Mass spectrometry for detection of 4‐hydroxy‐trans‐2‐nonenal (HNE) adducts with peptides and proteins

Abstract

Despite the great technical advancement of mass spectrometry, this technique has contributed in a limited way to the discovery and quantitation of specific/precocious markers linked to free radical-mediated diseases. Unsaturated aldehydes generated by free radical-induced lipid peroxidation of polyunsaturated fatty acids, and in particular 4-hydroxy-trans-2 nonenal (HNE), are involved in the onset and progression of many pathologies such as cardiovascular (atherosclerosis, long-term complications of diabetes) and neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, and cerebral ischemia). Most of the biological effects of HNE are attributed to the capacity of HNE to react with the nucleophilic sites of proteins and peptides (other than nucleic acids), to form covalently modified biomolecules that can disrupt important cellular functions and induce mutations. By considering the emerging role of HNE in several human diseases, an unequivocal analytical approach as mass spectrometry to detect/elucidate the structure of protein-HNE adducts in biological matrices is strictly needed not only to understand the reaction mechanism of HNE, but also to gain a deeper insight into the pathological role of HNE. This with the aim to provide intermediate diagnostic biomarkers for human diseases. This review sheds focus on the "state-of-the-art" of mass spectrometric applications in the field of HNE-protein adducts characterization, starting from the fundamental early studies and discussing the different MS-based approaches that can provide detailed information on the mechanistic aspects of HNE-protein interaction. In the last decade, the increases in the accessible mass ranges of modern instruments and advances in ionization methods have made possible a fundamental improvement in the analysis of protein-HNE adducts by mass spectrometry, and in particular by matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) tandem mass spectrometry. The recent developments and uses of combined analytical approaches to detect and characterize the type/site of interaction have been highlighted, and several other aspects, including sample preparation methodologies, structure elucidation, and data analysis have also been considered.

Peptide-phospholipid cross-linking reactions: Identification of leucine enkephalin-alka(e)nal-glycerophosphatidylcholine adducts by tandem mass spectrometry

“…Also, the product ions at m/z 1003.6 and 550.4 (filled diamond in Figure 1a) result from cleavage of the ␥-bond (Pathway a in Scheme 1), relative to the carboxylic group, occurring at the sn-1 and sn-2 residues, respectively, possibly through the 1,4 hydrogen elimination mechanism. Additionally, product ions formed by loss of N(CH 3 ) 3 The fragmentation pathways here described differ greatly from those identified for the lipid-peptide adducts [2], where only peptide chain cleavages are described. The dissimilarity between the fragmentation Scheme 1.…”

“…The peptide-lipid adducts occur by reaction of the primary amine group (1) with the terminal carbonyl group (named as Schiff adduct), or (2) with the double-bond present in unsaturated aldehydes (named as Michael adduct). Currently, the work published on the peptide-lipid covalent interactions has focused on the identification of peptide adducts formed with the 4-hydroxy-nonenal (4-HNE) [2], which is a secondary oxidation product of -6 lipids (such as linoleic and arachidonic acids) found in membrane phospholipids, in triglycerides, and in low density lipoprotein [3]. The studies published allowed to propose that the lipid interactions with key amino acids in proteins were responsible for major structure alterations, not just by modification of the catalytic site [4], but also by reaction with surface amino acids [2], which ultimately leads to structural (conformational) changes.…”

“…The studies published allowed to propose that the lipid interactions with key amino acids in proteins were responsible for major structure alterations, not just by modification of the catalytic site [4], but also by reaction with surface amino acids [2], which ultimately leads to structural (conformational) changes. Tandem mass spectrometry (MS/MS) studies performed on the peptidelipid Schiff and Michael adducts [2] report only product ions formed by peptide cleavages. Considering the formation of peptide/protein-lipid adducts, peptidephospholipid covalent interactions analogous to the ones described with 4-HNE could occur through reaction between peptide and phosphatidylcholinealka(e)nals.…”

Membrane-mediated Amyloidogenesis and the Promotion of Oxidative Lipid Damage by Amyloid β Proteins

“…Moreover, samples were not treated with a reducing agent to stabilize any Schiff bases that form. Michael adducts involving HNE may cyclize to form a hemiacetal (15,16,47,48); however, the mass shifts would remain 156 atomic mass units in both cases. Therefore, the most likely modification by HNE that would account for a 138-atomic mass unit shift is formation of the hemiacetal from the Michael adduct followed by dehydration to form a dihydrofuran (14).…”

Biomarkers of Oxidative Damage in Human Disease

“…Products of lipid peroxidation have therefore commonly been used as biomarkers of oxidative stress/damage. Lipid peroxidation generates a variety of relatively stable decomposition end products, mainly ␣,␤-unsaturated reactive aldehydes, such as malondialdehyde (MDA), 4-hydroxy-2-nonenal (HNE), and 2-propenal (acrolein) (21,22 ), and isoprostanes (23,24 ), which can then be measured in plasma and urine as an indirect index of oxidative stress. Compared with free radicals, the aldehydes are relatively stable and can diffuse within or even escape from the cell and attack targets far from the site of the original event.…”