Discovery of Acylated Glycine and Alanine by Integrating Chemical Derivatization-Based LC-MS/MS and Knowledge-Driven Prediction.

Acylated amino acids (acyl-AAs), which consist of an amino acid head and an organic acid tail, play vital roles in various biological processes. Glycine (Gly) is the most common substrate for acylation with the organic acid tails exhibiting considerable diversity. Alanine (Ala) also exists in multiple acylated forms, predominantly modified by long-chain fatty acids. However, the full scope of acylated Gly and Ala remains largely unexplored. In this study, we employed a knowledge-driven prediction approach to expand the spectrum of acylated Gly and Ala by incorporating 111 organic acids from five different classes as potential acyl donors, leading to the generation of 222 acylated Gly and Ala species. To enhance mass spectrometry (MS) response, we used a chemical derivatization-based LC-MS/MS approach, employing dimethylamino-naphthalene-1-sulfonyl piperazine (Dns-PP) and its stable isotope-labeled form (-Dns-PP) as labeling reagents. Moreover, in-source fragmentation (ISF) was utilized to increase the fragment diversity and utility, aiding in structure elucidation. This strategy resulted in the identification of 53 acylated Gly and Ala metabolites in rat biological matrices, including 17 novel metabolites with distinct tissue-specific distributions. Our approach offers a deeper understanding of the physiological and pathological roles of acylated Gly and Ala, while also opening avenues for the discovery of other modified metabolites.