On‐line reduction of insulin disulfide bonds with photoinduced radical reactions, upstream to nano liquid chromatography‐mass spectrometry

Abstract

AbstractA current focus of our research is dedicated to investigating tools that can be used to couple pancreas‐on‐a‐chip systems on‐line with mass spectrometry. To enable on‐line‐cleavage of insulin´s disulfide bonds, we coupled an inter‐chain cleavage reactor with a nano liquid chromatography‐mass spectrometry system. The reactor featured a high intensity 254 nm photochemical mercury quartz lamp radiating upon an ultraviolet transparent tubing where acetone and isopropanol form hydroxyalkyl radicals which cleave insulin disulfide bridges. The cleavage allowed for robust fragmentation mass spectra of insulin using nanospray/Q‐Exactive mass spectrometry. A nano liquid chromatography separation column was placed downstream to the reactor. We found that for the nano liquid chromatography format, a silica monolith provided substantially less carry‐over and improved chromatographic performance to the investigated particle‐packed columns. The photochemical reactor/silica monolithic nano liquid chromatography/nanospray/Q‐Exactive mass spectrometry was compatible with cell culture medium, and will be further investigated for on‐line coupling with pancreas‐on‐a‐chip systems.