Identification of the site of glycation of human insulin

Mass spectrometry characterization of the glycation sites of bovine insulin by tandem mass spectrometry

“…In vitro glycation was carried out by incubating insulin with D-glucose in a protocol adapted from O'Harte et al where reducing [23] and nonreducing conditions were used. In nonreducing conditions, insulin (200 g in HCl 2 mM) was incubated with D-glucose 220 mM in phosphate buffer 10 mM (pH 7.4) at 37°C for 30 d (toluene 5 mM was added as a bacteriostatic).…”

“…Peptide sequence B-chain [23]. This is most probably due to the high reactivity as this residue is situated at the N-terminal of the peptide chain.…”

“…More recently, independent reports generalized the view that there are three possible free amino groups in the insulin molecule, human or bovine, available for in vitro glucose binding [15,[21][22][23]. Nowadays, through ESI-MS/MS and MALDI-MS approaches, it is established the N-terminal Phe1 residue of the B-chain is the only glycation site of human [23] and bovine [21] insulin.…”

“…More recently, independent reports generalized the view that there are three possible free amino groups in the insulin molecule, human or bovine, available for in vitro glucose binding [15,[21][22][23]. Nowadays, through ESI-MS/MS and MALDI-MS approaches, it is established the N-terminal Phe1 residue of the B-chain is the only glycation site of human [23] and bovine [21] insulin. Moreover, O'Harte and coworkers determined, in a novel diglycated insulin (under hyperglycemic reducing conditions), the Nterminals Gly1 of A-chain and Phe1 of B-chain as the sites of glycation for human insulin [15], after Glu-C enzymatic digestion and identification of digested fragments by plasma desorption mass spectrometry (PDMS).…”

Demonstration of increased concentrations of circulating glycated insulin in human Type 2 diabetes using a novel and specific radioimmunoassay

“…Previous studies have shown that glycated insulin has a reduced ability to regulate plasma glucose homeostasis in vivo and to stimulate adipose tissue lipogenesis or glucose uptake and oxidation by isolated diaphragm and abdominal muscle in vitro [3,4,5]. Studies in healthy human volunteers using the hyperinsulinaemic-euglycaemic glucose clamp technique suggest that glycated insulin could contribute to insulin resistance in Type 2 diabetes mellitus [6].The site of glycation of human insulin has now been identified by electrospray tandem mass spectrometry as the N-terminal Phe 1 of the B-chain [7], enabling the development of a sensitive and specific radioimmunoassay to measure concentrations of glycated insulin and to identify glycated insulin in pancreatic islets using immunohistochemistry [8]. High performance liquid chromatography techniques used previously were neither sensitive nor reliable Formation of advanced glycation-end products (AGEs) plays an important role in long-term metabolic consequences of diabetes including ophthalmic, renal and atherosclerotic vascular complications.…”