High sensitivity analysis of phenylthiohydantoin amino acid derivatives by electrospray mass spectrometry

A new methodology has been developed for high sensitivity electrospray ionization mass spectrometric analyses of phenylthiohydantoin (PTH) amino acid derivatives. Key components of the methodology are the use of a solvent system consisting of methanol/dichloromethane (1:1 v/v) containing 5-mM lithium triflate, a stainless steel electrode having a relatively large surface area, and a microscale electrospray nozzle that provides for stable electrospray at flow rates in the range of 100–500 nL/min. A linear response for the absolute signal intensity of the protonated molecule was observed for a number of derivatives over the concentration range of 50–1000 fmol/µL. For all except the arginine derivative, there was a decrease in the signal intensity with increasing flow rate with 100–300 nL/min being optimum. Collision induced dissociation (CID) product ion spectra were obtained for 21 derivatives including carboxymethyl cysteine and dehydrothreonine. Leucine and isoleucine can be distinquished on the basis of their CID product ion spectra. A subfemtomole detection limit was demonstrated for the phenylalanine PTH derivative in a selected reaction monitoring (SRM) experiment. Samples from an automated Edman microsequencer run have been analyzed using the new technique and compared to results obtained by conventional high-performance liquid chromatography analysis with UV detection. This work demonstrates the feasibility of using mass spectrometry to identify and quantitate the products generated by automated protein microsequencing using standard Edman degradation chemistry.