N-terminal sequencing and PITC derivatives of proteins

In N-terminal sequencing of proteins phenylisothiocyanate (PITC) is used to modify the N-terminus of the targeted protein. This, combined with a number of steps that assure chain cleavage and phenylthiohydantoin amino acid adduct formation are used in N-terminal sequencing machines like the ABI Procise series of N-terminal sequencers. A schematic of the reaction is shown below. In the first step a base is used to bring the pH to 9.0 and the PITC is introduced in hexane. A phenylthiocarbamyl (PTC) adduct of the N-terminus is formed. Next, anhydrous trifluoroacetic acid (TFA) is introduced to initiate chain cleavage and the release of a thiazolinone derivative of the N-terminal amino acid. This derivative is mixed with aqueous trifluoroacetic acid in step three to from a stabile phenylthiohydantoin (PTH) amino acid. The derivative is injected onto a reversed phase HPLC column and the elution position of the PTH derivative is used to identify the N-terminal amino acid. The cycle is repeated and the next amino acid is determined. This is repeated until there is no more signal (circa 50 amino acids in a well behaved protein sample) and the amino acid sequence can be read from the chromatographic traces by a trained operator.

Figure 1: PITC derivitization used for N-terminal sequencing