Automated carboxy-terminal sequence analysis of peptides.
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 solution phase conditions for formation of the peptidylthiohydantoins with trimethylsilylisothiocyanate (TMS-ITC) and for hydrolysis of these peptidylthiohydantoins into an amino acid thiohydantoin derivative and a new shortened peptide capable of continued degradation (Bailey, J. M. & Shively, J. E., 1990, Biochemistry 29, 3145-3156). The current study is a continuation of this work and describes the construction of an instrument for automated C-terminal sequencing, the application of the thiocyanate chemistry to peptides covalently coupled to a novel polyethylene solid support (Shenoy, N. R., Bailey, J. M., & Shively, J. E., 1992, Protein Sci. I, 58-67), the use of sodium trimethylsilanolate as a novel reagent for the specific cleavage of the derivatized C-terminal amino acid, and the development of methodology to sequence through the difficult amino acid, aspartate. Automated programs are described for the C-terminal sequencing of peptides covalently attached to carboxylic acid-modified polyethylene. The chemistry involves activation with acetic anhydride, derivatization with TMS-ITC, and cleavage of the derivatized C-terminal amino acid with sodium trimethylsilanolate. The thiohydantoin amino acid is identified by on-line high performance liquid chromatography using a Phenomenex Ultracarb 5 ODS(30) column and a triethylamine/phosphoric acid buffer system containing pentanesulfonic acid. The generality of our automated C-terminal sequencing methodology was examined by sequencing model peptides containing all 20 of the common amino acids. All of the amino acids were found to sequence in high yield (90% or greater) except for asparagine and aspartate, which could be only partially removed, and proline, which was found not be capable of derivatization. In spite of these current limitations, the methodology should be a valuable new tool for the C-terminal sequence analysis of peptides.
Bailey
J M
JM
Beckman Research Institute of the City of Hope, Division of Immunology, Duarte, California 91010.
Shenoy
N R
NR
Ronk
M
M
Shively
J E
JE
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D001331:Automation; D002851:Chromatography, High Pressure Liquid; D008567:Membranes, Artificial; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D011095:Polyethylenes / Q000737:chemistry; D017421:Sequence Analysis / Q000295:instrumentation*; D012821:Silanes / Q000737:chemistry; D013861:Thiocyanates / Q000737:chemistry; D013867:Thiohydantoins / Q000737:chemistry
D000596:Amino Acids; D008567:Membranes, Artificial; D010455:Peptides; D011095:Polyethylenes; D012821:Silanes; D013861:Thiocyanates; D013867:Thiohydantoins