Extending the HSA-Cys34-Adductomics Pipeline to Modifications at Lys525.

We previously developed an adductomics pipeline that employed nanoflow liquid chromatography and high-resolution tandem mass spectrometry (nLC-HR-MS/MS) plus informatics to perform an untargeted detection of modifications to Cys34 in the tryptic T3 peptide of human serum albumin (HSA) (21ALVLIAFAQYLQQC34PFEDHVK41). In order to detect these peptide modifications without targeting specific masses, the pipeline interrogates MS2 ions that are signatures of the T3 peptide. The pipeline had been pilot-tested with archived plasma from healthy human subjects, and several of the 43 Cys34 adducts were highly associated with the smoking status. In the current investigation, we adapted the pipeline to include modifications to the ε-amino group of Lys525─a major glycation site in HSA─and thereby extend the coverage to products of Schiff bases that cannot be produced at Cys34. Because trypsin is generally unable to digest proteins at modified lysines, our pipeline detects miscleaved tryptic peptides with the sequence 525KQTALVELVK534. Adducts of both Lys525 and Cys34 are measured in a single nLC-HR-MS/MS run by increasing the mass range of precursor ions in MS1 scans and including both triply and doubly charged precursor ions for collision-induced dissociation fragmentation. For proof of principle, we applied the Cys34/Lys525 pipeline to archived plasma specimens from a subset of the same volunteer subjects used in the original investigation. Twelve modified Lys525 peptides were detected, including products of glycation (fructosyl-lysine plus advanced-glycated-end products), acetylation, and elimination of ammonia and water. Surprisingly, the carbamylated and glycated adducts were present at significantly lower levels in smoking subjects. By including a larger class of in vivo nucleophilic substitution reactions, the Cys34/Lys525 adductomics pipeline expands exposomic investigations of unknown human exposure to reactive electrophiles derived from both exogenous and endogenous sources.