Manual gas-phase isothiocyanate degradation

We describe a manual gas-phase isothiocyanate degradation procedure for the primary structure determination of proteins and peptides. The proteins and peptides are applied to a polybrene-coated glass fiber filter wedged into a small glass column. The phenylisothiocyanate is directly pipetted onto the filter disk. The coupling and cleavage reactions are performed in small desiccators containing trimethylamine and trifluoroacetic acid vapors, respectively. The wash and extraction steps are performed by allowing the suitable solvents to percolate through the filter disk. The extracted anilinothiazolinone is then converted to the phenylthiohydantoin and identified by any one of a number of described methods. Our results show that this method is very sensitive and that the reactions proceed faster than those of the published automated procedure. No expensive equipment is required and the manual degradation can be performed by a laboratory assistant. A large number of samples can be simultaneously subjected to the degradation under identical conditions, making this an ideal method for physicochemical investigations into the isothiocyanate degradation. We also use this method to screen HPLC fractions after enzymatic protein fragmentation. Manually sequenced glass filters can be transferred to the automated instrument for more extended degradations.