Characterization of glycation adducts on human serum albumin by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Abstract

Background-Non-enzymatic glycation of human serum albumin (HSA) is associated with the long-term complications of diabetes. We examined the structure and location of modifications on minimally glycated HSA and considered their possible impact on the binding of drugs to this protein.Methods-Minimally glycated and normal HSA (used as a control) were digested with trypsin, Glu-C or Lys-C, followed by fractionation of the resulting peptides and their analysis by matrixassisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) to determine the structures and locations of glycation adducts.Results-Several specific lysine and arginine residues were identified as modification sites in minimally glycated HSA. Residues K12, K51, K199, K205, K439 and K538 were found to be modified through the formation of fructosyl-lysine, while the modification of K159 and K286 involved the formation of pyrraline, N ε -carboxymethyl-lysine respectively. Lysine K378 was found to give N ε -carboxyethyl-lysine in some forms of glycated HSA but fructosyl-lysine in other forms. Residues R160 and R472 produced a modification based on N ε -(5-hydro-4-imidazolon-2-yl) ornithine. Lysine R222 was modified to produce argpyrimidine, N ε -[5-(2,3,4-trihydroxybutyl)-5-hydro-4-imidazolon-2-yl]ornithine or tetrahydropyrimidine.Conclusions-With the exception of K12, K199, K378, K439 and K525, all of the observed sites of modification for minimally glycated HSA were new to this current study. The fact that many of these glycation-related modifications are located at or near known drug binding sites on HSA explains why some differences have been previously noted in the binding of certain drugs to normal vs glycated HSA.

Structural Mechanism of Ring-opening Reaction of Glucose by Human Serum Albumin

“…Other glycation sites on HSA were identified previously in a number of independent studies (15)(16)(17)(18)30). Lys-525 was often observed to be glycated by glucose in a number of in vivo studies (15,16,18).…”

“…For serum albumin, the proportion of glycated albumin in healthy persons is in the range of 1-10% (5,8), which increases by 2-3-fold in the case of patients with diabetes mellitus (9). Glycation occurs on amine groups on the protein surface, commonly on lysine residues, but also on other residues (15,17,30).…”

Inhibitory effect of gold nanoparticles on the D-ribose glycation of bovine serum albumin

“…37,38 In the case of human and bovine albumin, this conjugation has been proposed to occur through the proteins' cysteine and lysine residues, 38,39 with the lysine residues also serving as the major sites of glycation. 40 Although the mechanism for the conjugation of these amino acids to GNPs remains unclear, to date, two mechanisms, one for cysteine 39 and one for lysine, 38,41 have been proposed. The interaction between cysteine residues and GNPs has been speculated to ensue through ligand exchange reactions, whereas those with lysine are suggested to depend on electrostatic forces.…”

Urinary Proteome Analysis using Capillary Electrophoresis Coupled to Mass Spectrometry: A Powerful Tool in Clinical Diagnosis, Prognosis and Therapy Evaluation

“…As outlined recently, any biomarker should be defined by its accurate molecular composition (39). The identification of a theoretical protein based on a few tryptic fragments may be quite misleading, as it generally does not allow accounting for posttranslational modifications, which may in fact confer »biomarker quality« to the protein: glycated albumin may serve as a biomarker for diabetes, while albumin precursor, which would be defined as biomarker based on several tryptic peptides, certainly does not (40). If albumin precursor was defined as biomarker based on a top-down 2DE-or LC-MS/MS approach, the subsequent validation of the results using an alternative technology for clinical application would have failed, due to the inaccurate definition of the original biomarker.…”