High-performance liquid chromatography of rat and mouse islet polypeptides: potential risk of oxidation of methionine residues during sample preparation

S Linde, B Hansen, B S Welinder, Jens Høiriis Nielsen

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

17 Citationer (Scopus)

Abstract

After preparative high-performance liquid chromatography of mouse islet culture medium, concentrated on disposable C18 cartridges (Sep-Pak), an unexpected insulin immunoreactive peak eluting earlier than mouse insulin I and II was detected. Molecular mass determination by mass spectrometry supported its suspected identity as methionine sulphoxide insulin II. We have examined the formation of Met-O derivatives of insulin II, glucagon and pancreatic polypeptide during sample preparation (Sep-Pak and Speed-Vac concentrating). The oxidation of methionine residues was found to depend very much on the buffer, the organic modifier and the procedure. In particular the use of methanol-trifluoroacetic acid resulted in extensive oxidation. The oxidation could be minimized by adding 2 mM dithiothreitol to the buffer and by degassing and/or nitrogen-bubbling of the buffer. Minimal formation of Met-O derivatives is important for the quantitation of methionine-containing polypeptides.
OriginalsprogEngelsk
TidsskriftJournal of Chromatography A
Vol/bind530
Udgave nummer1
Sider (fra-til)29-37
Antal sider9
ISSN0021-9673
StatusUdgivet - 24 aug. 1990

Citationsformater