Using Low Energy ESI Tandem MS To Differentiate α-and β-Aspatic Acids In The HeptaPeptides

Since mass spectrometry has its own advantages, so it is widely recognized as one of the best analytical tools for characterizing the primary structure of peptides and proteins. Proteins are consisted by various amino acids and control numerous biological processes. Aspartic acid (Asp) is an aliphatic, acidic amino acid and has a pair of isomers, which areα- andβ-aspartic acid. Theα-Asp is a common amino acid in proteins and theβ-Asp is a no-natural amino acid that presents in a protein or peptide by aβ-carboxyl group connecting polypeptide backbone and leaving aα-carboxyl group as side chain. Theβ-Asp has only one more methylene thanα-Asp in polypeptide backbone, but which can lead to the complete or partial loss of the biological activity of proteins. Recent progress and improvement have discovered that theβ-Asp relate to some diseases, such as Alzheimer's disease (AD). Furthermore, Numbers of synthetic peptides have been developed as drugs for pharmaceutical applications, Therefore fast and effective methods are required to tracking the presence and generation of the Asp isomer in protein or peptide are more important. In this work, we use low-energy ESI-MS/MS to differentiateα- andβ-Aspartic Acids in the heptaPeptides in these two aspects:1. The fragmentations of the 16 heptapeptides that contained twoα- orβ-Asp residues in the 2 and 6 positions were studied under low energy CAD conditions by ESI-MS/MS. When theα-Asps were replaced byβ-Asps in the peptides, the ratios of bn-H₂O/bn (n = 2 - 5) increased but the ratios of M+H-H₂O/M+H decreased. Therefore, we first used the ratios of bn-H₂O/bn (n = 2 - 5) and M+H-H₂O/M+H to discover the changes in the Asp isomer. Unfortunately, the two ratios were independent of the Asp position and could not differentiate the Asp isomer in a specific position. The ratios of bn/yl-n (where n = 2-5) were higher in the peptide that hadα-Asps. This was different from the work by Lehmann et al, which proposed that the b/y ratios only before and after Asp changed could be used for distinguishing the Asp isomers. Furthermore, close to the C terminal, the ratio shift of b₆/y₁ was affected by the peptide terminal group, and the ratio change of b₆-H₂O/b₆ was influenced by the sequence, which could not be tracked to the shift of the Asp isomer. Our results showed that the formation of b, y, b-H₂O, M+H-H₂O ions followed certain rules and involved several pathways that could be complex and competitive; however, the exact mechanism for these specific reactions needs further investigation.2. ESI ion trap MS/MS and ESI quadrupole MS/MS are used respectively to investigate the heptapeptides with CH₃COONa. Based on the results of the experiments, We suggest that the diagnostic peak bn+Na+OH (n represents Asp position) which generated by the sodium-cationized peptides containingα-Asp could identify Asp isomers at the C-terminal of the peptides which have the normal terminal and acylated N terminal. But this diagnostic peak ofα-Asp could not distinguish theα- andβ-Asp which are adjacent to the N terminals. However the reason for these specific results need to be further investigated.