Synthesis and characterization of carbohydrate-insulin conjugates using desorption ionization mass spectrometry

Abstract

Blood glycoproteins containing terminal non-reducing D-galactosyl groups have previously been shown to be rapidly cleared from the circulatory system of animals. This occurs by interaction at the asialoglycoprotein receptor of the hepatocytes. We prepared several carbohydrate–insulin conjugates, containing one or more terminal D-galactosyl residues, to study their binding to this receptor. Fast atom bombardment (FAB) and 252Cf plasma desorption (PD) mass spectrometry were used to characterize the conjugates formed when insulin was treated with N-hydroxysuccinimide lactobionate. The synthetic approach, using N-hydroxysuccinimide amino acid esters, is a well-known procedure for peptide synthesis. However, the coupling reaction did not yield the expected lactobionyl insulin products. Instead, each lactobionyl group was accompained by an additional mass increment (115 daltons) in the conjugate structure. PD mass spectrometry verified that the products were intact modified insulins, while FAB mass spectrometry, with computerized peak matching and tandem mass spectrometry, provided the exact mass and structure of the additional mass increment. The two desorption ionization methods provided complementary data enabling the rapid characterization of these unexpected synthetic products.