Comprehensive glycation profiling of Glargine by high-resolution mass spectrometry

Background: Methylglyoxal is a highly reactive α-oxoaldehyde metabolite formed during glycolysis that reacts with proteins in circulation leading to the formation of methylglyoxal-derived hydroimidazolones (MG-H) which can further be modified into advanced glycation end products (AGEs). AGEs activate the RAGE signaling pathway which leads to increased oxidative stress, inflammation, and insulin resistance. In diabetes, hyperglycemia and oxidative stress lead to increased plasma methylglyoxal concentrations and consequently, increased AGE formation. Glargine, the first long-acting insulin analog for diabetic therapy, has a prolonged plasma half-life that allows once a day injection. However, this also makes it susceptible to glycation which can affect the biological activity of the drug. Glargine being one of the most widely prescribed insulin analogs for diabetic therapy, it is crucial to determine how its function is affected by glycation. In this study, we have aimed to characterize glycated glargine to identify possible modifications.

Methods: Glargine was glycated in vitro with methylglyoxal at different pH for 4 days. Samples were collected every 24 hours and intact protein mass spectrometry was performed using a Q-Exactive High-Resolution Mass Spectrometer (HRMS) coupled to a UHPLC system. The mass spectra were deconvoluted using UniDec software to determine the mass shift and adduct formation. The observed mass shifts were validated by an extracted ion chromatogram (XIC).

Results: In this study, we observed that the acidic buffer condition was most suitable for the Glargine glycation study. 50% of glargine was found to be modified within 24 hours of methylglyoxal treatment with carboxyethyl lysine/arginine being the most abundant glycated proteoform. The amount of unmodified glargine gradually decreased to 18% after 96 hours of reaction. Day one did not show more than two sites of modification which increased to four on day two. Only after 72 hours of reaction, there is an appearance of five MG-H modifications.

Ours is the first study to identify the glycation of glargine by methylglyoxal. Glycation can lead to structural changes in glargine leading to reduced binding to insulin receptor and loss of activity. Furthermore, glycated glargine can bind to RAGE and aggravate insulin resistance.

References:

P. B. Walke, S. B. Bansode, N. P. More, A. H. Chaurasiya, R. S. Joshi, and M. J. Kulkarni, "Molecular investigation of glycated insulin-induced insulin resistance via insulin signaling and AGE-RAGE axis," Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease, 2021.

R. Hilgenfeld, G. Seipke, H. Berchtold, and D. R. Owens, "The evolution of insulin glargine and its continuing contribution to diabetes care," Drugs, 2014.

J. Xue et al., "The receptor for advanced glycation end products (RAGE) specifically recognizes methylglyoxal-derived AGEs," Biochemistry, 2014.