Parameter optimization for the analysis of underivatized protein amino acids by liquid chromatography and ionspray tandem mass spectrometry

The analysis of the 20 underivatized protein amino acids by liquid chromato graphy ionspray tandem mass spectrometry is investigated in positive ion mo de. First, by direct infusion, amino acid fragmentation was investigated ba sed on the product-ion mass spectrum of the parent compound in three differ ent collision energies (10, 20, 30 eV). Then, the relative abundance of fra gment ions was studied as a function of the collision energy in order to se lect the product ion with the highest abundance and to obtain the maximum s ensitivity for each amino acid, by using the optimum collision energy. Depe nding on the amino acid, the loss of H2O or NH3 or CH2O2 was selected as th e product ion from the molecular ion [M+H](+) in selective reaction monitor ing mode. 15 eV was chosen as a mean value of collision energy to obtain sa tisfactory sensitivity for the simultaneous determination of the 20 protein amino acids. In spite of the specificity of mass spectrometry, and in orde r to obtain maximum sensitivity, several pairs of amino acids had to be sep arated. The separation of these amino acids pairs was achieved in less than 20 min by using a porous graphitic carbon column and nonafluoropentanoic a cid as ion-pairing reagent. Detection limits depending on the amino acid va ried from 500 fmol to 40 pmol (using a 10 mul loop). (C) 2000 Elsevier Scie nce B.V. All rights reserved.