A Highly Specific Method for the Characterization of Glycation and Glyco‐oxidation Products of Globins

Abstract

A new method, based on matrix-assisted laser desorption/ionization (MALDI) measurements, has been developed for the evaluation of the glycation level of globins, a relevant parameter for diabetes control. It shows high levels of reproducibility and specificity, allowing the different glycated and glyco-oxidized products of both alpha- and beta-globins to be distinguished. Such specificity is reflected in its possible diagnostic use not only for the control of diabetes, but also for the occurrence of 'oxidative stress'. The comparison, for the same samples, of the obtained MALDI data with the related HbA1c values, determined by a high performance liquid chromatographic method, allowed it to be established that HbA1c percentages are not, as usually retained, related to the simply glycated beta-globin, but to the whole pool of glycated and glyco-oxidized alpha- and beta-globins.

The role of mass spectrometry in the study of non-enzymatic protein glycation in diabetes

“…Based on electrospray (ESI) mass spectrometry (Roberts et al, 1997), and applied to a large number of patients with differing degrees of metabolic control, this approach gave spectra from whole blood sample, mainly composed of glycated and unglycated a-and b-globins: ESI quantitative results plot linearly with HbA 1c measurements. The application of mass spectrometry to evaluation of glycated globins has the merit of demonstrating that both globins are glycated to similar extents (Lapolla et al, 1997b). MALDI can reveal the presence of glycooxidated products, which cannot be detected in ESI conditions 1999).…”

NMR Analysis of Synthetic Human Serum Albumin α-Helix 28 Identifies Structural Distortion upon Amadori Modification

“…Non-enzymatic glycation describes the initial products arising from the formation of Maillard reaction adducts due to the reaction between primary amino groups on a protein surface and reducing sugars such as glucose and fructose (1)(2)(3)(4). These non-enzymatic reactions are initiated with the reversible formation of a Schiff base adduct that undergoes rearrangement to form a more stable Amadori product (for glucose) (5) or Heyns product (for fructose) (6).…”

Analysis of glycated proteins by mass spectrometric techniques: qualitative and quantitative aspects

“…In contrast, MALDI-TOF mass spectrometry is able to distinguish between glycated and non-glycated species of specific proteins, as well as provide quantitative estimation of the level of glycation of the different serum proteins, such as human serum albumin (HSA) and the individual monomers of hemoglobin [47][48][49][50]. Equipped with "delayed extraction" ion source, the MALDI-TOF technique is able to reveal further degradation reactions of the sugar moieties of Amadori products of glycated proteins, such as dehydration and oxidation [51,52]. Coupled with enzymatic digests of glycated proteins, MALDI-TOF/MS of the partial hydrolysates of glycated proteins has been used to identify the glycation sites of the major intrinsic peptide (MIP) of the lens [53] and most susceptible glycation domain of immunoglobulin-G (IgG) [49].…”

“…With delayed extraction, MALDI-TOF can be used to observe further degradative reaction, such as dehydration and oxidation of the sugar moiety of Amadori products. Due to its high-mass range, TOF instruments also allow the observation of cross-linked proteins [51,54].…”