Quantification of prospective type 2 diabetes mellitus biomarkers by stable isotope dilution with bi-labeled standard glycated peptides

Abstract

Type 2 diabetes mellitus (T2DM) is a complex group of disorders, characterized by hyperglycemia, insulin resistance and insulin deficiency. In human blood, hyperglycemia ultimately results in the enhancement of glycation -a posttranslational modification formed by the interaction of protein amino groups with glucose. The resulting fructosamines (Amadori compounds) readily undergo further degradation resulting in advanced glycation end products (AGEs), known to be pro-inflammatory in humans. These compounds are highly heterogeneous and characteristic of advanced stages of the disease, whereas fructosamines are recognized markers of early diabetes stages (HbA 1C , glycated albumin). Recently, individual plasma protein glycation sites were proposed as promising T2DM biomarkers sensitive to short-term fluctuations of plasma glucose. However, corresponding absolute quantification strategies, applicable in regular clinical practice, are still not established. Therefore, here we propose a new analytical approach aiming at reproducible and precise quantification of multiple glycated peptides in human plasma tryptic digests. Thereby, the standard peptides comprised a 13 C, 15 N-labeled lysyl residue, a dabsyl moiety for determination of standard amounts, and a cleavable linker. Known amounts of these peptides were spiked to plasma samples prior to tryptic digestion, quantification relying on stable isotope dilution. The method was demonstrated to be applicable for quantification of individual glycated sites in T2DM patients and non-diabetic controls.

Probing Protein Glycation by Chromatography and Mass Spectrometry: Analysis of Glycation Adducts

“…However, the most important (if not the critical) improvement of the LC-MS/MS-based quantification methodology was done by combining it with the stable isotope dilution technique [ 139 ]. This approach ( Figure 4 A) allows direct determination of analyte concentration ( C A ) in experimental samples based on the ratio of analyte ( S A ) and internal standard ( S IS ) peak areas, multiplied by the concentration of the stable isotope-labeled internal standard ( IS ) spiked to the sample (C IS ) [ 159 ]: …”

“…Although the reagents, conventionally used in amino acid analysis might also produce such characteristic fragments (e.g., characteristic loss of 2,4,6-trinitrobenzene moiety from 2,4,6-trinitrobenzene sulfonate derivatives of amino acids [ 123 ]), unbiased profiling of protein enzymatic digests still needs to be performed. Recently, we successfully applied different variants of unbiased MS profiling to human plasma tryptic digests, aiming identification of specific protein glycation sites associated with type 2 diabetes mellitus (T2DM) [ 77 , 80 , 159 , 226 , 227 , 228 ]. We believe, that analogous experiments, performed at the amino acid level, might also reveal new disease-specific markers and characteristic indicators for the loss of food quality during storage and thermal treatment.…”

An Optimised Di-Boronate-ChemMatrix Affinity Chromatography to Trap Deoxyfructosylated Peptides as Biomarkers of Glycation

“…The same approach was followed to synthesise [Asn 7 As control peptides, we synthesised the phosphorylated analogue [Ser 7 (PO 3 H 2 )]CSF114 (7) [33] (bearing the O-phosphoryl moiety on Ser7) and the corresponding non-glycosylated peptide CSF114 (8). We selected O-phosphorylation as control modification, because in previous studies we evaluated that dysregulation in protein expression can be involved in diabetes immune dysfunction [33].…”

“…Reactive dicarbonyls, glyoxal, methylglyoxal, and 3-deoxyglucosone are potent glycation agents, highly reactive more than the glucose itself and thus originate glycation chain reactions on more and more protein sites [1][2][3]. AGEs and their concentration levels are considered, in the clinics, relevant for diagnosis of diabetes complications and proteomics technologies are proposed for their characterisation [4][5][6][7]. However the possibility to trap early glycation adducts (i.e., deoxyfructosylated proteins) and the corresponding peptides by efficient mass spectrometry technology is a challenge to anticipate AGEs formation.…”

“…Therefore, efficient methods for isolation, enrichment, and recognition of glycated proteins are of current research interest. Hereof, boronate affinity materials (BAMs) have gained increasing attention in recent years [9], because of the specificity of the reaction of boronic acid that has been demonstrated efficient for the separation of a wide variety of cis diol-containing compounds, including catechols, nucleosides, nucleotides, nucleic acids, carbohydrates [10], glycoproteins [6,7,11], and enzymes [12]. In particular, boronic acid forms with cis diols esters (in aqueous solution under basic conditions) that can be easily hydrolysed under acidic conditions.…”