Discovery of\nAcylated Glycine and Alanine by Integrating\nChemical Derivatization-Based LC-MS/MS and Knowledge-Driven Prediction

Acylated amino acids (acyl-AAs), which consist of an\namino acid\nhead and an organic acid tail, play vital roles in various biological\nprocesses. Glycine (Gly) is the most common substrate for acylation\nwith the organic acid tails exhibiting considerable diversity. Alanine\n(Ala) also exists in multiple acylated forms, predominantly modified\nby long-chain fatty acids. However, the full scope of acylated Gly\nand Ala remains largely unexplored. In this study, we employed a knowledge-driven\nprediction approach to expand the spectrum of acylated Gly and Ala\nby incorporating 111 organic acids from five different classes as\npotential acyl donors, leading to the generation of 222 acylated Gly\nand Ala species. To enhance mass spectrometry (MS) response, we used\na chemical derivatization-based LC-MS/MS approach, employing dimethylamino-naphthalene-1-sulfonyl\npiperazine (Dns-PP) and its stable isotope-labeled form (<i>d</i><sub>6</sub>-Dns-PP) as labeling reagents. Moreover, in-source fragmentation\n(ISF) was utilized to increase the fragment diversity and utility,\naiding in structure elucidation. This strategy resulted in the identification\nof 53 acylated Gly and Ala metabolites in rat biological matrices,\nincluding 17 novel metabolites with distinct tissue-specific distributions.\nOur approach offers a deeper understanding of the physiological and\npathological roles of acylated Gly and Ala, while also opening avenues\nfor the discovery of other modified metabolites.