The Kinetic Method: Determination of Proton Affinity of Proline by Cis and Trans Hydroxylation

Unknown Proton affinity (PA) of the amino acids were determined by comparing them with known PA of the reference standards. Initially we mixed 2 amino acids together [unknown (PA) and known (PA)] and introduce into mass spectrometer using direct infusion. After injecting we observe the peaks. The protonated dimer peak of known PA and unknown PA samples were isolated and fragmented. Once they were fragmented protonated dimer will produce two peaks corresponding to amino acid with unknown PA and amino acids with known PA. Based on the ratio of these amino acids we determine the unknown PA of an amino acid. Proline, trans-4-hydroxyproline and cis-4-hydroxyproline were used as amino acids with unknown PA and tryptophan, asparagine, tyrosine, methionine and phenylalanine as reference bases