Protective effect of thymoquinone on glycation of human myoglobin induced by d-ribose.

Nonenzymatic glycation and the subsequent accumulation of advanced glycation end-products (AGEs) in proteins are factors underlying long-term pathogenesis in diabetes. The study of protein glycation is crucial for elucidating their relationship with diabetes mellitus and related disorders. This study explores the interaction between d-ribose and human myoglobin (HMb), as well as the protective effect of thymoquinone (TQ) on glycation. A time-dependent in-vitro glycation study was performed to investigate the mechanism of d-ribose-induced structural interference of HMb in the absence and presence of TQ. Spectroscopic and proteomic analysis indicated that the presence of TQ significantly reduced the total amount of AGEs while maintaining structural characteristics of HMb. 14 glycated sites on HMb were further identified via liquid chromatography-tandem mass spectrometry (LC-MS/MS) after incubation with d-ribose for 12 h, predominantly interacting with lysine residues. TQ was found to disrupt this interaction, reducing the glycated sites from 14 to 12 sites and the percentage of glycated peptides from 26.50 % to 12.97 %. Additionally, there was a significant decrease in the degree of glycation at the same sites. In summary, our findings suggest that TQ has the potential to act as an anti-glycation agent and provide a comprehensive understanding underlying the inhibition mechanism of glycation.

The effect of B-type procyanidin on free radical and metal ion induced ß-lactoglobulin glyco-oxidation via mass spectrometry and interaction analysis.

Procyanidin is a group of dietary flavonoids abundant in berry fruits. In this study, the effects and underlying mechanisms of B type procyanidin (PC) on free radical and metal ion (H2O2, AAPH and Fe3+) induced milk protein ß-lactoglobulin (BLG) glyco-oxidation were investigated. The results indicated that PC protected BLG structure changes from cross-link and aggregation induced by free radicals and metal ion. Additionally, it effectively inhibited BLG oxidation by reducing approximately 21%-30% carbonyls and 15%-61% schiff base crosslink formation. Also, PC suppressed BLG glycation by inhibiting 48-70% advanced glycation end-products (AGEs) and reduced the accumulation of intermediate product methylglyoxal (MGO). The corresponding mechanisms were elucidated that PC exhibited great free radical scavenging and metal chelating properties; PC had non-covalent bind with the amino acid residues (preferably lysine and arginine) of BLG and blocked them from glycation; PC interrupted BLG glycation by forming procyanidin-MGO conjugates. Therefore, B type procyanidin was an effective glyco-oxidation inhibitor in milk products.

Effect of milk fat and its main fatty acids on oxidation and glycation level of milk.

Milk is a highly nutritional food rich in protein and fat that is prone to deterioration by oxidation and glycation reactions at storage and processing. In this study, glycation products and lipid oxidation products contents in skim milk, whole milk, and milk fat simulation groups were determined to evaluate the effect of milk fat components on glycation at 120 °C for 60 min. The increase rate of carbonyl compound, main advanced glycation end products (AGEs) levels, and glycation sites number of α-casein and ß-casein are higher in whole milk than that in skim milk, indicating that milk fat promoted protein glycation significantly. In milk fat simulation groups, oleic acid and linoleic acid (LA) were added to milk fat in skim milk proportionally, promoting the formation of glycation products; however, palmitic acid had no such effect. LA exhibited strong promotion on AGEs formation. Lipid oxidation radicals, protein carbonyl amine condensation, and carbonyl compound formation were critical factors for milk glycation, according to OPLS-DA results. Therefore, radicals of fat oxidation are speculated to trigger the early glycation, and carbonyl compounds of fat oxidation act as important intermediates of glycation, fat type, form, and its degradation rate, thus play essential roles in milk glycation. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-022-05658-z.

Comprehensive overview of human serum albumin glycation in diabetes mellitus.

The presence of excess glucose in blood is regarded as a sweet hurt for patients with diabetes. Human serum albumin (HSA) is the most abundant protein in human plasma, which undergoes severe non-enzymatic glycation with glucose in patients with diabetes; this modifies the structure and function of HSA. Furthermore, the advanced glycation end products produced by glycated HSA can cause pathological damage to the human body through various signaling pathways, eventually leading to complications of diabetes. Many potential glycation sites on HSA have different degrees of sensitivity to glucose concentration. This review provides a comprehensive assessment of the in vivo glycation sites of HSA; it also discusses the effects of glycation on the structure and function of HSA. Moreover, it addresses the relationship between HSA glycation and diabetes complications. Finally, it focuses on the value of non-enzymatic glycation of HSA in diabetes-related clinical applications.

Evaluation of glycation reaction of ovalbumin with dextran: Glycation sites identification by capillary liquid chromatography coupled with tandem mass spectrometry.

An important relationship exists between the changes in glycation sites and structure of proteins. A combination of liquid chromatography and tandem mass spectrometry was used to identify the glycation information from ovalbumin (OVA) after dextran (Dex) conjugation under water heating (WH) or microwave heating (MH) conditions. After Dex conjugation, 12O-linked glycation sites were identified in the OVA-Dex-MH conjugates, whereas 6O-linked, 2N-linked glycation sites were detected in the OVA-Dex-WH conjugates. These findings indicate that the amino acids at different positions in OVA molecular structure have different glycation reactivity under MH or WH induction systems. In addition, ß-sheet and ß-turn structures showed high glycation reactivity. The increased surface hydrophobicity of OVA-Dex conjugates was possibly attributed to the glycation sites that were mainly found in hydrophilic amino acids. Our study provides useful information for the glycation mechanism research of OVA and Dex.

Mechanism of Selenium Nanoparticles Inhibiting Advanced Glycation End Products.

Selenium nanoparticles (SeNPs) have been applied in fields of nanobiosensors, environment, nanomedicine, etc. as a result of their excellent characteristics. Early studies had shown that SeNPs have certain inhibition ability against glycation, but the inhibition mechanism, especially for the influence of SeNPs on the reaction activity of glycation sites, remains unclear. The aim of the presented research was to reveal the effects of SeNPs on the ß-lactoglobulin (ß-Lg)/d-ribose glycation system at the molecular level and explore the possible inhibitory mechanism of SeNPs on the formation of advanced glycation end products (AGEs) by analyzing the glycation sites via high-performance liquid chromatography (HPLC)-Orbitrap-tandem mass spectrometry (MS/MS). Changes in contents of AGE formation and free amino acid contents had indicated that SeNPs could significantly slow the glycation process, thus attenuating the formation of AGEs. HPLC-Orbitrap-MS/MS analysis revealed that, at 6, 12, and 24 h, the number of glycation sites of glycated ß-Lg decreased from 7, 7, and 9 to 5, 5, and 6 after the intervention of SeNPs, respectively. The glycation extent of each glycation site was controlled, and the dual-glycation ability of K8, K14, K47, K91, and K101 was changed. All of these results confirmed that SeNPs could indeed slow the process of protein glycation at the molecular level. This may be the reason for SeNPs reducing the formation of AGEs during glycation. Therefore, this study shed light on the insight of how SeNPs reduce the formation of AGEs.

Observation of the structural changes of α-lactalbumin induced by ultrasonic prior to glycated modification.

Bovine α-lactalbumin (BLA) was treated by ultrasonic at 150 W/cm2 for different times and subsequently glycated with mannose by dry-heating. Molecular weight, intrinsic fluorescence spectra, glycation sites and degree of modified BLA were observed. The proteinaceous high molecular weight components were formed after ultrasonic prior to glycated modification, while the conformational changes were obvious. Prior to ultrasonic pretreatment, K62, K114, and K122 of BLA were identified. After treated by ultrasound at 150 W/cm2 for 5, 10, 15, and 20 min, the sites were increased to four, four, five, and five, respectively. All glycated sites of modified BLA exhibited a higher degree of substitution per peptide (DSP) values compared to native BLA. Ultrasonic at 150 W/cm2 for 20 min revealed the most significant change in the BLA structure. Therefore, conformational changes, the intensified glycation site, and DSP value were responsible for the structural changes of BLA. Practical applications BLA is suitable as an ingredient for infant nutrition in food, and has immune-modulating, antioxidant, antibacterial, and antitumor activity etc. This study revealed that the structural changes of BLA induced by ultrasonic prior to glycated modification. It will be beneficial to understand the mechanism of the functional changes of modified BLA. Ultrasonic prior to glycated modification will be more likely to develop a practical technology to modify protein in the food industry, and improve the functional characteristics of food, such as produce hypo-allergenic cow's milk in future.

Multiple Glycation Sites in Blood Plasma Proteins as an Integrated Biomarker of Type 2 Diabetes Mellitus.

Type 2 diabetes mellitus (T2DM) is one of the most widely spread metabolic diseases. Because of its asymptomatic onset and slow development, early diagnosis and adequate glycaemic control are the prerequisites for successful T2DM therapy. In this context, individual amino acid residues might be sensitive indicators of alterations in blood glycation levels. Moreover, due to a large variation in the half-life times of plasma proteins, a generalized biomarker, based on multiple glycation sites, might provide comprehensive control of the glycemic status across any desired time span. Therefore, here, we address the patterns of glycation sites in highly-abundant blood plasma proteins of T2DM patients and corresponding age- and gender-matched controls by comprehensive liquid chromatography-mass spectrometry (LC-MS). The analysis revealed 42 lysyl residues, significantly upregulated under hyperglycemic conditions. Thereby, for 32 glycation sites, biomarker behavior was demonstrated here for the first time. The differentially glycated lysines represented nine plasma proteins with half-lives from 2 to 21 days, giving access to an integrated biomarker based on multiple protein-specific Amadori peptides. The validation of this biomarker relied on linear discriminant analysis (LDA) with random sub-sampling of the training set and leave-one-out cross-validation (LOOCV), which resulted in an accuracy, specificity, and sensitivity of 92%, 100%, and 85%, respectively.

Plasma levels of free fatty acids correlate with type 2 diabetes mellitus.

AIMS: Free fatty acids (FFA) mediate adverse metabolic effects such as downregulated carbohydrate metabolisms, providing causal links between obesity and the development of type 2 diabetes mellitus (T2DM). Here, we investigated the plasma concentrations of FFA alone and in combination with protein glycation as potential diagnostic and prognostic biomarkers of T2DM. MATERIALS AND METHODS: EDTA-plasma obtained from 48 newly diagnosed male T2DM patients, 48 long-term controlled (24 male and 24 female) T2DM patients, 20 prediabetic male T2DM patients and two age-matched control cohorts (48 non-diabetic (ND) men; 24 male and 24 female ND partipants) were analysed for a set of clinical parameters including FFA. Glycation sites were quantified after tryptic digestion using tandem mass spectrometry. RESULTS: Median plasma concentrations of FFA were almost three-fold higher in samples obtained from newly diagnosed (long-term controlled) T2DM patients than in those obtained from the control group, providing diagnostic sensitivity (SN) of 92% (85%) and specificity (SP) of 90% (88%). When combined with the glycation level of lysine-141 of haptoglobin, diagnostic accuracy improved further for newly diagnosed (SN, 94%; SP 96%) and long-term controlled (SN, 85%; SP, 94%) T2DM patients (HbA1c: SN, 88%; SP, 96%). A prospective pilot study evaluating the prognostic value revealed initially low FFA levels for pre-diabetic patients that increased in the following four years in patients whose prediabetic state worsened or who developed T2DM. CONCLUSIONS: FFA levels are elevated in newly diagnosed and long-term controlled T2DM patients, providing high diagnostic accuracy of 87% and 91%, respectively, which improved further when combined with the glycation degree of lysine-141 in haptoglobin. Additionally, FFA showed higher mean fold-changes than HbA1c or FPG in subjects developing T2DM, indicating higher sensitivity towards the progression of the disease.

Diagnostic Accuracy of Protein Glycation Sites in Long-Term Controlled Patients with Type 2 Diabetes Mellitus and Their Prognostic Potential for Early Diagnosis.

Current screening tests for type 2 diabetes mellitus (T2DM) identify less than 50% of undiagnosed T2DM patients and provide no information about how the disease will develop in prediabetic patients. Here, twenty-nine protein glycation sites were quantified after tryptic digestion of plasma samples at the peptide level using tandem mass spectrometry and isotope-labelled peptides as internal standard. The glycation degrees were determined in three groups, i.e., 48 patients with a duration of T2DM exceeding ten years, 48 non-diabetic individuals matched for gender, BMI, and age, and 20 prediabetic men. In long-term controlled diabetic patients, 27 glycated peptides were detected at significantly higher levels, providing moderate diagnostic accuracies (ACCs) from 61 to 79%, allowing a subgrouping of patients in three distinct clusters. Moreover, a feature set of one glycated peptides and six established clinical parameters provided an ACC of 95%. The same number of clusters was identified in prediabetic males (ACC of 95%) using a set of eight glycation sites (mostly from serum albumin). All patients present in one cluster showed progression of prediabetic state or advanced towards diabetes in the following five years. Overall, the studied glycation sites appear to be promising biomarkers for subgrouping prediabetic patients to estimate their risk for the development of T2DM.

Investigation into allergenicity reduction and glycation sites of glycated ß-lactoglobulin with ultrasound pretreatment by high-resolution mass spectrometry.

Ultrasound treatment could change the conformation of ß-lactoglobulin (ß-Lg) and improve the glycation reaction in aqueous solution under neutral condition. However, the effect of ultrasound pretreatment on glycation of ß-Lg with pentose at dry-state remains ambiguous, and the relationship between glycation and allergenicity of ß-Lg with ultrasound pretreatment is unclear. This study aimed to evaluate the effect of ultrasound pretreatment on glycation and allergenicity of ß-Lg. Markedly decreased allergenicity of ß-Lg was observed after glycation with ribose before and after ultrasound pretreatment with the minimum found at 400 W. Orbitrap LC-MS/MS showed that the glycation degree of some peptides in glycated ß-Lg with and without ultrasound pretreatment were different although the content of free amino group and molecular mass were insignificantly different. Therefore, ultrasound pretreatment promoted the reduction in allergenicity by improving the glycation extent of some glycation sites although it hardly enhanced the whole glycation degree of ß-Lg.

Glypre: In Silico Prediction of Protein Glycation Sites by Fusing Multiple Features and Support Vector Machine.

Glycation is a non-enzymatic process occurring inside or outside the host body by attaching a sugar molecule to a protein or lipid molecule. It is an important form of post-translational modification (PTM), which impairs the function and changes the characteristics of the proteins so that the identification of the glycation sites may provide some useful guidelines to understand various biological functions of proteins. In this study, we proposed an accurate prediction tool, named Glypre, for lysine glycation. Firstly, we used multiple informative features to encode the peptides. These features included the position scoring function, secondary structure, AAindex, and the composition of k-spaced amino acid pairs. Secondly, the distribution of distinctive features of the residues surrounding the glycation and non-glycation sites was statistically analysed. Thirdly, based on the distribution of these features, we developed a new predictor by using different optimal window sizes for different properties and a two-step feature selection method, which utilized the maximum relevance minimum redundancy method followed by a greedy feature selection procedure. The performance of Glypre was measured with a sensitivity of 57.47%, a specificity of 90.78%, an accuracy of 79.68%, area under the receiver-operating characteristic (ROC) curve (AUC) of 0.86, and a Matthews's correlation coefficient (MCC) of 0.52 by 10-fold cross-validation. The detailed analysis results showed that our predictor may play a complementary role to other existing methods for identifying protein lysine glycation. The source code and datasets of the Glypre are available in the Supplementary File.

Glycated lysine-141 in haptoglobin improves the diagnostic accuracy for type 2 diabetes mellitus in combination with glycated hemoglobin HbA1c and fasting plasma glucose.

BACKGROUND: Recent epidemiological studies indicate that only 30-50% of undiagnosed type 2 diabetes mellitus (T2DM) patients are identified using glycated hemoglobin (HbA1c) and elevated fasting plasma glucose (FPG) levels. Thus, novel biomarkers for early diagnosis and prognosis are urgently needed for providing early and personalized treatment. METHODS: Here, we studied the glycation degrees of 27 glycation sites representing nine plasma proteins in 48 newly diagnosed male T2DM patients and 48 non-diabetic men matched for age (range 35-65 years). Samples were digested with trypsin and enriched for glycated peptides using boronic acid affinity chromatography. Quantification relied on mass spectrometry (multiple reaction monitoring) using isotope-labelled peptides as internal standard. RESULTS: The combination of glycated lysine-141 of haptoglobin (HP K141) and HbA1c provided a sensitivity of 94%, a specificity of 98%, and an accuracy of 96% to identify T2DM. A set of 15 features considering three glycation sites in human serum albumin, HP K141, and 11 routine laboratory measures of T2DM, metabolic syndrome, obesity, inflammation, and insulin resistance provided a sensitivity of 98%, a specificity of 100%, and an accuracy of 99% for newly diagnosed T2DM patients. CONCLUSIONS: Our studies demonstrated the great potential of glycation sites in plasma proteins providing an additional diagnostic tool for T2DM and elucidating that the combination of these sites with HbA1c and FPG could improve the diagnosis of T2DM.

Higher degree of glycation of hemoglobin S compared to hemoglobin A measured by mass spectrometry: Potential impact on HbA1c testing.

BACKGROUND: Glycated hemoglobin (GHb), reported as HbA1c, is used as marker of long-term glycemia for diabetic patients. HbA1c results from boronate affinity methods are generally considered to be unaffected by most hemoglobin variants; this assumes comparable glycation of variant and non-variant (HbAA) hemoglobins. In this report, glycation of HbA beta chain (ßA) and HbS beta chain (ßS) for the most common Hb variant trait (HbAS) are examined. METHODS: We analyzed 41 blood samples from subjects with HbAS, both with and without diabetes. Using LC-MS, ratios of glycated HbS to glycated HbA were determined by comparison of areas under the curves from extracted ion chromatograms. RESULTS: Glycation of ßS chains was significantly higher (p<0.001) than ßA chains; this difference was consistent across subjects. Total (α+ß) glycated HbAS was theoretically estimated to be ~5% higher than glycated HbAA. CONCLUSION: This novel mass-spectrometric approach described allows for relative quantification of glycated forms of ßS and ßA. Although ßS glycation was significantly higher than that of ßA, the difference in total glycation of HbAS versus HbAA was smaller and unlikely to impact clinical interpretation of boronate affinity HbA1c results. These data support the continued use of boronate affinity to measure HbA1c in patients with HbAS.

Direct targeted glycation of the free sulfhydryl group of cysteine residue (Cys-34) of BSA. Mapping of the glycation sites of the anti-tumor Thomsen-Friedenreich neoglycoconjugate vaccine prepared by Michael addition reaction.

We present in this manuscript the characterization of the exact glycation sites of the Thomsen-Friedenreich antigen-BSA vaccine (TF antigen:BSA) prepared using a Michael addition reaction between the saccharide antigen as an electrophilic acceptor and the nucleophilic thiol and L-Lysine ε-amino groups of BSA using different ligation conditions. Matrix laser desorption ionization time-of-flight mass spectrometry of the neoglycoconjugates prepared with TF antigen:protein ratios of 2:1 and 8:1, allowed to observe, respectively, the protonated molecules for each neoglycoconjugates: [M + H](+) at m/z 67,599 and 70,905. The measurements of these molecular weights allowed us to confirm exactly the carbohydrate:protein ratios of these two synthetic vaccines. These were found to be closely formed by a TF antigen:BSA ratios of 2:1 and 8:1, respectively. Trypsin digestion and liquid chromatography coupled with electrospray ionization mass spectrometry allowed us to identify the series of released glycopeptide and peptide fragments. De novo sequencing affected by low-energy collision dissociation tandem mass spectrometry was then employed to unravel the precise glycation sites of these neoglycoconjugate vaccines. Finally, we identified, respectively, three diagnostic and characteristic glycated peptides for the synthetic glycoconjugate possessing a TF antigen:BSA ratio 2:1, whereas we have identified for the synthetic glycoconjugate having a TF:BSA ratio 8:1 a series of 14 glycated peptides. The net increase in the occupancy sites of these neoglycoconjugates was caused by the large number of glycoforms produced during the chemical ligation of the synthetic carbohydrate antigen onto the protein carrier.