Glycation Isotopic Labeling with 13C-Reducing Sugars for Quantitative Analysis of Glycated Proteins in Human Plasma

Abstract

Non-enzymatic glycation of proteins is a post-translational modification produced by a reaction between reducing sugars and amino groups located in lysine and arginine residues or in the N-terminal position. This modification plays a relevant role in medicine and food industry. In the clinical field, this undesired role is directly linked to blood glucose concentration and therefore to pathological conditions derived from hyperglycemia (>11 mM glucose) such as diabetes mellitus or renal failure. An approach for qualitative and quantitative analysis of glycated proteins is here proposed to achieve the three information levels for their complete characterization. These are: 1) identification of glycated proteins, 2) elucidation of sugar attachment sites, and 3) quantitative analysis to compare glycemic states. Qualitative analysis was carried out by tandem mass spectrometry after endoproteinase Glu-C digestion and boronate affinity chromatography for isolation of glycated peptides. For this purpose, two MS operational modes were used: higher energy collisional dissociation-MS2

Effect of selective breeding on collagen properties of Atlantic salmon ( Salmo salar L.)

“…The accumulation of reducing sugars has been associated with the formation of stable cross-links derived from non-enzymatic glycosylation of collagen, preferentially of Arg, and Lys, mainly at the telopeptide region (Priego-Capote et al, 2010). The present study showed an appreciable absorption of carbohydrates in the IR region between 1730 cm À1 and 1760 cm À1 (Fig.…”

“…** Hydrophobic amino acids: Thr + Ala + Val + Met + Met + Ile + Leu + Phe + Pro. of a Schiff base (Priego-Capote et al, 2010). Hence, both FP and WP appeared to show similar activity of the enzymes responsible for collagen glycosylation.…”

Characterization of the glycated human cerebrospinal fluid proteome

“…In the current work, we have identified glycated proteins in human CSF of aged-matched normal healthy subjects to create a database of identified glycated peptides and proteins as a source for the neurodegenerative disease research community to facilitate the discovery of potential novel markers of brain pathologies. The method, previously applied to human plasma and hemolysates samples, is based on the differential labeling of proteins with isotopically labeled-glucose ([ 13 C]), named Glycation Isotopic Labeling (GIL) [40,41]. The approach has enabled the identification of more than hundred glycated CSF proteins with many of them found specifically in high glucose perturbation in vitro.…”

A chemical and computational approach to comprehensive glycation characterization on antibodies

“…Other fragmentation strategies have also been employed including CID using MS 3 and multistage activation approaches, 13,14 electron transfer dissociation (ETD), 15-17 and higher energy collisional dissociation (HCD). 18 Given that CID is the most prevalent of the dissociation methods available to researchers, a method that relies upon derivatization of the glycated peptide to prevent neutral loss was developed, facilitating elucidation of the peptide backbone using CID. In this study glycation on a therapeutic IgG1 mAb was characterized.…”