Protein sequence analysis and the discovery of a new amino acid in prothrombin.

With large numbers of hydroxyl groups, the trimethylsilyl derivatives become too large in mass for the range of many quadrupole mass spectrometers, so methylation is used. However, this would cause problems in structural elucidation since those compounds in which demethylation of the methoxyl or dimethylamino group had taken place would be remethylated, thus obscuring these demethylating processes. This problem can be overcome by the use of deuteriomethylation. We originally carried this out by equilibrating the extract with 0-deuteriomethanol, to exchange all hydroxyl and amino hydrogens, and carrying out the methylation with diazomethane in deuteriomethanol. This, however, allows the formation of some undeuteriated methoxyl products. Commercial kits are now available for producing dideuteriodiazomethane of over 99 % purity. When monitoring of the methoxybenzyl ion at mle 121 was carried out on the deuteriomethylated extract at pH 9 from a volunteer taking mepyramine, the g.1.c.-m.s. run gave only one response, at the correct retention time for mepyramine. However, when mle 124 was monitored a response was again obtained at the same retention time as for mepyramine, and was about 10% of the response for mle 121. Since mle 124 corresponds to trideuteriomethoxybenzyl, this must have been formed from 0-demethylated mepyramine, which was present to the extent of about 10% of the level of unchanged drug. It was also expected that a metabolite would be formed in which the methoxybenzyl ring of compound (9) had become further hydroxylated. This hydroxylation could occur either before or after demethylation of the methoxyl group occurred. In the first case deuteriomethylation would lead to a methoxy deuteriomethoxybenzyl substituent, giving an ion of mle 154 whereas the second alternative would yield a bis deuteriomethoxy substitution on the benzyl ring, giving an ion of mle 157. Multiple-ion monitoring of both these ions on a g.1.c.-m.s. run gave a response only at mle 154. A mass spectrum obtained on this metabolite was identical with themass spectrum of deuteriomethylated compound (12), so that it can be assumed that hydroxylation has occurred at the 3 position of the methoxybenzyl substituent. Although mepyramine suffers 0-demethylation, no N-demethylation was seen to occur. Sidechain fission produces an ion of mle 58 due to CHz=NMez. Deuteriomethylation of an N-demethylated metabolite would shift this ion to mle 61, whereas deuteriomethylation of an N-didemethylated metabolite would shift this ion to mle 64. A g.1.c.-m.s. run monitoring these three ions produced responses only for m/e 58, thus ruling out any N-demethylation.