High-performance liquid chromatography of rat and mouse islet polypeptides: potential risk of oxidation of methionine residues during sample preparation
Research output: Contribution to journal › Journal article › Research › peer-review
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.
Original language | English |
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Journal | Journal of Chromatography A |
Volume | 530 |
Issue number | 1 |
Pages (from-to) | 29-37 |
Number of pages | 9 |
ISSN | 0021-9673 |
Publication status | Published - 24 Aug 1990 |
- Animals, Ascorbic Acid, Chromatography, High Pressure Liquid, Dithiothreitol, Glucagon, Insulin, Islets of Langerhans, Methionine, Mice, Molecular Weight, Oxidation-Reduction, Pancreatic Polypeptide, Peptides, Quality Control, Rats, Specimen Handling
Research areas
ID: 47974007