Heterogeneity of peptide adducts with carbonylated lipid peroxidation products

Abstract

Highly reactive lipid peroxidation-derived carbonyls (oxoLPP) modify protein nucleophiles via Michael addition or Schiff base formation. Once formed, Michael adducts can be further stabilized via cyclic hemiacetals with or without loss of water. Depending on the mechanism of their formation, peptide-oxoLPP can carry aldehyde or keto groups and thus be a part of the total protein carbonylation level. If a carbonyl function is lost during consecutive reactions, the oxoLPP-peptide adducts will not be detected using the common carbonyl labeling protocols. Because of the differences in adduct stabilities, it is possible to address the heterogeneity of peptide/protein-oxoLPP adducts by careful evaluation of tandem mass spectra of modified peptides. Here, we used hydrophilic interaction liquid chromatography-tandem mass spectrometry analysis of lysine, cysteine and histidine containing model peptides co-incubated with oxidized 1-palmitoyl-2-linoleoyl-sn-glycerophosphatidylcholine to characterize the collision-induced dissociation behavior of peptide-carbonyl adducts. Numerous modifications were detected based on the analysis of tandem mass spectra, including Schiff bases on lysine (two), Michael adducts on lysine (six), cysteine (eleven) and histidine (two), as well as 4-hydroxy-2-aldehydes derived dehydrated cyclic hemiacetals on cysteine (five) and histidine (one). Additionally, cysteine and histidine side chains were modified by lipid-bound aldehydes as Michael adducts and dehydrated hemiacetals. The tandem mass spectra revealed collision-induced dissociation characteristics specific for each class of oxoLPP-peptide adducts.

Protein lipoxidation: Detection strategies and challenges

“…Peptide/protein modifications by electrophilic lipids esterified to phospholipids have rarely been considered [3] . Recently, several groups have identified adducts of RCS derived from phosphatidylcholine with His, Cys and Lys in synthetic peptides of ApoA and ApoB-100 [75,76] . This is a field of research that should be explored further since adducts of proteins with oxidized phospholipids have been detected in HDL and in human platelets enriched with electrophilic truncated oxidized phosphatidylcholine (KODA-phospahtidylcholine) [77] .…”

Separation and characterization of oxidized isomeric lipid–peptide adducts by ion mobility mass spectrometry

“…The detection of protein‐oxoLPP in complex biological matrices relies on either specific immunoassays or sensitive mass spectrometry techniques, though the analysis of PL‐oxoLPP–protein adducts is challenged in reversed phase chromatography‐MS by the physicochemical properties, relatively low quantities in biological samples, and multiple positional isomers. Recently, we analyzed peptides modified by oxoLPP and PL‐bound oxoLPP, in mixtures containing also oxidized lipids and unmodified peptides using hydrophilic interaction liquid chromatography (HILIC) coupled on‐line to electrospray ionization (ESI)‐MS . Although this approach identified many peptide modifications resulting from PL oxoLPP, it was not possible to identify (positional) isomers most likely present in the mixture.…”

An Efficient Glycoblotting‐Based Analysis of Oxidized Lipids in Liposomes and a Lipoprotein

“…Small aldehydes generated by lipid oxidation, for example, malondialdehyde (MDA), acrolein (ACR), or 4‐hydroxynonenal (4‐HNE) are harmful substances upon formation of imines or other, more stable structures ,. Although several studies on small aldehydes have been reported, knowledge of lipid core aldehydes, that is, aldehydes still bound to the parent skeleton, has remained elusive .…”