Comprehensive identification of glycated peptides and their glycation motifs in plasma and erythrocytes of control and diabetic subjects

Nonenzymatic glycation of proteins sets the stage for formation of advanced glycation end-products and development of chronic complications of diabetes. In this report, we extended our previous methods on proteomics analysis of glycated proteins to comprehensively identify glycated proteins in control and diabetic human plasma and erythrocytes. Using immunodepletion, enrichment, and fractionation strategies, we identified 7749 unique glycated peptides, corresponding to 3742 unique glycated proteins. Semiquantitative comparisons showed that glycation levels of a number of proteins were significantly increased in diabetes and that erythrocyte proteins were more extensively glycated than plasma proteins. A glycation motif analysis revealed that some amino acids were favored more than others in the protein primary structures in the vicinity of the glycation sites in both sample types. The glycated peptides and corresponding proteins reported here provide a foundation for potential identification of novel markers for diabetes, hyperglycemia, and diabetic complications in future studies.     

Enrichment and analysis of nonenzymatically glycated peptides: boronate affinity chromatography coupled with electron-transfer dissociation mass spectrometry

Nonenzymatic glycation of peptides and proteins by d-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. However, no effective high-throughput methods exist for identifying proteins containing this low-abundance post-translational modification in bottom-up proteomic studies. In this report, phenylboronate affinity chromatography was used in a two-step enrichment scheme to selectively isolate first glycated proteins and then glycated, tryptic peptides from human serum glycated in vitro. Enriched peptides were subsequently analyzed by alternating electron-transfer dissociation (ETD) and collision induced dissociation (CID) tandem mass spectrometry. ETD fragmentation mode permitted identification of a significantly higher number of glycated peptides (87.6% of all identified peptides) versus CID mode (17.0% of all identified peptides), when utilizing enrichment on first the protein and then the peptide level. This study illustrates that phenylboronate affinity chromatography coupled with LC-MS/MS and using ETD as the fragmentation mode is an efficient approach for analysis of glycated proteins and may have broad application in studies of diabetes mellitus.     

Lucica MI urinary myoinositol kit: a new diagnostic test for diabetes mellitus and glucose intolerance

Diabetes mellitus is a growing healthcare problem internationally, and poses a major burden from both a individual and societal perspective. Diabetes causes potentially life-threatening complications that are preventable if the disease is detected early and appropriate interventions are put in place. Early detection is therefore imperative for preventing diabetes-related morbidity and mortality. Current methods of detection, including the oral glucose tolerance test (OGTT), and measures of fasting plasma glucose, glycated hemoglobin (HbA(1c)), or glycated albumin, can be time-consuming and uncomfortable for patients. Myoinositol can be measured in urine and has been found to be elevated in patients with diabetes and glucose intolerance; it has thus proven useful as a marker for the early detection of these conditions. Lucica MI is a diagnostic kit for the measurement of urinary myoinositol; it is used to detect glucose intolerance and diabetes mellitus at an early stage in disease progression. The test is based on an enzymatic method that uses liquid reagents requiring no preparation. Clinical trial results demonstrate that the test could be used to detect not only diabetes mellitus, but also to distinguish impaired fasting glucose and impaired glucose tolerance from normal glucose tolerance.     

Relative quantification of glycated Cu-Zn superoxide dismutase in erythrocytes by electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESIMS) was used for relative quantification of glycated Cu-Zn superoxide dismutase (SOD-1) in human erythrocytes. SOD-1 samples were prepared from erythrocytes by removing hemoglobin using hemoglobind gel followed by ethanol and chloroform extraction. The reproducibility in measurement of the relative percentage of glycated protein was good, and the standard deviation of each measurement was 4.0%. From the mass spectral analysis of a mixture of commercial SOD-1 and in vitro partially glycated SOD-1 in several ratios, it was found that free and glycated SOD-1 have the same ionization efficiencies. The percentage of glycation on SOD-1 was measured in 30 individuals, including patients with diabetes mellitus. The glycation levels ranged from 4.5% to below the detection limit. The SOD-1 sample extracted from erythrocytes was fractionated by Glyco-Gel B chromatography, and the separated fractions were analyzed by MS. The mass spectra of absorbed fraction showed significant amounts of non-specific binding of non-glycated proteins to Glyco-Gel B.     

Binding of long-term glycated low density lipoprotein and AGE-albumin by peripheral monocytes and endothelial cells

Modification of low density lipoprotein (LDL) and plasma or tissue proteins by non-enzymatic glycation culminating in the formation of advanced glycation endproducts (AGEs) is one of the essential pathomechanisms leading to diabetes-associated long-term complications. We compared binding of glycated, glycoxidated and oxidated LDL by peripheral monocytes in activated and quiescent form. Interaction via specific receptors was different for glycated as compared to (glyc)oxidated LDL-modifications. In addition, binding of glycated LDL to quiescent and activated human umbilical vein endothelial cells was studied. In patients with insulin-dependent diabetes mellitus (IDDM), AGE-binding was significantly increased as compared to healthy individuals. Specific and non-specific monocyte binding mechanisms were detected, and both were significantly increased in IDDM patients. Specific and non-specific binding strategies possibly act in concert to eliminate circulating AGEs, which are instrumental in the development and progress of microangiopathic and macroangiopathic complications of diabetes mellitus.     

Decreased interaction of fibronectin, type IV collagen, and heparin due to nonenzymatic glycation. Implications for diabetes mellitus

The nonenzymatic glycation of basement membrane proteins, such as fibronectin and type IV collagen, occurs in diabetes mellitus. These proteins are nonenzymatically glycated in vivo and can also be nonenzymatically glycated in vitro. After 12 days of incubation at 37 degrees C with 500 mM glucose, purified samples of human plasma fibronectin and native type IV collagen showed a 13.0- and 4.2-fold increase, respectively, in glycated amino acid levels in comparison to control samples incubated in the absence of glucose. Gelatin (denatured calfskin collagen) was glycated 22.3-fold under the same conditions. Scatchard analyses were performed on the binding of radiolabeled fibronectin to gelatin or type IV collagen. It was found that there is a 3-fold reduction in the affinity of fibronectin to type IV collagen due to the nonenzymatic glycation of fibronectin. The dissociation constant (KD) for the binding of control fibronectin to type IV collagen was 9.6 X 10(-7) M while the KD for glycated fibronectin and type IV collagen was 2.9 X 10(-6) M. This was similar to the 2.7-fold reduction in the affinity of fibronectin for gelatin found as a result of the nonenzymatic glycation of fibronectin (KD of 4.5 X 10(-7) M for the interaction of control fibronectin with gelatin vs. KD of 1.2 X 10(-6) M for the interaction of nonenzymatically glycated fibronectin with gelatin). The molecular association of control fibronectin or its glycated counterpart with [3H]heparin was also determined. Scatchard analyses of this interaction showed no difference between control fibronectin and glycated fibronectin in [3H]heparin binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

"Glycated" osteocalcin in human and bovine bone. The effect of age

Purified osteocalcin from cow and calf bone was analyzed for nonenzymatic glycosylation (glycation) by sodium [3H]borohydride reduction. Calf bone was found to be approximately 5% glycated, while bone from mature cows was 10% glycated. These results were confirmed by a second method which utilizes periodate oxidation followed by formaldehyde fluorescence. Osteocalcin in human bone was also found to be glycated. The content of glycated osteocalcin from the bones of 47 nondiabetic individuals, aged 0.6-97, was dependent upon age. The extent of glycation was lowest in children, was constant through the adult years, and increased linearly in bone taken from individuals aged 60-97. Glycated osteocalcin was purified by boronate affinity chromatography and subjected to one-step Edman degradation. It was established that the site of glycation was the amino-terminal tyrosine. Increases in the amount of glycated osteocalcin in the bones of older individuals may play a role in the pathogenesis of senile osteoporosis and in the osteopenia which may accompany diabetes mellitus.     

Peptide and amino acid glycation: new insights into the Maillard reaction.

Nonenzymatic glycation of proteins, peptides and other macromolecules (the Maillard reaction) has been implicated in a number of pathologies, most clearly in diabetes mellitus. but also in the normal processes of aging and neurodegenerative amyloid diseases such as Alzheimer's. In the early stage, glycation results in the formation of Amadori-modified proteins. In the later stages, advanced glycation end products (AGE) are irreversibly formed from Amadori products leading to the formation of reactive intermediates, crosslinking of proteins, and the formation of brown and fluorescent polymeric materials. Although, the glycation of structural proteins has been attributed a key role in the complications of diabetes, recent attention has been devoted to the physiological significance of glycated peptide hormones. This review focuses on the physico-chemical properties of the Amadori compounds of bioactive peptides of endogenous and exogenous origin, such as Leu-enkephalin and morphiceptin, investigated under different conditions as well as on novel pathways in the Maillard reaction observed from investigating intramolecular events in ester-linked glycopeptides.     

Effects of oxidative modifications induced by the glycation of bovine serum albumin on its structure and on cultured adipose cells

Non-enzymatic glycosylation (glycation) and oxidative damages represent major research areas insofar as such modifications of proteins are frequently observed in numerous states of disease. Albumin undergoes structural and functional alterations, caused by increased glycosylation during non insulin-dependent diabetes mellitus, which is closely linked with the early occurrence of vascular complications. In this work, we first characterized structural modifications induced by the glycation of bovine serum albumin (BSA). A pathophysiological effect of glycated BSA was identified in primary cultures of human adipocytes as it induces an accumulation of oxidatively modified proteins in these cells. BSA was incubated in the presence or absence of physiological, pathological or supra-physiological concentrations of glucose at 37 degrees C for 7 weeks. Enhanced BSA glycation percentages were determined using boronate affinity columns. The occurrence of oxidative modifications was found to be enhanced in glycated BSA, after determination of the free thiol groups content, electrophoretic migration and infrared spectrometry spectra. An accumulation of carbonyl-modified proteins and an increased release of isoprostane were observed in cell media following the exposure of adipocytes to glycated albumin. These results provide a new possible mechanism for enhanced oxidative damages in diabetes.     

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.     

Non-enzymatic glycation of antithrombin III in vitro

Non-enzymatic glycation of antithrombin III (AT-III) has been proposed as a significant contributor to the increased incidence of thrombo-occlusive events in diabetics. AT-III, isolated from normal human plasma by means of heparin affinity and ion-exchange chromatography, was incubated with 0-0.5 M glucose in neutral phosphate buffer at 37 degrees C. The extent of non-enzymatic glycation could be monitored by uptake of radioactivity as well as by binding to a phenylboronate affinity resin, which effectively retards AT-III containing ketoamine-linked glucose. Non-enzymatically glycated AT-III (approx. 1 mol glucose/mol protein) bound heparin nearly as efficiently as non-glycated AT-III. The two AT-III preparations were equally active in inhibiting thrombin cleavage of chromogenic substrate. Following incubation with [14C]glucose, structural analyses of cyanogen-bromide-cleaved peptides of enzymatically glycated AT-III showed that the [14C]glucose adducts were distributed over many sites on the molecule. This lack of specificity contrasts with the restricted sites of modification on hemoglobin, albumin and ribonuclease A, and explains why non-enzymatic glycation of AT-III has little if any effect on its function.     

Early glycation products of endothelial plasma membrane proteins in experimental diabetes

The participation of glucose and two intermediates of glucose metabolism: glucose-6-phosphate (G6P) and glyceraldehyde-3-phosphate (Gald3P) to the formation of early glycation products was comparatively evaluated in the endothelial plasma membrane of streptozotocin-induced diabetic rats. Antibodies risen to a carrier protein reductively glycated by each of the sugars mentioned above were used to probe by immunoblotting the proteins of the lung microvascular endothelium plasmalemma purified from normal and diabetic rats. The amount of glycated endothelial plasma membrane proteins was below the limit of detection in normoglycemic animals but increased dramatically in diabetic animals for glucose and G6P. In contrast, no signal was found in diabetic rats for Gald3P, indicating that either the contribution of this phosphotriose to the glycation of intracellular proteins is negligible in vivo, or the Schiff base generated by this sugar transforms very rapidly into products of advanced glycation. Globally, the endothelial plasma membrane proteins bound on average 300 times more glucose than G6P proving that, in spite of its low in vitro potency as glycating agent, glucose represents the main contributor to the intracellular formation of early glycation products. The most abundant glycated proteins of the lung endothelial plasma membrane were separated by two dimensional electrophoresis and identified by mass spectrometry.     

Detection of glycation sites in proteins by high-resolution mass spectrometry combined with isotopic labeling

The products of nonenzymatic glycation of proteins are formed in a chemical reaction between reducing sugars and the free amino group located either at the N terminus of the polypeptide chain or in the lysine side chain. Glycated proteins and their fragments could be used as markers of the aging process as well as diabetes mellitus and Alzheimer’s disease, making them an object of interest in clinical chemistry. In this article, we propose a new method for the identification of peptide-derived Amadori products in the mixtures obtained by enzymatic hydrolysis of glycated proteins. Two proteins, ubiquitin and human serum albumin (HSA), were modified with an equimolar mixture of glucose and [¹³C₆]glucose and were subjected to enzymatic hydrolysis. The obtained enzymatic digests were analyzed by high-resolution mass spectrometry (HRMS), and the peptide-derived Amadori products were identified on the basis of specific isotopic patterns resulting from ¹³C substitution. The number of glycated peptides in the digest of HSA detected by our procedure was in agreement with the data recently reported in the literature.     

Characterization of the glycated human cerebrospinal fluid proteome

Protein glycation is a nonenzymatic modification that involves pathological functions in neurological diseases. Despite the high number of studies showing accumulation of advanced end glycation products (AGEs) at clinical stage, there is a lack of knowledge about which proteins are modified, where those modifications occur, and to what extent. The goal of this study was to achieve a comprehensive characterization of proteins modified by early glycation in human cerebrospinal fluid (CSF). Approaches based on glucose diferential labeling and mass spectrometry have been applied to evaluate the glycated CSF proteome at two physiological conditions: native glucose level and in vitro high glucose content. For both purposes, detection of glycated proteins was carried out by HCD-MS2 and CID-MS3 modes after endoproteinase Glu-C digestion and boronate affinity chromatography. The abundance of glycation was assessed by protein labeling with (13)C(6)-glucose incubation. The analysis of native glycated CSF identified 111 glycation sites corresponding to 48 glycated proteins. Additionally, the in vitro high glucose level approach detected 265 glycation sites and 101 glycated proteins. The comparison of glycation levels under native and 15 mM glucose conditions showed relative concentration increases up to ten folds for some glycated proteins. This report revealed for the first time a number of key glycated CSF proteins known to be involved in neuroinflammation and neurodegenerative disorders. Altogether, the present study contains valuable and unique information, which should further help to clarify the pathological role of glycation in central nervous system pathologies. This article is part of a Special Issue entitled: Translational Proteomics.     

The assessment of protein glycation in human atherosclerotic plaques by affinity chromatography

SUMMARY

Human atherosclerotic plaques are characterized by a massive deposition of lipid within arterial walls. The lipids accumulated are partly oxidized, as assessed by gas chromatography of lipids and their oxidation products. Both advancing age and diabetes mellitus are associated with an increased prevalence and severity of atherosclerosis.

In diabetes mellitus the development of secondary complications appear to be increased by poor glucose control. Indeed, the post-translational modification of protein by non-enzymatic glycation may provide the link between abnormal glucose control and diabetic complications. For atherosclerosis however, the relationship between glucose control and disease is unclear, with evidence available to support and discount such a link. To study protein glycation in a condition associated with a significant level of lipid oxidation products poses several methodological problems, most of which are associated with interference by lipid-derived aldehydes. Many chemical assays of protein glycation monitor aldehydic products common to the chemistry of both protein glycation and lipid oxidation. Studies of protein glycation in human atheroma, obtained at necropsy, are presented which make use of a commercially available boronic acid affinity-based chromatographic assay of glycated protein. The commercially available affinity-based chromatographic assay of glycated protein appears to be free from such interference and may well prove useful in the study of other conditions in which the non-enzymatic glycation of protein is suspected.     

Glucose tolerance in patients with cystic fibrosis: five year prospective study.

OBJECTIVES--To study prevalence and incidence of diabetes mellitus in patients with cystic fibrosis. DESIGN--Five year prospective study with annual oral glucose tolerance tests. SETTING--CF Center Copenhagen, Denmark. SUBJECTS--191 patients with cystic fibrosis aged above 2 years. MAIN OUTCOME MEASURES--Glucose tolerance, plasma glucose concentrations after fasting and after glucose loading, and haemoglobin A1c levels. RESULTS--Prevalence of diabetes increased from 11% (n = 21) to 24% (n = 46) during study, with annual age dependent incidence of 4-9%. Diabetes was diagnosed at median age of 21 (range 3-40). At diagnosis of diabetes, symptoms of hyperglycaemia were present in 33% of patients, fasting hyperglycaemia (> or = 7.8 mmol/l) was seen in 16%, and increased haemoglobin A1c levels (> 6.4%) were seen in 16%. Impaired glucose tolerance implied higher risk for development of diabetes than normal glucose tolerance (odds ratio 5.6). In 58% of cases with impaired glucose tolerance, however, glucose tolerance was normal at next annual test. Normal glucose tolerance was found in only 37% of patients at all five tests. Within this group of patients, median plasma glucose concentrations after fasting and after glucose loading and haemoglobin A1c levels increased by 6-8% during study. CONCLUSIONS--Prevalence and incidence of diabetes in cystic fibrosis patients was high and increased with age. Since hyperglycaemic symptoms, fasting hyperglycaemia, and increased levels of glycated haemoglobin did not reliably identify diabetes mellitus, we recommend annual oral glucose tolerance tests in all cystic fibrosis patients aged over 10 years.     

Non-enzymatic glycation of proteins: From diabetes to cancer

Incubation of proteins with glucose leads to their non-enzymatic glycation and formation of Amadori products known as an early glycation product. Oxidative cleavage of Amadori products is considered as a major route to advanced glycation endproducts (AGEs) formation in vivo. Non-enzymatic glycation of proteins or Maillard reaction is increased in diabetes mellitus due to hyperglycemia and leads to several complications such as blindness, heart disease, nerve damage, and kidney failure. The early and advanced glycation products are accumulated in plasma and tissues of diabetic patients and cause production of autoantibodies against corresponding products. The advanced glycation products are also associated with other diseases like cancer. This review summarizes current knowledge of these stage specific glycated products as common and early diagnostic biomarkers for the associated diseases and the complications with the aim of a novel therapeutic target for the diseases.     

Action of pelargonidin on hyperglycemia and oxidative damage in diabetic rats: implication for glycation-induced hemoglobin modification

Glycation-modified hemoglobin in diabetes mellitus has been suggested to be a source of enhanced catalytic iron and free radicals causing pathological complications. The present study aims to verify this idea in experimental diabetes. Pelargonidin, an anthocyanidin, has been tested for its antidiabetic potential with emphasis on its role against pathological oxidative stress including hemoglobin-mediated free radical reactions. Male wistar rats were grouped as normal control, streptozotocin-induced diabetic control, normal treated with pelargonidin and diabetic treated with pelargonidin. Pelargonidin-treated rats received one time i.p injection of the flavonoid (3 mg/kg bodyweight). Biochemical parameters were assayed in blood samples of different groups of rats. Liver was used for histological examinations. Pelargonidin treatment normalized elevated blood glucose levels and improved serum insulin levels in diabetic rats. Glucose tolerance test appeared normal after treatment. Decreased serum levels of SOD and catalase, and increased levels of malondialdehyde and fructosamine in diabetic rats were reverted to their respective normal values after pelargonidin administration. Extents of hemoglobin glycation, hemoglobin-mediated iron release, iron-mediated free radical reactions and carbonyl formation in hemoglobin were pronounced in diabetic rats, indicating association between hemoglobin glycation and oxidative stress in diabetes. Pelargonidin counteracts hemoglobin glycation, iron release from the heme protein and iron-mediated oxidative damages, confirming glycated hemoglobin-associated oxidative stress in diabetes.     

Prevalence of glucose intolerance in free-ranging Macaca fascicularis of Mauritius

Oral glucose tolerance tests (OGTTs) were carried out on 30 free-ranging long-tailed macaques (Macaca fascicularis) on the island of Mauritius, following the suggestion that severe glucose intolerance and diabetes mellitus might be prevalent in this macaque population. OGTTs revealed no evidence of frank diabetes mellitus in the sample. However, 13% of individuals showed impaired glucose tolerance, with preserved insulin secretion, suggesting the presence of the target tissue resistance to insulin characteristic of human noninsulin-dependent diabetes mellitus (NIDDM). The macaques with impaired glucose tolerance were neither obese nor aged. Glucose levels at all time points of the OGTT in normal macaques in our free-ranging sample were lower than reported in captive populations, perhaps due to greater physical activity. Our observations demonstrate that a genetic predisposition to glucose intolerance does exist in M. fascicularis, and that this condition, well documented in laboratory macaques, is not simply an artifact of captivity.     

Deleterious effects of reactive aldehydes and glycated proteins on macrophage proteasomal function: Possible links between diabetes and atherosclerosis

People with diabetes experience chronic hyperglycemia and are at a high risk of developing atherosclerosis and microvascular disease. Reactions of glucose, or aldehydes derived from glucose (e.g. methylglyoxal, glyoxal, or glycolaldehyde), with proteins result in glycation that ultimately yield advanced glycation end products (AGE). AGE are present at elevated levels in plasma and atherosclerotic lesions from people with diabetes, and previous in vitro studies have postulated that the presence of these materials is deleterious to cell function. This accumulation of AGE and glycated proteins within cells may arise from either increased formation and/or ineffective removal by cellular proteolytic systems, such as the proteasomes, the major multi-enzyme complex that removes proteins within cells. In this study it is shown that whilst high glucose concentrations fail to modify proteasome enzyme activities in J774A.1 macrophage-like cell extracts, reactive aldehydes enhanced proteasomal enzyme activities. In contrast BSA, pre-treated with high glucose for 8 weeks, inhibited both the chymotrypsin-like and caspase-like activities. BSA glycated using methylglyoxal or glycolaldehyde, also inhibited proteasomal activity though to differing extents. This suppression of proteasome activity by glycated proteins may result in further intracellular accumulation of glycated proteins with subsequent deleterious effects on cellular function.