Quantitative screening of advanced glycation endproducts in cellular and extracellular proteins by tandem mass spectrometry

Abstract

Glycation of proteins forms fructosamines and advanced glycation endproducts. Glycation adducts may be risk markers and risk factors of disease development. We measured the concentrations of the early glycation adduct fructosyl-lysine and 12 advanced glycation endproducts by liquid chromatography with tandem mass spectrometric detection. Underivatized analytes were detected free in physiological fluids and in enzymic hydrolysates of cellular and extracellular proteins. Hydroimidazolones were the most important glycation biomarkers quantitatively; monolysyl adducts (N(epsilon)-carboxymethyl-lysine and N(epsilon)-1-carboxyethyl-lysine) were found in moderate amounts, and bis(lysyl)imidazolium cross-links and pentosidine in lowest amounts. Quantitative screening showed high levels of advanced glycation endproducts in cellular protein and moderate levels in protein of blood plasma. Glycation adduct accumulation in tissues depended on the particular adduct and tissue type. Low levels of free advanced glycation endproducts were found in blood plasma and levels were 10-100-fold higher in urine. Advanced glycation endproduct residues were increased in blood plasma and at sites of vascular complications development in experimental diabetes; renal glomeruli, retina and peripheral nerve. In clinical uraemia, the concentrations of plasma protein advanced glycation endproduct residues increased 1-7-fold and free adduct concentrations increased up to 50-fold. Comprehensive screening of glycation adducts revealed the relative and quantitative importance of alpha-oxoaldehyde-derived advanced glycation endproducts in physiological modification of proteins-particularly hydroimidazolones, the efficient renal clearance of free adducts, and the marked increases of glycation adducts in diabetes and uraemia-particularly free advanced glycation endproducts in uraemia. Increased levels of these advanced glycation endproducts were associated with vascular complications in diabetes and uraemia.

Degradation products of proteins damaged by glycation, oxidation and nitration in clinical type 1 diabetes

“…Glycation, oxidation and nitration adduct residues of plasma protein and haemoglobin were measured in exhaustive enzymatic digests (50 μg protein equivalent) prepared as described previously (with control subjects for protease autolysis) [11]. Samples were assayed by liquid chromatography with triple quadrupole mass spectrometric detection (LC-MS/MS) with stable isotope-substituted internal standardisation as described previously [5]. Pentosidine, NFK and trp were measured by liquid chromatography with modified fluorimetric detection: the mobile phase was 0.1% trifluoroacetic acid with isocratic 10% acetonitrile from 0 to 20 min and a linear gradient of 10-50% acetonitrile from 20 to 50 min eluted through column 1 only at a flow rate of 0.4 ml/min.…”

“…Protein glycation, as well as protein oxidation and nitration, is thought to contribute to vascular cell dysfunction and the development of microvascular diabetic complications (retinopathy, nephropathy and neuropathy) [2][3][4]. Recent research has shown that protein glycation, oxidation and nitration are increased in cellular and ex-tracellular proteins in diabetes [5]. Cells maintain the quality and functional integrity of proteins by degradation and replacement of damaged proteins; oxidation and glycation are major types of physiological protein damage [5,6].…”

“…Recent research has shown that protein glycation, oxidation and nitration are increased in cellular and ex-tracellular proteins in diabetes [5]. Cells maintain the quality and functional integrity of proteins by degradation and replacement of damaged proteins; oxidation and glycation are major types of physiological protein damage [5,6]. Cellular proteolysis liberates the glycated, oxidised and nitrated amino acids as free adducts.…”

“…Cellular proteolysis liberates the glycated, oxidised and nitrated amino acids as free adducts. These are released into blood plasma and excreted in urine [5]. The changes in plasma concentrations and urinary excretion of glycation, oxidation and nitration free adducts may reflect tissue damage in diabetes and provide new markers indicative of the damaging effects of hyperglycaemia.…”

“…Important AGEs, in a quantitative sense, are hydroimidazolones derived from arginine residues and modified by glyoxal, methylglyoxal and 3-DG (G-H1, MG-H1 and 3DG-H, respectively). Other important and widely studied AGEs are N " -carboxymethyl-lysine (CML), N " -carboxyethyl-lysine (CEL) and pentosidine [5]. Major quantitative markers of oxidative damage to proteins are methionine sulphoxide (MetSO) and N-formylkynurenine (NFK), formed by the oxidation of methionine and tryptophan respectively [7,8], and a widely studied marker of nitration damage to proteins is 3-nitrotyrosine (3-NT) [9] (Fig.…”