An Investigation into the Differentiation of Lactyllysine and Carboxyethyllysine Peptide Modifications by Liquid Chromatography/Mass Spectrometry

Post-translational modifications (PTMs) are key drivers in the regulation of protein activity. Therefore, the ability to measure and identify them accurately is critical to understanding the function and regulation of these modifications. <i>N</i>ε-carboxyethyllysine (CEL) and lactyllysine (LactylLys) are two modifications that share the same chemical composition and thus mass shift, making traditional LC-MS/MS approaches unsuitable for distinguishing them. Standard LC-MS/MS approaches utilizing HCD show that both modifications display extremely similar fragmentation, with no distinguishing features observed for either modification. Furthermore, cyclic and linear immonium ions, which have recently been reported as unique to LactylLys, were also observed in MS/MS from carboxyethylated peptides. We show that carboxyethylated and lactylated peptides can be chromatographically resolved on in-house packed and commercial C18 columns, and retention time alignment with isotopically labeled peptides can be used for discrimination. Furthermore, we observed differences in the MS/MS spectra obtained from EAD fragmentation of the two PTM-containing peptides. Our results highlight the analytical challenges associated with distinguishing CEL- and LactylLys-modified peptides and provide a proof of concept in which retention time alignment of endogenous peptides with isotopically labeled standards and electron activated dissociation (EAD) fragmentation is applied to accurately assign lactylation at K147 of aldolase A across various cell lines/tissues.