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) a mino acid derivatives. Key components of the methodology are the use o f a solvent system consisting of methanol/dichloromethane (1:1 v/v) co ntaining 5-mM lithium triflate, a stainless steel electrode having a r elatively large surface area, and a microscale electrospray nozzle tha t provides for stable electrospray at flow rates in the range of 100-5 00 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/mu L. For all except the arginine derivative, there was a decrease in the signal intensity with increasi ng flow rate with 100-300 nL/min being optimum. Collision induced diss ociation (CID) product ion spectra were obtained for 21 derivatives in cluding carboxymethyl cysteine and dehydrothreonine. Leucine and isole ucine can be distinguished on the basis of their CID product ion spect ra. A subfemtomole detection Limit was demonstrated for the phenylalan ine 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 conventio nal 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 mi crosequencing using standard Edman degradation chemistry. (C) 1997 Ame rican Society for Mass Spectrometry.