Background: Type 2 diabetes mellitus (T2DM) is the most common lifestyle disease affecting
all countries. Due to its asymptomatic onset, it is often diagnosed after irreversible vascular
complications have been initiated. Therefore, specific markers characteristic for very early disease
stages and suitable for early diagnostics are required. Glycation of plasma proteins, such as human
serum albumin (HSA), has been often suggested as marker. However, the total glycation degree of
HSA does not provide sufficient information about short-term fluctuations of blood glucose concentrations
due to the large number of glycation sites. Analysis of individual modification sites might be
more informative, but methods for reliable quantifications are still missing.
Objective: The main objective of this study was to establish and qualify a method of analysis applicable
to sensitive and precise quantification of glycations sites in plasma proteins.
Methods: Plasma samples obtained from diabetic patients and non-diseased individuals were separated
from low-molecular weight compounds, digested with trypsin, enriched for glycated peptides
by boronic acid affinity chromatography (BAC), desalted by solid phase extraction (SPE), and separated
by RP-HPLC coupled online to ESI-QqQ-MS. Quantification relied on multiple reaction monitoring
(MRM) of multiple glycation sites identified in plasma proteins using a stable isotope dilution
approach or internal standardization.
Results: The data presented here suggests high selectivity and precision (relative standard deviations
below 10%) of the overall approach appearing to be well suited for the identification of prospective
biomarkers. Six glycated peptides corresponding to different glycation sites of HSA were present in
plasma samples obtained from T2DM patients at significantly higher levels than in non-diabetic men
matched for age. Additionally, each of the studied glycation site of HSA appeared to be affected at
Conclusion: The presented approach enables the sensitive and robust quantification of prospective
T2D biomarkers promising for clinical diagnostics.