Automated Carboxy-Terminal Sequence Analysis of Polypeptides Containing C-Terminal Proline

• Published in: Analytical biochemistry Vol. 224; no. 2; pp. 588 - 596
• Main Authors: Bailey, J.M., Tu, O., Issai, G., Ha, A., Shively, J.E.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 20-01-1995
• Subjects: Acetic Anhydrides - chemistry; Amino Acids - analysis; Chromatography, High Pressure Liquid; Hydrolysis; Isothiocyanates; Magnetic Resonance Spectroscopy; Mass Spectrometry; Peptides - analysis; Proline - analogs & derivatives; Proline - analysis; Proline - chemical synthesis; Proline - chemistry; Pyridines - chemistry; Sequence Analysis - methods; Spectrophotometry; Thiocyanates - chemistry; Thiohydantoins - chemical synthesis
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.1995.1091

Proteins and peptides can be sequenced from the carboxy-terminus with isothiocyanate reagents to produce amino acid thiohydantoin derivatives. Previous studies in our laboratory have focused on automation of the thiocyanate chemistry using diphenyl phosphoroisothiocyanatidate (DPP-ITC) and pyridine to derivatize the C-terminal amino acid to a thiohydantoin and sodium trimethylsilanolate for specific hydrolysis of the derivatized C-terminal amino acid (Bailey, J. M., Nikfarjam, F., Shenoy, N. S., and Shively, J. E. (1992) Protein Sci. 1, 1622-1633). A major limitation of this approach was the inability to derivatize C-terminal proline. We now describe chemistry based on the DPP-ITC/pyridine reaction which is capable of derivatizing C-terminal proline to a thiohydantoin. The reaction of DPP-ITC/pyridine with C-terminal proline rapidly forms an acyl isothiocyanate which is capable of forming a quaternary amine containing thiohydantoin. Unlike formation of peptidylthiohydantoins with the other 19 commonly occurring amino acids in which cyclization to a thiohydantoin is concomitant with loss of a proton from the amide nitrogen, proline has no amide proton and as a result the newly formed proline thiohydantoin contains an unprotonated ring nitrogen. This cyclic structure if left unprotonated will regenerate C-terminal proline during the cleavage reaction. However, if protonated by the addition of acid, the proline thiohydantoin ring is stabilized and can be readily hydrolyzed to proline thiohydantoin and a shortened peptide by the addition of water vapor or alternatively by sodium or potassium trimethylsilanolate, the reagent normally used for the cleavage reaction. By introducing vapor-phase trifluoroacetic acid (TFA) for the protonation reaction and water vapor for the hydrolysis reaction we have been able to automate the chemistry required for derivatization of C-terminal proline. Since the TFA/water steps have no effect on peptidylthiohydantoins 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 sequencing program which permits the C-terminal sequence analysis of all 20 of the commonly occurring amino acids. Automated programs are described for the C-terminal sequencing of peptides covalently attached to carboxylic acid-modified polyethylene and larger polypeptides noncovalently applied to Zitex (porous Teflon).