Extent Of Glycation Research Articles

Combined incubation of platelets and endothelial cells with glycated albumin: Altered thrombogenic and inflammatory responses.

Diabetes mellitus is a salient risk factor for the development of cardiovascular diseases (CVDs). Part of this risk is associated with the presence of advanced glycation end products (AGEs), which have been shown to up-regulate platelet or endothelial cell inflammatory and thrombogenic responses that are associated with CVDs. However, platelets perform mechanisms that alter endothelial cell inflammatory and thrombogenic responses, and endothelial cells perform similar mechanisms on platelets. Thus, our goal was to evaluate platelet and endothelial cell inflammatory and thrombogenic reactions that AGEs elicit during concurrent exposure. Endothelial cells were incubated with AGEs for 5 days, after which platelets were added. A time course for CVD inflammatory and thrombogenic responses was quantified as a function of extent of glycation. In general, the presence of platelets reduced AGE-induced endothelial cell responses associated with CVD progression and the presence of endothelial cells reduced platelet adhesion and activation responses, as compared with individual exposures. In general, the presence of irreversibly glycated albumin promoted CVD development to a greater extent than reversibly glycated albumin. This suggests that under diabetic conditions, platelets and endothelial cells can negatively feedback on each other, likely via enhanced adhesion, to elicit a reduced response associated with CVD progression.

Improved Glycation after Ultrasonic Pretreatment Revealed by High-Performance Liquid Chromatography–Linear Ion Trap/Orbitrap High-Resolution Mass Spectrometry

The glycation extent of bovine serum albumin (BSA) before and after ultrasonication was evaluated by MALDI-TOF and Orbitrap mass spectrometry. Ultrasonic pretreatment significantly improved the incorporation of galactose to BSA. Prior to ultrasonic pretreatment, only 12 sites (11 lysines and 1 arginine) were glycated, whereas the number of glycation sites was increased to 42, including 39 lysines and 3 arginines, after treatment. Average degree of substitution per peptide molecule of BSA (DSP) was used to evaluate the glycation level for each glycation site. The ultrasonic pretreatment significantly improved the DSP value of all glycation sites. The prevalently promoted glycation by ultrasonic pretreatment suggests that ultrasonication improves glycation through altering the structure of BSA throughout all three domains. An ultrahigh-resolution linear ion trap Orbitrap mass spectrometer facilitates unambiguous localization of glycation sites, allowing an in-depth analysis of the nature and extent of protein glycation at the molecular level. High-intensity ultrasonication can greatly improve protein glycation and, therefore, opens new routes to modify the functionality of proteins in a positive way.

Effect of Maillard reaction conditions on the degree of glycation and functional properties of whey protein isolate – Maltodextrin conjugates

The Maillard reaction has been used as a natural alternative to improve protein functionality by covalent coupling with saccharides. However, if reaction conditions are not properly selected, glycation can lead to a loss in functional properties. The objective of our research was to study the effect of temperature, time, water activity and reactants molar ratio on the degree of glycation and color development in whey protein isolate conjugated with maltodextrins. Three different levels of glycation (low, medium and high) were selected to investigate functional properties. The extent of glycation was assessed by quantifying the loss of amino groups using the o-phthaldialdehyde technique. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to evaluate the molecular weight of the glycoproteins. Color changes were determined using a Minolta colorimeter and calculating the browning index. Functional properties evaluated were solubility, rheological behavior, foam overrun and foam stability. Temperature and water activity were the most influential factors determining the degree of glycation and color change. The correlation coefficient between blocked amino groups and color was of 0.743. Whey protein isolate exhibited lower solubility at pH 5 and conjugates at pH 4. Consistency index and foaming properties improved according to the level of glycation achieved.

Glycation Accelerates Fibrillization of the Amyloidogenic W7FW14F Apomyoglobin

Neurodegenerative diseases are associated with misfolding and deposition of specific proteins, either intra or extracellularly in the nervous system. Advanced glycation end products (AGEs) originate from different molecular species that become glycated after exposure to sugars. Several proteins implicated in neurodegenerative diseases have been found to be glycated in vivo and the extent of glycation is related to the pathologies of the patients. Although it is now accepted that there is a direct correlation between AGEs formation and the development of neurodegenerative diseases, several questions still remain unanswered: whether glycation is the triggering event or just an additional factor acting on the aggregation pathway. To this concern, in the present study we have investigated the effect of glycation on the aggregation pathway of the amyloidogenic W7FW14F apomyoglobin. Although this protein has not been related to any amyloid disease, it represents a good model to resemble proteins that intrinsically evolve toward the formation of amyloid aggregates in physiological conditions. We show that D-ribose, but not D-glucose, rapidly induces the W7FW14F apomyoglobin to generate AGEs in a time-dependent manner and protein ribosylation is likely to involve lysine residues on the polypeptide chain. Ribosylation of the W7FW14F apomyoglobin strongly affects its aggregation kinetics producing amyloid fibrils within few days. Cytotoxicity of the glycated aggregates has also been tested using a cell viability assay. We propose that ribosylation in the W7FW14F apomyoglobin induces the formation of a cross-link that strongly reduces the flexibility of the H helix and/or induce a conformational change that favor fibril formation. These results open new perspectives for AGEs biological role as they can be considered not only a triggering factor in amyloidosis but also a player in later stages of the aggregation process.

Glycation of α-lactalbumin with different size saccharides: Effect on protein structure and antigenicity

The effect of the size of saccharides on protein structure and antigenicity after glycation was studied. α-Lactalbumin (α-LA) was incubated in a dry state with glucose, maltose or maltooligosaccharides (up to maltopentaose), at 55 °C and 65% relative humidity. The extent of glycation, aggregation processes, antigenicity, glycation sites and structural changes were investigated. Structural changes were observed when the protein was incubated with larger saccharides. Furthermore, the degree of glycation decreased with an increase in the size of the saccharide. The antigenicity of all samples significantly decreased when glycation increased, but the greatest reduction of antigenicity was observed with maltotriose-α-LA. By comparing the glycation sites of all the glycated samples, glycation of Lys58 was dominant in glucose-α-LA and maltotriose-α-LA, whose antigenicity reductions were the highest. These results suggest that when the main epitope was glycated, the degree of antigenicity reduction increased with the increase in the size of the saccharide.

Dietary flaxseed oil and fish oil modulates expression of antioxidant and inflammatory genes with alleviation of protein glycation status and inflammation in liver of streptozotocin–nicotinamide induced diabetic rats

Beneficial effects of dietary flaxseed oil or fish oil on streptozotocin–nicotinamide induced diabetic rats were investigated. Rats were divided into three diabetic and three non-diabetic groups and received control, flaxseed oil or fish oil diets (10%w/w). Both diets reduced blood glucose, TBARS and hepatic NO. The extent of glycation measured in terms of glycated albumin and hemoglobin was reduced significantly with both diets. Flaxseed oil diet up-regulated hepatic catalase (CAT) (activity and expression), superoxide dismutase (SOD) (activity and expression) and glutathione peroxidase (GPx) expression. Fish oil diet up-regulated hepatic CAT (activity and expression), paraoxonase-1 (PON-1) expression and down-regulated heme oxygenase-1 (HO-1) expression. Furthermore, both diets down-regulated the expression of hepatic inflammatory genes TNF-α, IL-6, MCP-1, INF-γ and NF-κB. These results were supported by histopathological observations which showed better tissue preservation in both the diets. Thus, both the diets proved to be beneficial in preventing tissue injury and alleviating diabetic insults in the livers of STZ–NIC diabetic rats.

Characterization of glycated lysozyme with galactose, galactooligosaccharides and galactan: Effect of glycation on structural and functional properties of conjugates

Glycated lysozyme (LZM) with galactose, galactooligosaccharides (GOSs) and galactan, through the Maillard reaction, were produced and characterized structurally and functionally. The extent of glycation of LZM was evaluated by the measurement of the blocked lysine and the formed furosine. The results indicated a high initial reactivity of galactose as compared to GOSs and galactan. In the presence of GOSs, the oxidative and cross-linking side reactions competed with the initial formation of Amadori product. The longer chain carbohydrate galactan exhibited the lowest initial rate of glycation with no significant cross-linking side reaction. Electrospray ionization mass spectrometry revealed the formation of glycated LZM containing up to eight galactose moieties; while only mono- to tetra-glycated LZM with GOSs were detected. 2–3 mol of galactan were conjugated to 1 mol of LZM. Galactan:LZM conjugates exhibited higher solubility, thermal stability and emulsion stability as compared to the unmodified LZM and LZM:galactose conjugates. LZM:GOS conjugates demonstrated the most improvement in the emulsion stability than the other conjugates. Furthermore, the glycation of LZM with galactose/GOSs/galactan decreased its immunoreactivity.

Characterization of soy β-conglycinin–dextran conjugate prepared by Maillard reaction in crowded liquid system

The conjugation reaction between soy β-conglycinin and dextran via Maillard reaction in crowded liquid system was studied. Apparent viscosity indicated that environment became more and more crowded with the increase of dextran concentration. The covalently linked conjugate was produced by incubating aqueous solution containing soy β-conglycinin and dextran at pH7.0 and 95°C for 6h.Free amino group content and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profile showed that soy β-conglycinin–dextran conjugate was successfully formed and macromolecular crowding enhanced the extent of glycation. The results of atomic force microscopy indicated that glycation inhibited the thermal aggregate of protein. Meanwhile, the tertiary structure of protein in conjugate changed with increasing number of aromatic side-chains exposed in heating environment. All data showed that the introduction of macromolecular crowding in soy β-conglycinin–dextran conjugate was an effective and promising method for linking polysaccharides to proteins.

Chemical modifications of casein occurring during industrial manufacturing of milk protein powders

The effect of milk acidification at different pH levels applied during the processing of various types of milk protein powders was first evaluated in the experimental samples of caseins, caseinates and milk protein concentrates. The absence of inorganic colloidal phosphorus was detected when milk was acidified to pH 4.60, whereas low levels (0.36–0.72 g/100 g of protein) were detected when mild milk acidification occurred. The extent of protein glycation and the occurrence of protein cross-linking were assessed in the same samples by measuring their furosine (5.7–427 mg/100 g of protein) and lysinoalanine (0.4–35.2 mg/100 g of protein) levels, respectively. In addition to heat treatments, the contents of these markers strictly depend on the presence of lactose and consequently on the effectiveness of the washing step applied during processing before drying to remove non-protein soluble components. The determination of these chemical markers in the samples permitted the identification of different technological conditions applied in the manufacturing of commercial milk protein powders with the same class.

The in vitro inhibition effect of 2 nm gold nanoparticles on non-enzymatic glycation of human serum albumin

The reaction of amino groups of protein and the carbonyl groups of reducing sugar molecules, non-enzymatically induce a series of chemical reactions that form a heterogeneous group of compounds known as advanced glycation end products (AGEs). The accumulation of AGEs is associated with various disease conditions that include complications in diabetes, Alzheimer’s disease and aging.The current study monitored the extent of non-enzymatic glycation of human serum albumin (HSA) in order to estimate the formation of HSA related AGEs in the presence of 2nm gold nanoparticles. The rate of glycation was evaluated using several analytical methods. Physiological concentrations of HSA and glyceraldehyde mixtures, incubated with various concentrations of negatively charged 2nm gold nanoparticles, resulted in a lower reaction rate than mixtures without 2GNP. Moreover, increasing concentrations of gold nanoparticles exhibited a pronounced reduction in AGE formation. High performance liquid chromatography, UV–visible spectroscopy and circular dichroism analytical methods provide reliable techniques for evaluating AGE formation of HSA adducts.

Glycated Albumin and Pathological Shear Stress Alters Endothelial Cell Thrombogenic Potential, Pro-Inflammatory State and Cytoskeletal Dynamics

Background: Diabetes mellitus is a disease that is characterized by a high blood glucose concentration, which leads to advanced glycation end product (AGE) formation. AGEs have various effects on the vasculature and this may be dependent on the extent and reversibility of glycation. AGEs present in the vasculature can promote cardiovascular diseases through modifications to circulating proteins and endothelial cells. Furthermore, cardiovascular diseases are characterized by altered shear stress, including both high magnitude short duration shear and low oscillatory shear. However, the combined role of high magnitude shear stress and the presence of AGEs on endothelial cell functions have not been elucidated. Our objective was to evaluate changes to endothelial cell responses under these conditions. Methods: To accomplish this, albumin was glycated for up to 8 weeks and endothelial cells were subjected to glycated albumin for up to 5 days. Endothelial cells were then exposed to shear stress in a cone-and-plate shearing device. Endothelial cell metabolic activity, surface expression of intracellular adhesion molecule-1, thrombomodulin, tissue factor, connexin-43, and caveolin-1, cytoskeletal organization and morphology were investigated. Results: In general, the combination of pathological shear stress and irreversibly glycated albumin deteriorated endothelial cell culture conditions and cytoskeletal organization, while enhancing pro-inflammatory and pro-thrombotic markers. The expression of connexin-43 and caveolin-1, was independent of shear stress, but was markedly enhanced after exposure to irreversibly glycated albumin. Conclusions: Our data suggests that the presence of irreversible glycated albumin diminishes endothelial cell culture conditions and that this is exacerbated by the application of high magnitude shear stress. It is therefore possible that the combination of altered shear stress and glycated albumin may accelerate the pathologies seen during diabetes mellitus to promote cardiovascular diseases.

Cross-Linking Behavior and Foaming Properties of Bovine α-Lactalbumin after Glycation with Various Saccharides

α-Lactalbumin was glycated via the Maillard reaction in the dry state using various mono- and oligosaccharides. The reaction resulted not only in coupling of the saccharides to α-lactalbumin but also in cross-linked proteins. The glycation rate and the extent of cross-link formation were highly dependent on the saccharide used. Glycation by arabinose and xylose led to a very fast protein cross-link formation, whereas glucose showed a relatively low protein cross-linking ability. The stability of foams, created using the various glycated protein samples, depended on the type of saccharide used, the extent of glycation, and possibly the amount of cross-linked protein. Compared to nonmodified α-lactalbumin, glycation with rhamnose and fucose improved foam stability, whereas application of glucose, galacturonic acid, and their oligosaccharides did not exert a clear effect. Mass spectrometric analysis revealed that dehydration of the Amadori products is an indicator of the formation of protein cross-links.

Controlling glycation of recombinant antibody in fed‐batch cell cultures

Protein glycation is a non-enzymatic glycosylation that can occur to proteins in the human body, and it is implicated in the pathogenesis of multiple chronic diseases. Glycation can also occur to recombinant antibodies during cell culture, which generates structural heterogeneity in the product. In a previous study, we discovered unusually high levels of glycation (>50%) in a recombinant monoclonal antibody (rhuMAb) produced by CHO cells. Prior to that discovery, we had not encountered such high levels of glycation in other in-house therapeutic antibodies. Our goal here is to develop cell culture strategies to decrease rhuMAb glycation in a reliable, reproducible, and scalable manner. Because glycation is a post-translational chemical reaction between a reducing sugar and a protein amine group, we hypothesized that lowering the concentration of glucose--the only source of reducing sugar in our fed-batch cultures--would lower the extent of rhuMAb glycation. When we decreased the supply of glucose to bioreactors from bolus nutrient and glucose feeds, rhuMAb glycation decreased to below 20% at both 2-L and 400-L scales. When we maintained glucose concentrations at lower levels in bioreactors with continuous feeds, we could further decrease rhuMAb glycation levels to below 10%. These results show that we can control glycation of secreted proteins by controlling the glucose concentration in the cell culture. In addition, our data suggest that rhuMAb glycation occurring during the cell culture process may be approximated as a second-order chemical reaction that is first order with respect to both glucose and non-glycated rhuMAb. The basic principles of this glycation model should apply to other recombinant proteins secreted during cell culture.

Proteomic Analysis of Glycated Proteins from Streptozotocin-Induced Diabetic Rat Kidney

Glycation of proteins leading to formation of advanced glycation end products (AGEs) has been considered as one of the important causes of diabetic nephropathy. Therefore, in this study, glycated proteins were detected by anti-AGE antibodies from kidney of streptozotocin-induced diabetic rat showing nephropathic symptoms, by using two dimensional electrophoresis and western blot analysis. These glycated proteins were identified and characterized by using combination of peptide mass finger printing and tandem mass spectrometric approaches. Glycated proteins identified included proteins from metabolic pathways, oxidative stress, cell signaling, and transport. Several of the proteins modified by glycation were involved in glucose metabolism. The extent of glycation was higher in diabetes compared to control, in the glycated proteins that were common to both control and diabetic kidney. Two dimensional electrophoresis proteins profiling of glycated proteins suggest that four of the glycated proteins were significantly up regulated in diabetes.

English

Non-enzymatic glycation of proteins, leading to chemical modification and cross-linking are of importance in the pathology of diabetic complications. In our early reported we showed that, camel milk possesses antihyperglycemic, antihyperlipidemic and exhibit beneficial role on membrane-bound ATPases in streptozotocin-diabetic rats after 45 days of treatment of camel milk at the optimum dose of 250 ml/day. In the present study, the effect of camel milk on the glycation and cross-linking of collagen from tail tendon of streptozotocin (STZ)-diabetic rats. Diabetes was induced in adult male albino rats of the Wistar strain, weighing 180 to 200 g, by administration of streptozotocin (STZ) (40 mg/kg) intraperitoneally. The animals were randomly divided into five groups of six animals each. Rats of groups II and IV were fed 250 ml of raw camel milk daily through watering bottle instead of water. Whereas animals in groups I, III and V were given tap water, and rats of group V were given 600 μg/kg body weight of glibenclamide orally, once in a day in the morning for 45 days. The levels of hydroxylproline and total collagen content elevated in the tail tendon of the diabetic control. The levels of extent of glycation and fluorescence of collagen increased while decreased levels of acid, neutral and pepsin soluble collagens were observed in the tail tendon of diabetic rats. These changes were alleviated by the simultaneous ingestion of camel milk. Our results demonstrate that intake of camel milk has a positive influence on tail tendon collagen glycation and other variables in STZ-diabetic rats and its effect was comparable with glibenclamide.   Key words: Camel milk, glycation, collagen, streptozotocin, diabetes.

Multiphoton spectral microscopy for imaging and quantification of tissue glycation

Tissue glycation from diabetes and aging can result in complications such as renal failure, blindness, nerve damage and vascular diseases. In this work, we applied multiphoton microscopy for imaging and characterizing the extent of tissue glycation. The characteristic features of multiphoton autofluorescence (MPAF) and second harmonic generation (SHG) images as well as MPAF spectra of glycated bovine skin, cornea and aorta were acquired. The analysis of MPAF intensity change accompanying the glycation process shows that collagen is more responsive to the formation of autofluorescent advanced glycation endproducts (AGE) than elastic fibers. Changes in spectral features were also used to estimate the rate of glycation in tissues with intrinsic AF. Our study shows that multiphton imaging may be used for the in vitro investigation of the effects of tissue glycation and that this approach may be used for monitoring AGE formation in the clinical setting.

Comparison of modification sites formed on human serum albumin at various stages of glycation

Background Many of the complications encountered during diabetes can be linked to the non-enzymatic glycation of proteins, including human serum albumin (HSA). However, there is little information regarding how the glycation pattern of HSA changes as the total extent of glycation is varied. The goal of this study was to identify and conduct a semi-quantitative comparison of the glycation products on HSA that are produced in the presence of various levels of glycation. Methods Three glycated HSA samples were prepared in vitro by incubating physiological concentrations of HSA with 15 mmol/l glucose for 2 or 5 weeks, or with 30 mmol/l glucose for 4 weeks. These samples were then digested and examined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify the glycation products that were formed. Results It was found that the glycation pattern of HSA changed with its overall extent of total glycation. Many modifications including previously-reported primary glycation sites (e.g., K199, K281, and the N-terminus) were consistently found in the tested samples. Lysines 199 and 281, as well as arginine 428, contained the most consistently identified and abundant glycation products. Lysines 93, 276, 286, 414, 439, and 524/525, as well as the N-terminus and arginines 98, 197, and 521, were also found to be modified at various degrees of HSA glycation. Conclusions The glycation pattern of HSA was found to vary with different levels of total glycation and included modifications at the 2 major drug binding sites on this protein. This result suggests that different modified forms of HSA, both in terms of the total extent of glycation and glycation pattern, may be found at various stages of diabetes. The clinical implication of these results is that the binding of HSA to some drug may be altered at various stages of diabetes as the extent of glycation and types of modifications in this protein are varied.

Advanced glycation end products in diabetic and non-diabetic human subjects suffering from cataract

Advanced glycation end products (AGEs) play a pivotal role in loss of lens transparency, i.e., cataract. AGEs formation occurs as a result of sequential glycation and oxidation reaction between reducing sugars and protein. AGEs production takes place throughout the normal aging process but its accumulation is found to be more rapid in diabetic patients. In this study, we quantified AGEs and N-(carboxyethyl) lysine (CEL) in human cataractous lenses from non-diabetic (n=50) and diabetic patients (n=50) using ELISA. We observed significantly higher (p<0.001) levels of lens AGEs and CEL in diabetic patients with cataract as compared with their respective controls. The presence of AGEs and CEL was also determined by western blotting and immuno-histochemical analysis. Furthermore, isolated β-crystallin from cataractous lenses of non-diabetic and diabetic patients was incubated with different sugars to evaluate the extent of glycation in a time dependent manner. Our data indicated more pronounced glycation in patients suffering from diabetes as compared to non-diabetics subjects demonstrating the need to focus on developing normoglycemic approaches. Such studies may provide an insight in developing therapeutic strategies and may have clinical implications.

Counterpoint: The Reporting of Estimated Glucose with Hemoglobin A1c

Hemoglobin A1c (Hb A1c)1 is a key parameter for understanding glycemic control in diabetes. It is used by both clinicians and patients to trigger adjustments in behavior and treatment. In general, clinicians understand Hb A1c and its limitations well, but evidence suggests that patients have difficulty understanding what Hb A1c means and how it relates to glucose. This situation may be particularly applicable for patients who regularly monitor their own glucose. We know a relationship exists between Hb A1c levels and average glucose concentrations over approximately 4 months, and it seems intuitive that it should be possible to express Hb A1c in terms of average glucose to provide a measure that patients more easily understand. Therefore, following completion and publication of the A1c-Derived Average Glucose (ADAG) study (1), a proposal was made to report Hb A1c as estimated average glucose (eAG) concentration by means of an equation derived from the ADAG study data set, as well as in conventional units (percent and millimoles per mole hemoglobin) (2). Although all clinicians and laboratory specialists will support the desire to promote understanding of Hb A1c, many have challenged whether converting Hb A1c to eAG is scientifically justified from the available evidence and whether it is likely to be helpful in practice. This Counterpoint summarizes the case against eAG. No single relationship exists between average glucose and Hb A 1c ; therefore, it is not possible to have a single equation that expresses Hb A 1c as eAG and is applicable to all patients. The proposal to express Hb A1c as eAG is underpinned by a number of assumptions. For such an approach to be valid, a consistent mathematical relationship between glucose concentration and the extent of glycation is necessary, …

Glycation Isotopic Labeling with 13C-Reducing Sugars for Quantitative Analysis of Glycated Proteins in Human Plasma

Non-enzymatic glycation of proteins is a post-translational modification produced by a reaction between reducing sugars and amino groups located in lysine and arginine residues or in the N-terminal position. This modification plays a relevant role in medicine and food industry. In the clinical field, this undesired role is directly linked to blood glucose concentration and therefore to pathological conditions derived from hyperglycemia (>11 mm glucose) such as diabetes mellitus or renal failure. An approach for qualitative and quantitative analysis of glycated proteins is here proposed to achieve the three information levels for their complete characterization. These are: 1) identification of glycated proteins, 2) elucidation of sugar attachment sites, and 3) quantitative analysis to compare glycemic states. Qualitative analysis was carried out by tandem mass spectrometry after endoproteinase Glu-C digestion and boronate affinity chromatography for isolation of glycated peptides. For this purpose, two MS operational modes were used: higher energy collisional dissociation-MS2 and CID-MS3 by neutral loss scan monitoring of two selective neutral losses (162.05 and 84.04 Da for the glucose cleavage and an intermediate rearrangement of the glucose moiety). On the other hand, quantitative analysis was based on labeling of proteins with [(13)C(6)]glucose incubation to evaluate the native glycated proteins labeled with [(12)C(6)]glucose. As glycation is chemoselective, it is exclusively occurring in potential targets for in vivo modifications. This approach, named glycation isotopic labeling, enabled differentiation of glycated peptides labeled with both isotopic forms resulting from enzymatic digestion by mass spectrometry (6-Da mass shift/glycation site). The strategy was then applied to a reference plasma sample, revealing the detection of 50 glycated proteins and 161 sugar attachment positions with identification of preferential glycation sites for each protein. A predictive approach was also tested to detect potential glycation sites under high glucose concentration.