We report here on the first analysis of polymerase chain reaction (PCR) products amplified from a small amount of human blood DNA by electrospray ionization mass spectrometry (ESI-MS). Adenomatous polyposis coli (APC) gene fragment of about 50 base pairs (bp) with a relative molecular mass (M(r)) of approximately 15,000 u was amplified from human blood DNA by PCR. The accurate molecular mass of the PCR products was determined with an accuracy of approximately 0.005% by ESI-MS. The amount of DNA used was only 100 ng (approximately 50 zmol; the theoretically required amount of blood is therefore less than 1 microliter for PCR). The ESI-MS measurement of the PCR products proved to be a new accurate, sensitive and fast tool for gene diagnosis.

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Genes encoding pre-protein and prepro-protein of wheat germ agglutinin isolectin 2 (WGA2) were chemically synthesized and expressed in the yeast Saccharomyces cerevisiae under the control of the E N 0 1 promoter. Yeast harboring either a pre-WGA2 or a prepro-WGA2 gene expression plasmid secreted a mature form of WGA2 into the culture medium. The amount of WGA2 secreted by the strain KS58-2Dde1, which has a ssll mutation causing a supersecretion of human lysozyme [Suzuki, [58][59][60][61][62][63][64], was 20-fold grcater than that secreted by the wild-type strain KK4. The recombinant WGA2 from the cells containing the prepro-WGA2 gene expression plasmid was purified to homogeneity by a three-step ion-exchange chromatography scheme. As in wheat, the N-terminal signal peptide of recombinant WGA2 purified from yeast culture was processed to form an N-terminal5-oxoprolyl (pyroglutamyl) residue. Likewise, we found that the C-terminal pro-region of recombinant WGA2 had also been processed in yeast. Using electrospray ionization mass spectrometry, we found thc proccssed C-tcrminus to be heterogeneous in both recombinant WGA2 purified from yeast and in authentic WGA2. The major component of the recombinant WGA2 contained two additional amino acids at its C-terminus compared to that of authentic WGA2. In spite of this difference in the C-terminus, the recombinant WGA2 exhibited a sugar binding activity that was indistinguishable from that of authentic WGA2.

This paper proposes an efficient method for computer-aided peptide sequencing based on fast atom bombardment mass spectrometry. The method allows us to analyse peptides much larger than loo0 u within a reasonable cpu time and to have a set of amino acid sequences which are consistent with the observed fast atom bombardment mass spectrum to varying degrees. Another feature is that there is no need to pick sequence ion peaks out of the spectrum; all the ions observed are equally taken into account in the analysis so that one can exhaust the possibilities expected from the whole spectrum. For most samples of penta-to tridecapeptides, parts of an amino acid sequence could he reproduced satisfactorily without prior knowledge of amino acid composition. The degree of success in sequence elucidation depends entirely on the spectrum used in the analysis. When fragment ion peaks are clearly observed, the sequence can be predicted successfully. The result obtained for a given amino acid composition was always satisfactory.