TY - JOUR

T1 - High-performance liquid chromatography of rat and mouse islet polypeptides

T2 - potential risk of oxidation of methionine residues during sample preparation

AU - Linde, S

AU - Hansen, B

AU - Welinder, B S

AU - Nielsen, Jens Høiriis

PY - 1990/8/24

Y1 - 1990/8/24

N2 - 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.

AB - 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.

KW - Animals

KW - Ascorbic Acid

KW - Chromatography, High Pressure Liquid

KW - Dithiothreitol

KW - Glucagon

KW - Insulin

KW - Islets of Langerhans

KW - Methionine

KW - Mice

KW - Molecular Weight

KW - Oxidation-Reduction

KW - Pancreatic Polypeptide

KW - Peptides

KW - Quality Control

KW - Rats

KW - Specimen Handling

M3 - Journal article

C2 - 2277117

VL - 530

SP - 29

EP - 37

JO - Journal of Chromatography

JF - Journal of Chromatography

SN - 0301-4770

IS - 1

ER -