Discrimination of Threonine Isomers by Multiple-Stage Tandem Mass Spectrometry with Collision-Induced Dissociation

Fragmentation of protonated threonine isomers was investigated by multiple-stage tandem mass spectrometry (MS<i>ⁿ</i>) with ion-trap collision-induced dissociation (CID) and density functional theory (DFT) calculations. Protonated molecules containing oxygen or nitrogen often produce heterocyclic fragments by CID. DFT calculations revealed that H₂O loss from threonine isomers produced cyclic amines, lactams, and lactones. The transition-state barriers and rate constant for the formation of these fragments are highly dependent on the ring size. Although 3-membered cyclic amines are observed in the product ion spectrum, lactams and lactones are formed only as rings larger than four and five members, respectively. H₂O loss from the protonated threonine side chain produced a 3-membered cyclic amine containing a carboxyl group, which undergoes loss of the combined elements of H₂O and CO upon CID. H₂O loss from protonated homoserine and β-homoserine produced 5-membered lactone and 4-membered lactam, respectively. Further dissociation of the corresponding lactone and lactam results in fragment ions formed via the loss of CO and CH₂CO, respectively. In contrast, H₂O loss from the protonated γ-amino-β-hydroxybutyric acid provides 5-membered lactams, which undergo only further H₂O loss upon CID. MS³ analysis of protonated threonine isomers through dehydrated precursor ions produced different fragment ions. The threonine isomers could be distinguished by characteristic fragment ions, and the molar ratio of the isomers in the mixture can be estimated from the relative abundances of their fragment ions. These results demonstrate MS<i>ⁿ</i> with ion-trap CID to be a useful method for the identification and semiquantification of threonine isomers.