posted on 2023-11-30, 20:04authored byZhijun Zhu, Shuling Xu, Zicong Wang, Daniel G. Delafield, Michael J. Rigby, Gaoyuan Lu, Ting-Jia Gu, Peng-Kai Liu, Min Ma, Luigi Puglielli, Lingjun Li
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 pairisobaric 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.