Mass spectrometric studies on protonated and alkali metal cationized [beta]-amino acids and [beta]-peptides

Abstract

b-alanine (b-Ala) and peptides containing b-Ala are the most commonly found b-amino acid/b-peptides in nature, b-peptides are more resistant to enzymatic degradations than their a-amino acid analogues, and hold the key to a new generation of bacteria-resistance drugs. Mass spectrometry is the preferred analytical technique for identification and differentiation of b-Ala and b-peptides from their a-analogues at the micro- to picomole level. However, the intrinsic properties related to mass spectrometric characterization, e.g., proton (H+) and alkali metal cation (M+) binding affinities in the gas phase, and their mass spectrometric fragmentation characteristics have not been systematically investigated. In the present study, the alkali metal cation binding affinities of b-Ala and the proton affinities of model b-dipeptides were determined by the mass spectrometric kinetic method. The M+ affinities (M+ = Li+, Na+ and K+) of b-Ala and (b-Ala)OMe (in kJ mol-1) were determined to be: Li+-(b-Ala) (247.0), Li+-(b-Ala)OMe (256.0), Na+-(b-Ala) (165.1), Na+-(b-Ala)OMe (173.9), K+-(b-Ala) (133.8) and K+-(b-Ala)OMe (131.0). The experimental Li+ / Na+ and K+ affinities are in very good agreement with our theoretical values obtained at G2(MP2,SVP) level and B3-LYP/6-311+G(3df,2p) //B3-LYP/6-31G+(d) level, respectively, with a mean-absolute-deviation (MAD) of +- 5.2 kJ mol-1 only. The most stable Li+and Na+ bound structures of b-Ala is the charge-solvated CS1 form (Li+/Na+ binding to the NH2 and C=O sites), while the CS2 form (K+ binding to two carboxylic oxygens) is the most stable binding mode for K+-(b-Ala). For M+-(b-Ala)OMe, methylation leads to the CS1 mode to be the most stable binding mode for all the alkali cations (Li+, Na+ and K+). The proton affinities of four model b-dipeptides has been determined (in kJ mol-1): Gly(b-Ala) 942.0, (b-Ala)Gly 971.3, Ala(b-Ala) 947.8, (b-Ala)Ala 970.3. The experimental values are found to be in very good agreement with theoretical affinities calculated at the B3-LYP/6-311+G(3df, 2p)//B3-LYP/6-31G+ level, with MAD of +- 6.4 kJ mol-1. The more stable proton binding site is found at the N-terminal nitrogen. Unlike a-dipeptides, the proton affinity is noticeably greater when b-Ala is located at the N-terminal than at the C-terminal of the b-dipeptide. b-Ala and b-dipeptides are found to have higher alkali metal cation affinities and proton affinities, respectively, than that of the corresponding a-Ala and a-dipeptides. The sole difference between a-Ala and b-Ala is the absence and presence, respectively, of a b-methylene -CH2-) unit between the N- and C-terminus of the ammo acid. The enhanced alkali metal cation affinities of b-Ala and proton affinities of b-dipeptides is attributed to the enhanced flexibility of the carbon chain in b-Ala, allowing (i) the alkali metal cation to approach the O/N heteratom sites at closer binding distances, and (ii) enhanced intramolecular hydrogen bonding in protonated b-dipeptides. The collision-induced dissociation of protonated b-Ala yields a dominant b1 (protonated b-lactam) fragment ion at m/z 72 by loss of H2O, which is not able to be formed from protonated a-Ala. This indicates that the additional -CH2- unit in b-Ala does have an effect on the fragmentation mechanisms of b-Ala. Other energetically preferred pathways are loss of CH2CO, and (CH2CO + H2O), while loss of NH=CH2, NH3 and (NH3 + CO) neutrals are observed under more energetic CID conditions. The stable intermediates and transitional structures of the dissociation pathways of b-Ala are found by high level density functional theory calculations at the B3-LYP/6-311+G(3df,2p)//B3-LYP/6-31+G(d) level. The calculated free energy changes (as indicated by the calculated G298 values) can be suitably applied to rationalize the ion trap appearance threshold voltages and relative abundances of different fragment ions in the MS/MS spectrum of protonated b-Ala. The CID of protonated b-dipeptides with b-Ala at the C-terminus (Xxx(b-Ala), Xxx = Gly, Leu, Phe and Met) showed competitive formation of y1 and b'2(oxazinone) fragment ions, and the further dissociation of b'2(oxazinone) to yield preferably fragment ions due to (i) loss of NH3 and loss of NH=CH2 (29 Da). Aside from y1 ion formation, protonated b-dipeptides with b-Ala at the C-terminus ((b-Ala)Xxx, Xxx =Gly, Phe, Trp and His) also showed characteristic and competitive formation of b1 ion (protonated b-lactam) and fragment ions due to loss of NH3. Furthermore, (b-Ala)-containing b2(oazolone) ions dissociate further by loss of NH=CH2 and other neutrals specific to b-Ala, thus providing confirmatory evidence in the differentiation of isomeric a- and b-dipeptides.