To elucidate the mechanism of the exceptional behavior of lysine for the ionization (protonation) yields in matrix‐assisted laser desorption/ionization (MALDI) observed by Nishikaze and Takayama [Rapid Commun. Mass Spectrom. 2006, 20, 376], the temperature dependences of proton affinity (PA) and gas phase basicity for 20 amino acids are theoretically analyzed with correlated ab initio molecular orbital method under ideal gas condition. We have found that two different conformations, the linear structure with elongation of the side chain and the folded one having intramolecular hydrogen bonding, play important roles for the exceptional behavior of lysine. At low temperatures of around 298 K, the most stable conformation of the protonated lysine is the folded structure due to the formation of intramolecular hydrogen bonding. Meanwhile, at high temperatures, the Gibbs free energy of linear structure of protonated lysine becomes lower than that of the folded one because of the increment of vibrational entropic contribution. To explicitly take account of the contribution of the free energies, we have proposed the effective PA values thermally averaged using the ratio of Boltzmann distributions for two conformations. Since the effective PA value for lysine drastically decreases as the temperature increases above 1000 K, the linear correlation is clearly obtained between our effective PA values at high temperature and the ion yields in MALDI. Copyright © 2011 John Wiley & Sons, Ltd.