Microsequence analysis of peptides and proteins: Trimethylsilylisothiocyanate as a reagent for COOH-terminal sequence analysis

Abstract

A reinvestigation of the isothiocyanate-based chemistry for cyclic degradations of peptides and proteins revealed that the reagent trimethylsilylisothiocyanate (TMS-ITC) gives superior results in terms of coupling efficiency and lack of complicating side reactions. Acetic anhydride (10 min at various temperatures) was used to activate the carboxyl terminus, and 6 n HCl (30 min at room temperature) was used for cleavage as originally described by G. R. Stark ( Biochemistry, 8, 4735, 1968). Reaction conditions for efficient coupling were explored using subtractive chemistry on bradykinin, a nonapeptide, and separation of the reaction products by reverse-phase high-performance liquid chromatography. The products were analyzed by fast atom bombardment-mass spectrometry and shown to be the N-acetylated starting material and the N-acetylated des-Arg 9 derivative of bradykinin. The pseudo-first-order rate constants measured at 50, 70, and 90°C were 5.6 × 10 −5, 5.1 × 10 −4, and 8.6 × 10 −4 s −1, respectively. In order to obtain complete couplings within 30–40 min at 50°C, the effect of pyridine catalysis was studied. The addition of 0.225 m pyridine resulted in roughly doubling the rates at 50 and 70°C. In the case of bradykinin, the reaction with TMS-ITC in the presence of the pyridine catalyst at 50°C was complete in 15 min. In order to apply this methodology to the analysis of proteins, the thiohydantoin derivatives of amino acids were synthesized and separated by reverse-phase HPLC. The derivatives were also characterized by mass spectrometry. The above reaction conditions were tested on 3 nmol of sperm whale apomyoglobin for three cycles of degradation. The sample was first coupled to p-phenylene diisothiocyanate-derivatized aminopropyl glass with a 90% yield. The approximate initial yield of glycine at cycle one was 30%. The first three cycles corresponded exactly to the predicted carboxy-terminal sequence of myoglobin. These results demonstrate the feasibility of a new Stark reagent for automated carboxy-terminal chemistry.