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Title: Mass spectrometric studies on protonated and alkali metal cationized β-amino acids and β-peptides
Authors: Chan, On-ying
Keywords: Hong Kong Polytechnic University -- Dissertations
Mass spectrometry
Amino acid sequence
Alkali metals
Issue Date: 2006
Publisher: The Hong Kong Polytechnic University
Abstract: β-alanine (β-Ala) and peptides containing β-Ala are the most commonly found β-amino acid/β-peptides in nature, β-peptides are more resistant to enzymatic degradations than their α-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 β-Ala and β-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 β-Ala and (β-Ala)OMe (in kJ mol⁻¹) were determined to be: Li⁺-(β-Ala) (247.0), Li⁺-(β-Ala)OMe (256.0), Na⁺-(β-Ala) (165.1), Na⁺-(b-Ala)OMe (173.9), K⁺-(β-Ala) (133.8) and K⁺-(β-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 β-Ala is the charge-solvated CS1 form (Li⁺/Na⁺ binding to the NH₂ and C=O sites), while the CS2 form (Ki⁺ binding to two carboxylic oxygens) is the most stable binding mode for Ki⁺-(β-Ala). For M⁺-(β-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 β-dipeptides has been determined (in kJ mol⁻¹): Gly(β-Ala) 942.0, (β-Ala)Gly 971.3, Ala(β-Ala) 947.8, (β-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 α-dipeptides, the proton affinity is noticeably greater when β-Ala is located at the N-terminal than at the C-terminal of the β-dipeptide.
β-Ala and β-dipeptides are found to have higher alkali metal cation affinities and proton affinities, respectively, than that of the corresponding α-Ala and α-dipeptides. The sole difference between α-Ala and β-Ala is the absence and presence, respectively, of a β-methylene -CH₂-) unit between the N- and C-terminus of the ammo acid. The enhanced alkali metal cation affinities of β-Ala and proton affinities of b-dipeptides is attributed to the enhanced flexibility of the carbon chain in β-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 β-dipeptides. The collision-induced dissociation of protonated β-Ala yields a dominant b₁ (protonated β-lactam) fragment ion at m/z 72 by loss of H₂O, which is not able to be formed from protonated α-Ala. This indicates that the additional -CH₂- unit in β-Ala does have an effect on the fragmentation mechanisms of β-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 β-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 ΔG₂₉₈ 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 β-Ala. The CID of protonated β-dipeptides with β-Ala at the C-terminus (Xxx(β-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 β-dipeptides with β-Ala at the C-terminus ((β-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, (β-Ala)-containing b₂(oazolone) ions dissociate further by loss of NH=CH₂ and other neutrals specific to β-Ala, thus providing confirmatory evidence in the differentiation of isomeric a- and β-dipeptides.
Description: xx, 176 p. : ill. ; 30 cm.
PolyU Library Call No.: [THS] LG51 .H577M ABCT 2006 Chan
Rights: All rights reserved.
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