Maillard Proteomics: Opening New Pages

Abstract

Protein glycation is a ubiquitous non-enzymatic post-translational modification, formed by reaction of protein amino and guanidino groups with carbonyl compounds, presumably reducing sugars and α-dicarbonyls. Resulting advanced glycation end products (AGEs) represent a highly heterogeneous group of compounds, deleterious in mammals due to their pro-inflammatory effect, and impact in pathogenesis of diabetes mellitus, Alzheimer's disease and ageing. The body of information on the mechanisms and pathways of AGE formation, acquired during the last decades, clearly indicates a certain site-specificity of glycation. It makes characterization of individual glycation sites a critical pre-requisite for understanding in vivo mechanisms of AGE formation and developing adequate nutritional and therapeutic approaches to reduce it in humans. In this context, proteomics is the methodology of choice to address site-specific molecular changes related to protein glycation. Therefore, here we summarize the methods of Maillard proteomics, specifically focusing on the techniques providing comprehensive structural and quantitative characterization of glycated proteome. Further, we address the novel break-through areas, recently established in the field of Maillard research, i.e., in vitro models based on synthetic peptides, site-based diagnostics of metabolism-related diseases (e.g., diabetes mellitus), proteomics of anti-glycative defense, and dynamics of plant glycated proteome during ageing and response to environmental stress.

Keywords: advanced glycation end products (AGEs); bottom-up proteomics; glycation; glyoxalase; model synthetic peptides; plant glycation; post-translational modifications; proteomics.

Figure 1

Pathways of early and advanced glycation (oxidative glycosylation [12], Namiki pathway [13], enolization [14], oxidative [15] and non-oxidative (enolization and dehydration stages are not mentioned) [16] degradation of early glycation products, polyol pathway [17] and lipid peroxidation [18] (a) and structures of major AGEs detected in vivo and in thermally processed foods (b)). R₁, R₂, R₃, R₄, R₅, polypeptide chains; R₁′, R₂′, R₃′, fatty acid residues; R₁′′ = H, R₂′′ = H for glyoxal; R₁′′ = H, R₂′′ = CH₃ for methylglyoxal; R1′′ = H, R2′′ = C₄H₉O₃ for 3-DG. * Ketoses form so-called Heyns products.

Figure 2

The overview of the gel- and LC-based workflows in bottom-up proteomics. The scissors denote enzymatic proteolysis.

Figure 3

Enrichment of glycated (Amadori) peptides by BAC [127]. The experimental workflow relies on m-aminophenylboronic acid-agarose, filled in polypropylene columns, and gravity flow design.

Figure 4

Synthesis of Amadori-modified peptides by global glycation approach and building block strategy. After selective deprotection of the site to be glycated, Amadori moiety can be introduced directly by incubation with reducing sugar [273] or via the Lobry de Bruyn reaction with acetonide-protected hexodiulose (2,3:4,5-di-O-isopropylidene-aldehydo-β-d-arabino-hexos-2-ulo-2,6-pyranose) in presence of cyanoborohydride in methanol-isopropanol-water mixture (2:2:1 by volume) [274]. Alternatively, glycated moiety can be introduced with an acetonide-protected N^ε-Boc-N^ε-fructosyl-N^α-Fmoc-lysine building block [272].

Figure 5

Tandem mass spectrometric (MS/MS) fragmentation patterns of: glucose-derived Amadori (a); and fructose-derived Heyns (b) peptides, represented by characteristic signals, corresponding to oxonium (-H₂O and -2H₂O), pyrylium (-3H₂O) and furylium/immonium (-3H₂O-HCHO) ions. The Heyns products can be distinguished by the presence of 2(hydroxymethyl)pyrylium (2-HNP) ion and specific mass increment of the Heyns-derived furylium signal (96 u).

Figure 6

Quantification of individual protein glycation sites as Amadori-modified peptides in blood plasma tryptic digests by targeted and untargeted LC-MS approaches. LC-QqQ-MS/MS: green, white, black and grey circles are precursor ions, different shapes in green denote product ions; LC-LIT-Orbitrap-MS, LC-QqTOF-MS: green, white, black and grey circles denote quasi-molecular ions.

Figure 7

The experimental workflow for analysis of plant protein glycation and characterization of underlying mechanisms. The experiments relied on a combination of proteomics and metabolomics approaches.