High Sensitivity of Zn²⁺ Insulin to Metal-Catalyzed Oxidation: Detection of 2-Oxo-Histidine by Tandem Mass Spectrometry

Abstract

Purpose. To establish the sensitivity of Zn²⁺ insulin (Zn²⁺-INS) to metal-catalyzed oxidation (MCO) and to use tandem mass spectrometry/mass spectroscopy (MS/MS) for the identification and quantification of 2-oxo-histidine at ⁵His_B and ¹⁰His_B upon the MCO of INS.

Methods. Zn²⁺-INS was exposed to Cu²⁺/ascorbate-induced MCO. Products were analyzed by reverse-phase high-performance liquid chromatography, electrospray ionization mass spectrometry (ESI-MS) and MS/MS, sodium dodecylsulfate polyacrylamide gel electrophotesis (SDS-PAGE), and CD spectroscopy.

Results. A maximal loss of 40% INS was achieved when 20 μM INS/8.8μM Zn²⁺ were exposed to 8 μM Cu²⁺ and 50 μM ascorbate. MCO was completely inhibited by ethylenediaminetetraacetic acid or native catalase but not with a 1000-fold molar excess of Zn²⁺ over Cu²⁺. MCO did not alter the aggregation state of INS. High-performance liquid chromatography-fractionated products contained portions of oxidized and native INS monomers. Oxidation selectively targeted the B chain of INS, where MS/MS sequencing revealed 2-oxo-His formation at both His residues at a relative ratio of ¹⁰His_B-ox/⁵His_B-ox = 2.8 ± 1.3 (SD).

Conclusions. At a Zn²⁺/INS molar ratio comparable to that in regular INS preparations, Zn²⁺-INS was susceptible to MCO. Both His residues of INS were converted partially to 2-oxo-His, with ¹⁰His_B possessing ca. three times greater susceptibility to MCO than ⁵His_B.