High-performance liquid chromatography of rat and mouse islet polypeptides: potential risk of oxidation of methionine residues during sample preparation
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High-performance liquid chromatography of rat and mouse islet polypeptides : potential risk of oxidation of methionine residues during sample preparation. / Linde, S; Hansen, B; Welinder, B S; Nielsen, Jens Høiriis.
I: Journal of Chromatography A, Bind 530, Nr. 1, 24.08.1990, s. 29-37.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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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 -
ID: 47974007