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.
| Original language | English |
|---|---|
| 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 |
Keywords
- 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