Protein Sequencing

The chemical process used in the Procise system is derived from the technique developed by Pehr Edman in the 1950’s for the sequential degradation of proteins and peptides. In the Edman degradation, a protein’s N-terminal amino acid is specifically reacted with phenylisothiocyanate (PITC). This derivatized aminoacid is then selectively removed, leaving the rest of the peptide chain intact. Each cycle of the degradation removes the new N-terminal amino acid from the peptide chain. The resulting PTHamino acids are analyzed sequentially (by RP HPLC) to determine the amino acid sequence of the protein or peptide. The Laboratory operates the Procise 491 HT Protein Sequencing System and the Procise 494 cLC Protein Sequencing System (high sensitivity analyses at sub-picomole level).

Amino Acid Analysis

Our Laboratory conducts several hundreds of analyses of sample concentration, composition, and purity annually, thus aiding the research teams in their proteomics, protease inhibition, recombinant protein, and incorporation studies. Typically, amino acids bound in peptide or protein are hydrolyzed in a vacuum for 20 hours by means of 6N HCl, the hydrolysate is vacuum evaporated, reconstituted in loading buffer (pH 2.2) containing the internal standard norleucine, and injected onto the Biochrom 20 Amino Acid Analyzer. Amino acids are separated primarily according to their charge and secondarily according to their size and hydrophobicity. Effluent amino acids mix on-line with the ninhydrin detection reagent, react at elevated temperature, and their derivatives are detected photometrically at 570 and 440 nm. Hydrolysis and analysis usually require about two business days. The estimated amount of protein required for an accurate analysis is 0.1 to 0.5 mg. Generally, pure samples are required as the presence of salts, buffers (phosphate, sulphate, amine buffers), carbohydrates, glycerol, and detergents is potentially deleterious. Samples containing heavy metals are unacceptable. The average internal error is between 5 and 10%.