AUTOMATED CARBOXY-TERMINAL SEQUENCE-ANALYSIS OF POLYPEPTIDES CONTAINING C-TERMINAL PROLINE

Proteins and peptides can be sequenced from the carboxy-terminus with isothiocyanate reagents to produce amino acid thiohydantoin derivative s. Previous studies in our laboratory have focused on automation of th e thiocyanate chemistry using diphenyl phosphoroisothiocyanatidate (DP P-ITC) and pyridine to derivatize the C-terminal amino acid to a thioh ydantoin and sodium trimethylsilanolate for specific hydrolysis of the derivatized C-terminal amino acid (Bailey, J. M., Nikfarjam, F., Shen oy, N. S., and Shivery, J, E. (1992) Protein Sci. 1, 1622-1633). A maj or limitation of this approach was the inability to derivatize C-termi nal proline. We now describe chemistry based on the DPP-ITC/pyridine r eaction which is capable of derivatizing C-terminal proline to a thioh ydantoin. The reaction of DPP-ITC/pyridine with C-terminal proline rap idly forms an acyl isothiocyanate which is capable of forming a quater nary amine containing thiohydantoin, Unlike formation of peptidylthioh ydantoins with the other 19 commonly occurring amino acids in which cy clization to a thiohydantoin is concomitant with loss of a proton from the amide nitrogen, proline has no amide proton and as a result the n ewly formed proline thiohydantoin contains an unprotonated ring nitrog en. This cyclic structure if left unprotonated will regenerate C-termi nal proline during the cleavage reaction. However, if protonated by th e addition of acid, the proline thiohydantoin ring is stabilized and c an be readily hydrolyzed to proline thiohydantoin and a shortened pept ide by the addition of water vapor or alternatively by sodium or potas sium trimethylsilanolate, the reagent normally used for the cleavage r eaction. By introducing vapor-phase trifluoroacetic acid (TFA) for the protonation reaction and water vapor for the hydrolysis reaction we h ave been able to automate the chemistry required for derivatization of C-terminal proline. Since the TFA/water steps have no effect on pepti dylthiohydantoins formed from the other 19 amino acids, the additional steps required for proline were readily integrated into the automated sequencing program, providing for the first time an automated sequenc ing program which permits the C-terminal sequence analysis of all 20 o f the commonly occurring amino acids. Automated programs are described for the C-terminal sequencing of peptides covalently attached to carb oxylic acid-modified polyethylene and larger polypeptides noncovalentl y applied to Zitex (porous Teflon). (C) 1995 Academic Press, Inc.