JOURNAL OF MASS SPECTROMETRY J. Mass Spectrom. 2005; 40: 1300–1308 Published online 3 October 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jms.905 Improved proton affinity measurements for proline and modified prolines using triple quadrupole and ion trap mass spectrometers S. Mezzache, 1 N. Bruneleau, 1 K. Vekey, 2 C. Afonso, 1 P. Karoyan, 1 F. Fournier 1 and J.-C. Tabet 1* 1 Synth ` ese, Structure et Fonction de Mol ´ ecules Bioactives, CNRS, UMR 7613, Universit ´ e Pierre et Marie Curie, 4 place Jussieu, 75252 Paris cedex 05, France 2 Chemical Research Center, Hungarian Academy of Sciences, Pusztaszeri 59-67, H-1025 Budapest, Hungary Received 25 April 2005; Accepted 11 July 2005 Proton affinity (PA) of compounds such as proline, cis-3-methylproline, cis-3-ethylproline, cis-3- isopropylproline and cis-3-isopentanylproline was determined by kinetic method with amines as the reference bases. The effective temperatures determined using ion trap and triple quadrupole mass spectrometers were found to be significantly different. In the case of the triple quadrupole instruments, the effective temperature depends significantly on the collision energy. The influence of the apparent basicity (GB app ) on the effective temperature may be used to estimate the difference in protonation entropy (11S° ) between the sample and reference compounds. In case of the ion trap mass spectrometer, the variation of the effective temperature as a function of the excitation amplitude is small, so it is difficult to account for the contribution of the entropy effects to the proton affinity value. A better estimation of the PA and 11S° values for the investigated molecules is obtained by combining the GB app and T eff data pairs that are obtained from both the mass spectrometers. Copyright  2005 John Wiley & Sons, Ltd. KEYWORDS: amino acids; kinetic method; proton affinity; effective temperature; entropy INTRODUCTION The biological role of certain noncovalent complexes depends on their conformation and structure. Therefore, the charac- terization and study of noncovalent 1 associations is very important for the understanding of biochemical mecha- nisms. In order to rationalize these noncovalent associations, knowledge of the physical and chemical properties of the noncovalent complexes in a solvent-free environment might be required. Mass spectrometry allows the determination of the intrinsic thermochemical properties of noncovalent com- plexes in the absence of external chemical factors such as solvent, salt presence and the pH value. One of the widely used experimental approaches to determine the thermochemical data such as basicities and acidities is the kinetic method developed by Cooks et al. 2,3 more than 25 years ago. In the kinetic method, 2,3 determination of proton affinities (PA) is based upon the comparison of the relative rate constants of the competitive dissociations (k o and k i ⊳ of selected hydrogen-bonded heterodimers [(B o H C B i ⊳], where B o is a base with unknown proton affinity value (PA(B o )) and B i , is one with known L Correspondence to: J.-C. Tabet, Synth` ese, Structure et Fonction de Mol´ ecules Bioactives, CNRS, UMR 7613, Universit´ e Pierre et Marie Curie, 4 place Jussieu, 75252 Paris cedex 05, France. E-mail: tabet@ccr.jussieu.fr Contract/grant sponsor: CEB, CNRS and UPMC. proton affinity value (PA(B i )) (Eqn (1)). (B i + B o )H + B i + B o H + B o + B i H + k i k o (a) (b) ⊲1⊳ Such competitive dissociations can occur either by metastable dissociation (with sector instruments) or using collision-induced dissociation (ion trap, FTICR and triple quadrupole mass spectrometers). The k i /k o ratio (Eqn (2)) therefore allows the determination of the proton affinity of B o relative to the B i base. The k i /k o ratio is approximately equal to the abundance ratio of the product ions I(B i H C ) and I(B o H C ) measured from the tandem mass spectrum of (B i C B o )H C and is related to the proton affinity difference as shown in Eqn (2). ln⊲k i /k o ⊳ ³ ln[I⊲B i H C ⊳/I⊲B o H C ⊳] D [PA⊲B i ⊳  PA⊲B o ⊳]/RT eff ⊲2⊳ The effective temperature (T eff ⊳ is a measure of the mean internal energy of ions which dissociate within the time- window characteristic of a particular analyzer and operating conditions. 4–6 The effective temperature depends 4–6 upon the molecular [the activation energy (E o ⊳, the degrees of Copyright  2005 John Wiley & Sons, Ltd.