, in human serum is described. Rapid and easy serum sample preparation, well separated Apo A-I peaks in the human serum electropherogram and good linearity of the peak area vs. concentration plot, covering the range of the clinically relevant Apo A-I serum contents, suggest the introduction of this method routinely in clinical laboratories.