CHRISTMAS: Chiral Pair Isobaric Labeling Strategy for Multiplexed Absolute Quantitation of Enantiomeric Amino Acids

• Published in: Analytical chemistry (Washington) Vol. 95; no. 50; pp. 18504 - 18513
• Main Authors: Zhu, Zhijun, Xu, Shuling, Wang, Zicong, Delafield, Daniel G., Rigby, Michael J., Lu, Gaoyuan, Gu, Ting-Jia, Liu, Peng-Kai, Ma, Min, Puglielli, Luigi, Li, Lingjun
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 19.12.2023
• Subjects: Alzheimer's disease; Amines; Amino acids; Amino Acids - analysis; Animals; Chromatography, Liquid - methods; Enantiomers; Ionic mobility; Isobars; Isomers; Labeling; Leucine; Leucine - chemistry; Mass spectrometry; Mass spectroscopy; Mice; Multiplexing; Neurodegenerative diseases; Proteomics - methods; Quantitation; Reagents; Separation
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/acs.analchem.3c03847

Amino acids (AAs) in the d-form are involved in multiple pivotal neurological processes, although their l-enantiomers are most commonly found. Mass spectrometry-based analysis of low-abundance d-AAs has been hindered by challenging enantiomeric separation from l-AAs, low sensitivity for detection, and lack of suitable internal standards for accurate quantification. To address these critical gaps, N,N-dimethyl-l-leucine (l-DiLeu) tags are first validated as novel chiral derivatization reagents for chromatographic separation of 20 pairs of d/l-AAs, allowing the construction of a 4-plex isobaric labeling strategy for enantiomer-resolved quantification through single step tagging. Additionally, the creative design of N,N-dimethyl-d-leucine (d-DiLeu) reagents offers an alternative approach to generate analytically equivalent internal references of d-AAs using d-DiLeu-labeled l-AAs. By labeling cost-effective l-AA standards using paired d- and l-DiLeu, this approach not only enables absolute quantitation of both d-AAs and l-AAs from complex biological matrices with enhanced precision but also significantly boosts the combined signal intensities from all isobaric channels, greatly improving the detection and quantitation of low-abundance AAs, particularly d-AAs. We term this quantitative strategy CHRISTMAS, which stands for chiral pair isobaric labeling strategy for multiplexed absolute quantitation. Leveraging the ion mobility collision cross section (CCS) alignment, interferences from coeluting isomers/isobars are effectively filtered out to provide improved quantitative accuracy. From wild-type and Alzheimer’s disease (AD) mouse brains, we successfully quantified 20 l-AAs and 5 d-AAs. The significant presence and differential trends of certain d-AAs compared to those of their l-counterparts provide valuable insights into the involvement of d-AAs in aging, AD progression, and neurodegeneration.