C-terminal sequence analysis of peptides using triphenylgermanyl isothiocyanate.
The Schlack-Kumpf degradation, also called the isothiocyanate method, is thought to be a promising approach to chemical C-terminal sequencing of peptides and proteins. The derivatizing reagent is most crucial to this method. A new derivatizing reagent, triphenylgermanyl isothiocyanate (TPG-ITC), has been synthesized and applied to C-terminal peptide sequencing. The chemistry involves activation with acetic anhydride, derivatization with TPG-ITC, and cleavage of the derivatized C-terminal amino acid thiohydantoin with sodium hydroxide. A series of reaction conditions, including activation reagent volume, activation time, and derivatization temperature and time, have been investigated using a model peptide covalently attached to 1,4-phenylene diisothiocyanate (DITC)-glass beads. This procedure has been successfully used to sequence eight C-terminal residues of a model peptide at low nanomole levels. TPG-ITC is a white solid with relatively long shelf-life. According to our previous article (B. Mo, J. Li, and S. P. Liang, 1997, Anal. Biochem. 252, 169-176), TPG-ITC is a type II derivatizing reagent. Compared with acetyl isothiocyanate and trimethylsilyl isothiocyanate, TPG-ITC is much more stable and efficient for use in peptide C-terminal sequencing.
Li
Junyong
J
College of Life Science, Hunan Normal University, Changsha, Hunan, 410081, People's Republic of China.
Liang
Songping
S
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D017879:Isothiocyanates / Q000737:chemistry*; D009942:Organometallic Compounds / Q000737:chemistry*; D020539:Sequence Analysis, Protein / Q000379:methods*
D017879:Isothiocyanates; D009942:Organometallic Compounds; C456582:triphenylgermanyl isothiocyanate; C037152:isothiocyanic acid