Biochemical Society Transactions

679th Meeting of the Biochemical Society

Quantitative screening of protein biomarkers of early glycation, advanced glycation, oxidation and nitrosation in cellular and extracellular proteins by tandem mass spectrometry multiple reaction monitoring

N. Ahmed, P.J. Thornalley


Glycation of proteins forms fructosamines and AGEs (advanced glycation end products). Oxidative and nitrosative stress leads to the formation of oxidative and nitrosative modifications. The modified amino acid residues formed in these processes are biomarkers of protein damage: some are risk markers and some may be risk factors for disease development. We developed a method for the concurrent quantitative measurement of 16 biomarkers indicative of protein glycation, oxidation and nitrosation damage using LC-MS/MS (LC with tandem MS detection). Underivatized analytes were detected free in physiological fluids and in enzymatic hydrolysates of cellular and extracellular proteins. Hydroimidazolones were the most important glycation biomarkers, and methionine sulphoxide was the most important oxidative biomarker quantitatively; 3-nitrotyrosine was the biomarker of nitrosation. Quantitative screening showed high levels of AGEs in cellular protein and moderate levels in protein of blood plasma. Levels of 3-nitrotyrosine were typically 100-fold lower than this. The major glycation adducts in blood plasma had high renal clearances in normal healthy human subjects, whereas methionine sulphoxide and 3-nitrotyrosine had low renal clearances due to further metabolism. Physiological AGEs in blood plasma were eliminated from the circulation in the kidney and not in the liver. LC-MS/MS peptide mapping was also used to locate the protein biomarkers. These studies reveal that advanced glycation is a significant modification of cellular and extracellular protein. The enzymatic defences against glycation, antioxidants and proteasomal protein degradation inside cells are probable factors regulating biomarker levels of cellular protein.

  • fructosamine
  • glycation
  • methionine sulphoxide
  • mass spectrometry (MS)
  • nitrotyrosine
  • oxidative stress


  • 679th Meeting of the Biochemical Society held at the University of Essex, Colchester, 2–4 July 2003

  • Abbreviations used: AGE, advanced glycation end product; CML, N-carboxymethyl-lysine; 3DG-H, 3-deoxyglucosone; 3DG-H1, NΔ-[5-hydro-5-(2,3,4-trihydroxybutyl)-4-imidazolon-2-yl]-ornithine; 3DG-H2, 5-[2-amino-5-hydro-5-(2,3,4-trihydroxybutyl)-4-imidazolon-1-yl]norvaline; 3DG-H3, 5-[2-amino-4-hydro-4-(2,3,4-trihydroxybutyl)-5-imidazolon-1-yl]norvaline; DOLD, 3-deoxyglucosone-derived lysine dimer [1,3-di(N-lysino)-4-(2,3,4-trihydroxybutyl)imidazolium salt]; ESRD, end-stage renal disease; GOLD, glyoxal-derived lysine dimer [1,3-di(N-lysino)imidazolium salt]; MetSO, methionine sulphoxide; MetSO2, methionine sulphone; LC-MS/MS, LC with tandem MS detection; MG-H1, NΔ-(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine; MG-H2, 5-(2-amino-5-hydro-5-methyl-4-imidazolon-1-yl)norvaline; MG-H3, 5-(2-amino-4-hydro-4-methyl-5-imidazolon-1-yl)norvaline; MOLD, methylglyoxal-derived lysine dimer [1,3-di(N-lysino)-4-methylimidazolium salt]; MRM, multiple reaction monitoring; 3-NO2Tyr, 3-nitrotyrosine.