Multiple Glycation Sites in Blood Plasma Proteins as an Integrated Biomarker of Type 2 Diabetes Mellitus

Abstract

Type 2 diabetes mellitus (T2DM) is one of the most widely spread metabolic diseases. Because of its asymptomatic onset and slow development, early diagnosis and adequate glycaemic control are the prerequisites for successful T2DM therapy. In this context, individual amino acid residues might be sensitive indicators of alterations in blood glycation levels. Moreover, due to a large variation in the half-life times of plasma proteins, a generalized biomarker, based on multiple glycation sites, might provide comprehensive control of the glycemic status across any desired time span. Therefore, here, we address the patterns of glycation sites in highly-abundant blood plasma proteins of T2DM patients and corresponding age- and gender-matched controls by comprehensive liquid chromatography-mass spectrometry (LC-MS). The analysis revealed 42 lysyl residues, significantly upregulated under hyperglycemic conditions. Thereby, for 32 glycation sites, biomarker behavior was demonstrated here for the first time. The differentially glycated lysines represented nine plasma proteins with half-lives from 2 to 21 days, giving access to an integrated biomarker based on multiple protein-specific Amadori peptides. The validation of this biomarker relied on linear discriminant analysis (LDA) with random sub-sampling of the training set and leave-one-out cross-validation (LOOCV), which resulted in an accuracy, specificity, and sensitivity of 92%, 100%, and 85%, respectively.

Keywords: Amadori compounds; biomarkers; glycation; glycation sites; label-free quantification; linear discriminant analysis; mass spectrometry; plasma proteins; type 2 diabetes mellitus.

Figure 1

Early and advanced glycation in human blood plasma. The major pathways of advanced glycation end product (AGE) formation: monosaccharide autoxidation (Wolff-pathway), autoxidation of aldimins (Namiki-pathway), polyol pathway, autoxidation of Amadori products (Hodge-pathway), and non-oxidative pathway.

Figure 2

Overview of the experimental workflow.

Figure 3

Tandem mass spectra acquired at m/z 673.66 (A) and 463.56 (B) corresponding to the [M + 3H]³⁺ ions of the peptides, EQLK_AmAVM_oxDDFAAFVEK and FK_AmDLGEENFK (both human serum albumin). The spectra were acquired with the LTQ-Orbitrap Velos Pro mass spectrometer operated in the positive ion mode as a part of targeted RP-HPLC-Orbitrap-LIT-MS/MS DDA workflow. The inserts represent extracted ion chromatograms (m/z ± 0.02) acquired for the m/z 673.66 (A) and 463.56 (B). K_Am and M_Ox denote glycated lysyl and oxidized methionyl residues, respectively.

Figure 3

Tandem mass spectra acquired at m/z 673.66 (A) and 463.56 (B) corresponding to the [M + 3H]³⁺ ions of the peptides, EQLK_AmAVM_oxDDFAAFVEK and FK_AmDLGEENFK (both human serum albumin). The spectra were acquired with the LTQ-Orbitrap Velos Pro mass spectrometer operated in the positive ion mode as a part of targeted RP-HPLC-Orbitrap-LIT-MS/MS DDA workflow. The inserts represent extracted ion chromatograms (m/z ± 0.02) acquired for the m/z 673.66 (A) and 463.56 (B). K_Am and M_Ox denote glycated lysyl and oxidized methionyl residues, respectively.

Figure 4

Results of the principle component analysis (PCA) performed for T2DM patient (opened diamonds) and normoglycemic (closed circles) groups. The analysis was based on all 42 differentially glycated peptides and indicated clear separation between the groups.

Figure 5

Values of the linear discriminant analysis (LDA) response variable plotted for T2DM (opened diamonds) and control (closed circles) individuals, calculated using selected predictive variable sets (as summarized in Table 3). (A) Estimated via leave-one-out cross-validation; (B) estimated from original samples, with LDA trained on generated samples.

Figure 6

Peak areas of the signals at 38.7 min (A), and 48.9 min (B), integrated in the extracted ion chromatograms (XICs) of m/z 698.56 ± 0.02 (A) and 691.09 ± 0.02 (B), corresponding to the [M + 4H]⁴⁺ ions of glycated peptides LVNEVTEFAK_AmTCCAMVADESAENCCAMDK (human serum albumin, A) and QNCCAMELFEQLGEYK_AmFQNALLVR (human serum albumin, B), respectively. The data were acquired in the RP-HPLC-QqTOF-MS experiments performed with boronic acid affinity chromatography (BAC) enriched tryptic digests obtained from individual T2DM and control plasma samples. Statistical significance was estimated by the Mann-Whitney U-test, and p values below 0.05 were considered to be significant. K_Am and C_CAM denote glycated lysyl and carbamidomethylatedcysteinyl residues, respectively.

Figure 7

The level of HbA_1c in plasma samples obtained from patients with T2DM and healthy persons. Statistical significance was estimated by the Mann-Whitney U-test, p < 0.0001.