Evaluation of aminolysis of anilinothiazolinones to phenylthiocarbamoyl amino acid methyl amides as an alternative conversion method in protein sequencing.

The aminolysis of products of sequential degradation of proteins and peptides by methylamine is an alternative method of conversion of the unstable 5-alkyl-2-anilino-4-thiazolinones into the stable methyl amides of N alpha-phenylthiocarbamoyl amino acids. The volatility of methylamine permits use in the gas phase during both manual and automatic sequential degradation. Two procedures were studied: (mode A) aminolysis by methylamine in the sequencer reaction chamber after liberation of the thiazolinones by trifluoroacetic acid and (mode B) aminolysis by methylamine vapors passed through a 1-chlorobutane solution of thiazolinones in the conversion flask of the sequencer. The sequencing program was modified for both procedures by making use of the standard sequencer functions. The yields of aminolysis in the conversion flask (mode B) are comparable to those obtained by standard conversion in 25% trifluoracetic acid and the procedure does not affect the repetitive yield. Aminolysis on the glass filter (mode A) requires a major modification of the degradation process, yet gives higher yields of the degraded amino acid derivatives. A disadvantage of both procedures, especially of mode A, is the presence of N-methyl-N'-phenylthiourea in the methyl amide samples. We have not been able to achieve the expected improvement of the yields of degraded hydroxy amino acids. Therefore the replacement of acid conversion of anilinothiazolinones to phenylthiohydantiones by aminolysis for routine degradation cannot be recommended. High yields of methyl amides make aminolysis a promising candidate for the incorporation of fluorescent or other labels in the products of sequencing degradation.