Abstract
Protein glycation refers to the reversible reaction between aldoses (or ketoses) and amino groups yielding relatively stable Amadori (or Heyns) products. Consecutive oxidative cleavage reactions of these products or the reaction of amino groups with other reactive substances (e.g. α-dicarbonyls) yield advanced glycation end products (AGEs) that can alter the structures and functions of proteins. AGEs have been identified in all organisms, and their contents appear to rise with some diseases, such as diabetes and obesity. Here, we report a pilot study using highly sensitive and specific proteomics approach to identify and quantify AGE modification sites in plasma proteins by reversed phase HPLC mass spectrometry in tryptic plasma digests. In total, 19 AGE modification sites corresponding to 11 proteins were identified in patients with type 2 diabetes mellitus under poor glycemic control. The modification degrees of 15 modification sites did not differ among cohorts of normoglycemic lean or obese and type 2 diabetes mellitus patients under good and poor glycemic control. The contents of two amide-AGEs in human serum albumin and apolipoprotein A-II were significantly higher in patients with poor glycemic control, although the plasma levels of both proteins were similar among all plasma samples. These two modification sites might be useful to predict long term, AGE-related complications in diabetic patients, such as impaired vision, increased arterial stiffness, or decreased kidney function.
Highlights
The reaction between reducing sugars and amines is termed glycation or nonenzymatic glycosylation [1]
Quantification of Amide-advanced glycation end products (AGEs) in Plasma—Seventeen of the identified amide-AGE-modified peptides could be quantified by the peak areas displayed in the extracted ion chromatograms in all plasma samples with relative standard deviations among the technical replicates typically below 60%, except for DRQCdioxKacetylYIWoxGQK, which was not detected in samples 17, 19, and 22
SKformylEQLTPLIK from apolipoprotein A-II (Fig. 3B) was able to discriminate poorly controlled patients with type 2 diabetes mellitus (T2DM) from all other cohorts. To ensure that these higher levels in poorly controlled patients with T2DM were not associated with an elevated level of the protein, nonmodified peptides corresponding to both proteins were quantified as well
Summary
Materials—Chemicals and materials were from Carl Roth (Karlsruhe, Germany): tris(2-carboxyethyl)phosphine (Ն98%), glycerin (Ն99.5%); Biosolve (Valkenswaard, Netherlands): ace-. Tryptic Digestion—Plasma samples (100 l) were depleted of lipids by centrifugation (9168 ϫ g, 30 min, 4 °C), an aliquot (20 l) diluted 10-fold with ammonium hydrogen carbonate buffer (0.1 mol/liter, pH 8.0), and desalted using ultrafiltration (Vivaspin filter devices, 0.5 ml, 5-kDa cut-off). Plasma aliquots corresponding to a protein content of 25 g were complemented with SDS (10% (w/v) in water, 2 l) and tris(2-carboxyethyl)phosphine (50 mmol/liter in water, 2 l), diluted with aqueous ammonium hydrogen carbonate buffer (50 mmol/liter, pH 8.0) to obtain a final volume of 20 l, and incubated at 60 °C for 15 min. An aliquot (2 g protein) of the sample was diluted with sample buffer (0.05% (w/v) bromphenol blue, 62.5 mmol/liter Tris-HCl, pH 6.8, 20% (v/v) glycerin, 2% (w/v) SDS, 5% (v/v) -mercaptoethanol) at least 2-fold and heated to 95 °C for 5 min.
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