Unveiling the Fragmentation Mechanisms of Modified Amino Acids as the Key for Their Targeted Identification

The alteration of modified amino acid (MAA) profiles in biological samples is related to important cellular, physiological, and pathological processes. To achieve the interpretation of their biochemical relevance, it is critical to define their whole chemical spectrum using metabolomic research works. We present a detailed in-source fragmentation (ISF) study based on the mechanisms of the major fragmentation reactions observed of diagnostic ions (DIs) generated in positive electrospray ionization for 57 amino acid standard compounds using capillary electrophoresis coupled with high-resolution mass spectrometry. The DIs presented and our in-house fragment library allowed us to establish a workflow for targeted extraction of MAAs. We present key examples showing successful findings such as the identification of N₂-methyl-l-lysine, which provides insight into the lysine methylome. The experimental results presented prove that the use of ISF data, when combined with a thorough study of the fragmentation mechanisms, constitutes an informative source of accurate molecular identity.