RESUMO
Glycation is a post-translational modification resulting from the interaction of protein amino and guanidino groups with carbonyl compounds. Initially, amino groups react with reducing carbohydrates, yielding Amadori and Heyns compounds. Their further degradation results in formation of advanced glycation end products (AGEs), also originating from α-dicarbonyl products of monosaccharide autoxidation and primary metabolism. In mammals, AGEs are continuously formed during the life of the organism, accumulate in tissues, are well-known markers of aging, and impact age-related tissue stiffening and atherosclerotic changes. However, the role of AGEs in age-related molecular alterations in plants is still unknown. To fill this gap, we present here a comprehensive study of the age-related changes in the Arabidopsis thaliana glycated proteome, including the proteins affected and specific glycation sites therein. We also consider the qualitative and quantitative changes in glycation patterns in terms of the general metabolic background, pathways of AGE formation, and the status of plant anti-oxidative/anti-glycative defense. Although the patterns of glycated proteins were only minimally influenced by plant age, the abundance of 96 AGE sites in 71 proteins was significantly affected in an age-dependent manner and clearly indicated the existence of age-related glycation hot spots in the plant proteome. Homology modeling revealed glutamyl and aspartyl residues in close proximity (less than 5 Å) to these sites in three aging-specific and eight differentially glycated proteins, four of which were modified in catalytic domains. Thus, the sites of glycation hot spots might be defined by protein structure that indicates, at least partly, site-specific character of glycation.
Assuntos
Arabidopsis/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Proteômica , Sequência de Aminoácidos , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Glicosilação , Modelos Moleculares , Oxirredução , Conformação Proteica , Proteólise , Fatores de Tempo , Tripsina/metabolismoRESUMO
In the present work, we describe the synthesis and the temperature-dependent behavior of photoreactive membrane lipids as well as their capability to study peptide/lipid interactions. The modified phospholipids contain an azide group either in the middle part or at the end of an alkyl chain and also differ in the linkage (ester vs ether) of the second alkyl chain. The temperature-dependent aggregation behavior of the azidolipids was studied using differential scanning calorimetry (DSC), Fourier-transform infrared (FTIR) spectroscopy, and small-angle X-ray scattering (SAXS). Aggregate structures were visualized by stain and cryo transmission electron microscopy (TEM) and were further characterized by dynamic light scattering (DLS). We show that the position of the azide group and the type of linkage of the alkyl chain at the sn-2 position of the glycerol influences the type of aggregates formed as well as their long-term stability: P10AzSPC and r12AzSHPC show the formation of extrudable liposomes, which are stable in size during storage. In contrast, azidolipids that carry a terminal azido moiety either form extrudable liposomes, which show time-dependent vesicle fusion (P15AzPdPC), or self-assemble in large sheet-like, nonextrudable aggregates (r15AzPdHPC) where the lipid molecules are arranged in an interdigitated orientation at temperatures below Tm (LßI phase). Finally, a P10AzSPC:DMPC mixture was used for photochemically induced cross-linking experiments with a transmembrane peptide (WAL-peptide) to demonstrate the applicability of the azidolipids for the analysis of peptide/lipid interactions. The efficiency of photo-cross-linking was monitored by attenuated total reflection infrared (ATR-IR) spectroscopy and mass spectrometry (MS).
Assuntos
Azidas/química , Varredura Diferencial de Calorimetria , Lipídeos de Membrana , Espalhamento a Baixo Ângulo , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios XRESUMO
Chemical cross-linking/mass spectrometry (MS) is gradually developing into a routine method to investigate protein conformation and to decipher protein interaction networks. To increase identification rates of the frequently low abundant cross-linked products in LC/MS/MS experiments, fast and reliable sample preparation protocols are indispensable. We present simplified solid phase extraction methods using C18/SCX StageTips and mixed-mode OASIS MCX cartridges for a single-step enrichment of cross-linked products prior to LC/MS/MS analysis. Our improved protocols result in 3.5 to 4.6 times higher numbers of cross-link identifications for the model protein bovine serum albumin compared to non-processed samples.
Assuntos
Conformação Proteica , Mapeamento de Interação de Proteínas , Proteínas/química , Cromatografia Líquida/métodos , Reagentes de Ligações Cruzadas/química , Espectrometria de Massas em Tandem/métodosRESUMO
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.
Assuntos
Reação de Maillard , Peptídeos/química , Proteoma/química , Proteômica/métodos , Sequência de Aminoácidos , Animais , Produtos Finais de Glicação Avançada/química , Produtos Finais de Glicação Avançada/metabolismo , Glicosilação , Humanos , Espectrometria de Massas/métodos , Peptídeos/metabolismo , Proteoma/metabolismoRESUMO
Bacteria have acquired resistance mechanisms to overcome antibiotic treatments, triggering major concerns about the return of epidemic infections. Antimicrobial peptides identified in insects, animals, and plants represent a huge pool of promising lead structures that can be further developed for medical applications. Short proline-rich antimicrobial peptides (PrAMPs) have gained much attention due to their clinically interesting activity spectrum, serum protease stability, efficacy in murine infection models, and low adverse effects. Here we induced resistances by incubating Escherichia coli with increasing concentrations of apidaecin 1b, a PrAMP isolated from honeybees, and quantitatively evaluated the proteomes between wild-type and resistant strains. Surprisingly, 2D differential gel electrophoresis did not reveal differences, indicating that the expression levels of dominant proteins were very similar. Reversed-phase chromatography coupled online to a mass spectrometer identified 2131 proteins in the soluble fraction (cytosolic fraction) and 1296 proteins in the nonsolubilized pellet (membrane fraction). Overall 29 proteins showed a statistically significant upregulation in the resistant E. coli strain, whereas 18 proteins were downregulated. Interestingly, periplasmic chaperone FimC, fimbrial protein FimA, and mannose-binding domain protein FimH, which are part of the fimbrial complex, were not detected in the resistant strain that was also unable to form biofilms. Furthermore, the expression of a few other proteins known as virulence factors was downregulated. Additionally, the expression level of isochorismatase hydrolase (YcaC) decreased in the membrane fraction of the resistant strain to 35%, and the corresponding knockout mutant of E. coli BW25113 was eight times less susceptible to apidaecin 1b and the related designer peptide Api88.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Farmacorresistência Bacteriana , Escherichia coli/química , Proteoma/efeitos dos fármacos , Proteômica/métodos , Animais , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/farmacologia , Abelhas/química , Cromatografia em Gel , Cromatografia de Fase Reversa , Farmacorresistência Bacteriana/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/fisiologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Espectrometria de MassasRESUMO
OBJECTIVES: To evaluate the efficacy of antimicrobial peptide Onc112 in a lethal Escherichia coli infection model and the pharmacokinetics of Onc72 and Onc112 administered intravenously or intraperitoneally in mice. METHODS: Onc72, Onc112 and their major metabolites in blood, kidneys, liver, brain and urine were quantified by MS using multiple reaction monitoring (MRM) and isotope-labelled peptides. RESULTS: Onc112 rescued all animals when administered intraperitoneally at a dose of 2.5 mg/kg and was thus slightly more efficient than Onc72. The MRM method provided limits of quantification in plasma, urine and kidney, liver and brain homogenates of 7-80 µg/L, well below the MICs of 2-4 mg/L. Onc72 and Onc112 reached all organs within 10 min when administered intraperitoneally (5 mg/kg). Their initial concentrations in plasma were 11.9 and 22.6 mg/L, respectively, with elimination t1/2 values of â¼14 and 21 min. The peptide concentrations in blood remained above their MICs for 20 min for Onc72 and 80 min for Onc112. The highest peptide concentrations were detected in kidney homogenates, which also contained the highest content of metabolites, indicating, together with the results from analysis of urine samples, that both peptides are cleared through the kidneys. CONCLUSIONS: Onc72 and Onc112 reach organs, including the brain, within 10 min after intravenous and intraperitoneal administration. Onc112 remained in blood at concentrations above its MIC for 80 min. The pharmacokinetic profiles explain the high in vivo efficacies in models of systemic infection and indicate the potential use of these agents for the treatment of urinary tract infections.
Assuntos
Antibacterianos/farmacocinética , Peptídeos Catiônicos Antimicrobianos/farmacocinética , Animais , Antibacterianos/administração & dosagem , Peptídeos Catiônicos Antimicrobianos/administração & dosagem , Modelos Animais de Doenças , Monitoramento de Medicamentos , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/mortalidade , Feminino , Injeções Intraperitoneais , Camundongos , Sepse/tratamento farmacológico , Sepse/microbiologia , Sepse/mortalidade , Fatores de Tempo , Distribuição Tecidual , Resultado do TratamentoRESUMO
Among the environmental alterations accompanying oncoming climate changes, drought is the most important factor influencing crop plant productivity. In plants, water deficit ultimately results in the development of oxidative stress and accumulation of osmolytes (e.g. amino acids and carbohydrates) in all tissues. Up-regulation of sugar biosynthesis in parallel to the increasing overproduction of reactive oxygen species (ROS) might enhance protein glycation, i.e. interaction of carbonyl compounds, reducing sugars and α-dicarbonyls with lysyl and arginyl side-chains yielding early (Amadori and Heyns compounds) and advanced glycation end-products (AGEs). Although the constitutive plant protein glycation patterns were characterized recently, the effects of environmental stress on AGE formation are unknown so far. To fill this gap, we present here a comprehensive in-depth study of the changes in Arabidopsis thaliana advanced glycated proteome related to osmotic stress. A 3 d application of osmotic stress revealed 31 stress-specifically and 12 differentially AGE-modified proteins, representing altogether 56 advanced glycation sites. Based on proteomic and metabolomic results, in combination with biochemical, enzymatic and gene expression analysis, we propose monosaccharide autoxidation as the main stress-related glycation mechanism, and glyoxal as the major glycation agent in plants subjected to drought.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Pressão Osmótica , Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiologia , Desidratação , Produtos Finais de Glicação Avançada/metabolismo , Glicosilação , Monossacarídeos/metabolismo , Oxirredução , Proteoma/metabolismo , TranscriptomaRESUMO
The leptin receptor antagonist peptide Allo-aca exhibits picomolar activities in various cellular systems and sub-mg/kg subcutaneous efficacies in animal models making it a prime drug candidate and target validation tool. Here we identified the biochemical basis for its remarkable in vivo activity. Allo-aca decomposed within 30 min in pooled human serum and was undetectable beyond the same time period from mouse plasma during pharmacokinetic measurements. The C max of 8.9 µg/mL at 5 min corresponds to approximately 22% injected peptide present in the circulation. The half-life was extended to over 2 h in bovine vitreous fluid and 10 h in human tears suggesting potential efficacy in ophthalmic diseases. The peptide retained picomolar anti-proliferation activity against a chronic myeloid leukemia cell line; addition of a C-terminal biotin label increased the IC50 value by approximately 200-fold. In surface plasmon resonance assays with the biotin-labeled peptide immobilized to a NeutrAvidin-coated chip, Allo-aca exhibited exceptionally tight binding to the binding domain of the human leptin receptor with ka = 5 × 10(5) M(-1) s(-1) and kdiss = 1.5 × 10(-4) s(-1) values. Peptides excel in terms of high activity and selectivity to their targets, and may activate or inactivate receptor functions considerably longer than molecular turnovers that take place in experimental animals.
Assuntos
Leptina/antagonistas & inibidores , Peptídeos/química , Receptores para Leptina/química , Animais , Bovinos , Linhagem Celular Tumoral , Desenho de Fármacos , Feminino , Meia-Vida , Humanos , Cinética , Leptina/química , Leptina/metabolismo , Camundongos , Peptídeos/sangue , Peptídeos/metabolismo , Peptídeos/farmacocinética , Receptores para Leptina/metabolismoRESUMO
Apidaecins are cationic, proline-rich antimicrobial peptides originally isolated from honeybees and exhibit high Gram-negative activity by inhibiting bacterial protein translation. Pharmacokinetics of apidaecin derivative Api137 was studied using single and multiple intravenous or subcutaneous injections as well as continuous subcutaneous infusion and correlated to its efficacy in a lethal murine Escherichia coli infection model. Survival rates of infected CD-1 mice were monitored and Api137 and its metabolites were quantified in plasma of uninfected CD-1 mice and Sprague Dawley rats using reversed-phase chromatography coupled online to mass spectrometry. The highest Api137 plasma levels of 23 mg/L were obtained after a single intravenous injection of 20 mg/kg body weight, which declined fast over the next 120 min (half-life time < 30 min). In contrast, continuous subcutaneous infusion of a similar dose over an hour (19.2 mg/kg/h) lead to stable plasma levels of â¼6 mg/L, which was above the minimal inhibitory concentration against E. coli ATCC 25922 (4 mg/L). The increased exposure by continuous subcutaneous administration of Api137 at 19.2 mg/kg/h over 48 h improved efficacy in the murine intraperitoneal sepsis model with survival rates of 67% over 5 days compared to 33% after intravenous and subcutaneous administration in different dosing schemes. To the best of our knowledge, continuous subcutaneous infusion using osmotic pumps was successfully utilized for delivery of an antimicrobial peptide for the first time. Additionally, the potential of apidaecin analogs as novel antibiotics is demonstrated even in a scenario where the infection site is clearly separated from the route of administration.
RESUMO
Proline-rich antimicrobial peptides (PrAMPs) kill bacteria in a multimodal mechanism by inhibiting the 70S ribosome (i.e., protein translation) as dominant lethal mechanism besides inhibition of several other proteins, such as chaperone DnaK. PrAMPs pass the outer membrane of Gram-negative bacteria, probably by a self-promoted uptake followed by a transporter-mediated uptake from the periplasm. Mutation of transporter protein SbmA is a well-studied resistance mechanism observed in vitro by resistance induction with PrAMPs. Here, we compared the membrane compositions of Escherichia coli BL21AI and BL21AI Apir, which was obtained by resistance induction with PrAMP apidaecin 1b. Lipid A was partially modified by phosphatidylethanolamine, 4-aminoarabinose, or both groups, but the relative contents of these and further unidentified species did not differ much between wild-type and resistant strains, indicating that resistance was not related to lipid A modifications. The same was true for 20 glycerophospholipids identified, i.e., 11 phosphatidylethanolamines and 9 phosphatidylglycerols. However, glycerophospholipids in BL21AI Apir contained much lower levels of cyclopropane-modified acyl groups, which probably alter the biophysical properties of the inner membrane and the inner leaflet of the outer membrane. Indeed, when cyclopropane-fatty-acyl-phospholipid synthase was knocked out in E. coli BW25113, the resulting BW25113 Δcfa was less susceptible against apidaecin 1b.
Assuntos
Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Membrana Celular/química , Escherichia coli/química , Escherichia coli/efeitos dos fármacos , Fosfolipídeos/análise , Fenômenos BiofísicosRESUMO
Chemical cross-linking in combination with mass spectrometric analysis of the created cross-linked products is an emerging technology aimed at deriving valuable structural information from proteins and protein complexes. The goal of our protocol is to obtain distance constraints for structure determination of proteins and to investigate protein-protein interactions. We present an integrated workflow for cross-linking/mass spectrometry (MS) based on protein cross-linking with MS-cleavable reagents, followed by enzymatic digestion, enrichment of cross-linked peptides by strong cation-exchange chromatography (SCX), and LC/MS/MS analysis. To exploit the full potential of MS-cleavable cross-linkers, we developed an updated version of the freely available MeroX software for automated data analysis. The commercially available, MS-cleavable cross-linkers (DSBU and CDI) used herein possess different lengths and react with amine as well as hydroxy groups. Owing to the formation of two characteristic 26-u doublets in their MS/MS spectra, many fewer false positives are found than when using classic, non-cleavable cross-linkers. The protocol, exemplified herein for BSA and the whole Escherichia coli ribosome, is robust and widely applicable, and it allows facile identification of cross-links for deriving spatial constraints from purified proteins and protein complexes. The cross-linking/MS procedure takes 2-3 days to complete.
Assuntos
Reagentes de Ligações Cruzadas/química , Proteínas/química , Software , Espectrometria de Massas em Tandem/métodos , Animais , Bovinos , Reagentes de Ligações Cruzadas/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Conformação Proteica , Mapeamento de Interação de Proteínas/métodos , Proteínas/metabolismo , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Fluxo de TrabalhoRESUMO
Proline-rich antimicrobial peptides (PrAMPs) represent promising alternative therapeutic options for the treatment of multidrug-resistant bacterial infections. PrAMPs are predominantly active against Gram-negative bacteria by inhibiting protein expression via at least two different modes of action, i.e., blocking the ribosomal exit tunnel of 70S ribosomes (oncocin-type binding) or inhibiting the assembly of the 50S ribosomal subunit (apidaecin-type binding). The in vivo efficacy and favorable biodistribution of oncocins confirmed the therapeutic potential of short PrAMPs for the first time, whereas the in vivo evaluation of apidaecins is still limited despite the promising efficacy of apidaecin-analog Api88 in an intraperitoneal murine infection model. Here, the in vivo efficacy of apidaecin-analog Api137 was studied, which rescued all NMRI mice from a lethal intraperitoneal infection with E. coli ATCC 25922 when administered three times intraperitoneal at doses of 0.6 mg/kg starting 1 h after infection. When Api88 and Api137 were administered intravenous or intraperitoneal at doses of 5 and 20 mg/kg, their plasma levels were similarly low (<3 µg/mL) and four-fold lower than for oncocin-analog Onc72. This contradicted earlier expectation based on the very low serum stability of Api88 with a half-life time of only ~5 min compared to ~6 and ~3 h for Api137 and Onc72, respectively. Pharmacokinetic data relying on a sensitive mass spectrometry method utilizing multiple reaction monitoring and isotope-labeled peptides revealed that Api88 and Api137 were present in blood, urine, and kidney, and liver homogenates at similar levels accompanied by the same major metabolites comprising residues 1-16 and 1-17. The pretended discrepancy was solved, when all peptides were incubated in peritoneal lavage. Api137 was rapidly degraded at the C-terminus, while Api88 was rather stable despite releasing the same degradation products. Onc72 was very stable explaining its higher plasma levels compared to Api88 and Api137 after intraperitoneal administration illuminating its good in vivo efficacy. The data indicate that the degradation of therapeutic peptides should be studied in serum and further body fluids. Moreover, the high efficacy in murine infection models and the fast clearance of Api88 and Api137 within ~60 min after intravenous and ~90 min after intraperitoneal injections indicate that their in vivo efficacy relates to the maximal peptide concentration achieved in blood.
RESUMO
Glycation is a non-enzymatic reaction of protein amino and guanidino groups with reducing sugars or dicarbonyl products of their oxidative degradation. Modification of arginine residues by dicarbonyls such as glyoxal and methylglyoxal results in formation of advanced glycation end-products (AGEs). In mammals, these modifications impact in diabetes mellitus, uremia, atherosclerosis and ageing. However, due to the low abundance of individual AGE-peptides in enzymatic digests, these species cannot be efficiently detected by LC-ESI-MS-based data-dependent acquisition (DDA) experiments. Here we report an analytical workflow that overcomes this limitation. We describe fragmentation patterns of synthetic AGE-peptides and assignment of modification-specific signals required for unambiguous structure retrieval. Most intense signals were those corresponding to unique fragment ions with m/z 152.1 and 166.1, observed in the tandem mass spectra of peptides, containing glyoxal- and methylglyoxal-derived hydroimidazolone AGEs, respectively. To detect such peptides, specific and sensitive precursor ion scanning methods were established for these signals. Further, these precursor ion scans were incorporated in conventional bottom-up proteomic approach based on data-dependent acquisition (DDA) LC-MS/MS experiments. The method was successfully applied for the analysis of human serum albumin (HSA) and human plasma protein tryptic digest with subsequent structure confirmation by targeted LC-MS/MS (DDA). Altogether 44 hydroimidazolone- and dihydroxyimidazolidine-derived peptides representing 42 AGE-modified proteins were identified in plasma digests obtained from type 2 diabetes mellitus (T2DM) patients.
Assuntos
Arginina/análise , Arginina/química , Produtos Finais de Glicação Avançada/análise , Fragmentos de Peptídeos/análise , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Arginina/sangue , Arginina/metabolismo , Diabetes Mellitus/sangue , Produtos Finais de Glicação Avançada/sangue , Produtos Finais de Glicação Avançada/química , Produtos Finais de Glicação Avançada/metabolismo , Glicosilação , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Tripsina/metabolismoRESUMO
Glycation is a ubiquitous nonenzymatic reaction of carbonyl compounds with amino groups of peptides and proteins, resulting in the formation of advanced glycation end-products (AGEs) and thereby affecting the properties and quality of thermally processed foods. In this context, mechanisms of the Maillard reaction of proteins need to be understood; that is, glycation products and intermediates (α-dicarbonyls and sugars) need to be characterized. Although the chemical analysis of proteins, peptides, and α-dicarbonyls is well established, sensitive and precise determination of multiple sugars in glycation mixtures is still challenging. This paper presents a gas chromatography-mass spectrometry (GC-MS) method for absolute quantitation of 22 carbohydrates in the model of phosphate-buffered glycation systems. The approach relied on the removal of the phosphate component by polymer-based ion exchange solid phase extraction (SPE) followed by derivatization of carbohydrates and subsequent GC-MS analysis. Thereby, baseline separation for most of the analytes and detection limits of up to 10 fmol were achieved. The method was successfully applied to the analysis of in vitro glycation reactions. Thereby, at least seven sugar-related Maillard reaction intermediates could be identified and quantified. The most abundant reaction product was d-fructose, reaching 2.70 ± 0.12 and 2.38 ± 0.66 mmol/L after 120 min of incubation in the absence and presence of the model peptide, respectively.
Assuntos
Carboidratos/química , Cromatografia Gasosa-Espectrometria de Massas/métodos , Produtos Finais de Glicação Avançada/química , Glicosilação , Reação de Maillard , Espectrometria de Massas , Estrutura Molecular , Peptídeos/químicaRESUMO
Glycation refers to the reaction of amino groups, for example in proteins, with reducing sugars. Intermediately formed Amadori products can be degraded by oxidation (Maillard reactions) leading to a heterogeneous class of advanced glycation end-products (AGEs), especially during exposure to heat. AGEs are considered to be toxic in vivo due to their pronounced local and systemic inflammatory effects. At high temperatures, these reactions have been mostly investigated at the amino acid level. Here, we studied the formation of arginine-related AGEs in peptides under conditions simulating household cooking at physiological d-glucose concentrations. High quantities of AGE-modified peptides were produced within 15 min, especially glyoxal-derived products. The intermediately formed dihydroxy-imidazolidine yielded glyoxal- (Glarg) and methylglyoxal-derived hydro-imidazolinones (MG-H), with Glarg being further degraded to carboxymethyl-l-arginine (CMA). Carboxyethyl-l-arginine was not detected. The formation rates and yields were strongly increased in the presence of physiologically relevant concentrations of Fe(II)-ions and ascorbate. A nearby histidine residue increased the content of AGEs, whereas glutamic acid significantly reduced the CMA levels.