A New Strategy for Early Diagnosis of Type 2 Diabetes by Standard-Free, Label-Free LC-MS/MS Quantification of Glycated Peptides

Abstract

The early diagnosis of diabetes, one of the top three chronic incurable diseases, is becoming increasingly important. Here, we investigated the applicability of an 18O-labeling technique for the development of a standard-free, label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the early diagnosis of type 2 diabetes mellitus (T2DM). Rather than attempting to identify quantitative differences in proteins as biomarkers, glycation of the highest abundance protein in human plasma, human serum albumin (HSA), was monitored through quantitative analysis of HSA characteristic peptides using the 18O-labeling technique. Eight glucose-sensitive peptides and one glucose-insensitive peptide were discovered. The glucose-insensitive peptide served as the internal standard, and a standard-free, label-free LC-MS/MS method was developed. This method was then used to select putative biomarkers for T2DM in a clinical trial with 389 human plasma samples. As a result, three of the eight glucose-sensitive peptides (FKDLGEENFK, LDELRDEGK, and KVPQVSTPTLVEVSR) were selected and could be used as potential biomarkers for the early diagnosis of T2DM.

Development of Diagnostic Fragment Ion Library for Glycated Peptides of Human Serum Albumin: Targeted Quantification in Prediabetic, Diabetic, and Microalbuminuria Plasma by Parallel Reaction Monitoring, SWATH, and MSE

“…Among these sites, K549, K438, K490, K88, and K375 were highly sensitive for glycation modification as they had both AML and CML modifications, and K549 had additional CEL modification, while K375 (m/z 886.4) showed the highest significant fold change in prediabetes and diabetic plasma. These sites were also found to be sensitive in a previous study, where glycation sensitive peptides of albumin were determined (31). Although it is intriguing that only one lysine site i.e.…”

“…Certain lysine residues of HSA are more prone to undergo glycation modification, which are also called glycation/glucose-sensitive sites (31). As these sites are continuously exposed to higher glucose concentration, arguably, these sites can possibly undergo sequential AGE modifications followed by initial Amadori rearrangement.…”

“…In this regard, a stable-isotope-dilution tandem mass spectrometry method was employed for simultaneous analysis of CML and CEL in hydrolysates of plasma proteins (28), and 13 C6-glucose was utilized to quantify glycated proteins in the plasma and erythrocytes (29,30). In a recent study, the glycationsensitive peptides of HSA that could serve as markers for early diagnosis of type 2 diabetes were quantified by using an MS-based 18 O-labeling technique (31). However, most of the previous studies have focused on AML modification, rather than other AGE modification.…”

Proteome wide reduction in AGE modification in streptozotocin induced diabetic mice by hydralazine mediated transglycation

“…Furthermore, the extent of decrease in AGE modification by hydralazine was studied. In a recent study, the extent of glycation of eight glucose sensitive peptides of human serum albumin was monitored for early diagnosis of Type 2 diabetes20 (supplementary Table 1). In this study, a similar approach was used albeit with a slight modification, which is described in Material and Methods.…”

“…The extent of glycation modification was determined by analyzing the AGE modification of glucose sensitive peptides of serum albumin as described20 with a slight modification (supplementary Table 1). We have monitored the AGE modification of all the peptides containing the Glucose Sensitive Amino acid Residues (GSARs) (K44, R168, K210, K264, K438, R452) since trypsin digestion can generate different peptides containing these amino acid residues.…”