Analysis of glycated insulin by MALDI-TOF mass spectrometry.
Non-enzymatic glycation of protein is mediated via an interaction between the aldehyde group of a reducing sugar and available alpha- or epsilon-amino moieties of the protein. The above event can alter the biological activity of the protein and therefore, it is of particular interest to monitor the glycation of proteins having important functional roles in metabolism. In the present study, matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) has been used to determine the non-enzymatic glycation of bovine insulin. The degree of insulin glycation was increased in both concentration- and time-dependent manner in relation to exposure to glucose, and the event was more pronounced for monoglycation reaction than that noticed for the diglycation of the hormone. Enzymatic digestion of insulin preparations with endoproteinase Glu C has revealed that each of the B 1-13 and B 22-30 peptide fragments of glycated insulin contains a site of binding of a single glucose molecule. Finally, attempt has been made in order to increase the sensitivity of the glycation assay through efficient enrichment of the glycated insulin on magnetic beads containing immobilized 3-aminophenylboronic acid (APBA) on their surface.
Farah
M Abul
MA
Proteonik Inc., Gyeonggi Technopark, Sangnok-Gu, Ansan City, 425-170, South Korea.
Bose
Shambhunath
S
Lee
Jeong-Heon
JH
Jung
Ho-Chul
HC
Kim
Yangsun
Y
D000478:Alkylation; D000818:Animals; D001897:Boronic Acids; D002417:Cattle; D005947:Glucose / Q000737:chemistry; D006031:Glycosylation; D007328:Insulin / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry*; D008280:Magnetics; D008863:Microspheres; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000032:analysis; D012697:Serine Endopeptidases / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001897:Boronic Acids; D007328:Insulin; D010446:Peptide Fragments; C039522:insulin, glycosylated; C028592:3-aminobenzeneboronic acid; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D005947:Glucose