Poster Presentation 28th Annual Lorne Proteomics Symposium 2023

Differentiation of lactyllysine and carboxyethyllysine modified peptides (#114)

Leigh Donnellan 1 , Clifford Young 1 , Peter Hoffmann 1
  1. Clinical Health Sciences, University of South Australia, Adelaide, South Australia, Australia

Post-translational modifications (PTMs) of proteins play a critical role in biological events by regulating activity, interactions, stability and degradation. The gold standard for identifying PTMs is high-resolution mass spectrometry, which relies on specific mass shifts caused by the modification, in relation to the parent peptide. Unfortunately, some PTMs can share identical mass shifts, making their differentiation difficult. Such PTMs include the recently discovered Lactyllysine (Kla) and Nε-carboxyethyllysine (CEL) which share the exact same mass shift of (72.02113031 Da). Kla is derived from S-lactoylglutathione or lactyl-CoA and CEL primarily from methylglyoxal (MGO). In recent years, several methods have been employed to confirm presence of Kla sites, including metabolic tracing using 13C6-glucose, however, all substrates responsible for Kla and CEL formation form as by-products of glycolysis. Therefore, the observed mass shift of 3.0101 Da in lactylated peptides would also be observed for those modified with CEL. Furthermore, several studies have used pan antibodies to generate an enriched lactylproteome. However, it was recently shown that these antibodies cross react with CEL modified proteins, suggesting the lactylproteome would also contain CEL modified peptides.

To investigate any differences occurring from Kla or CEL modifications, we generated peptides in-house by modifying BSA with S-lactoylglutathione and MGO, respectively. Initially, we found that each substrate preferentially modified different lysine residues, suggesting each modification may have different signalling consequences, further confirming the importance of their differentiation. Based on MS and MS/MS alone, there were no identifying features unique to either modification. Moreover, we also found that CEL modified peptides produced a cyclic immonium ion (156.103 Da), which was recently reported to be unique to lactylated peptides.  Interestingly, we found that Kla and CEL modified peptides have different retention times on C18 columns, with the former being more hydrophobic, eluting from the column ~6 min later than the latter when using a 105-minute gradient. Therefore, chromatographic separation of the two modified peptides may allow their differentiation.