Relative quanti¢cation of glycated Cu-Zn superoxide dismutase in
erythrocytes by electrospray ionization mass spectrometry
Mandapati Saraswathi, Toyofumi Nakanishi, Akira Shimizu *
Department of Clinical Pathology, Osaka Medical College, 2-7 Daigaku-cho, Takatsuki-city, Osaka 569-8686, Japan
Received 24 August 1998; received in revised form 30 November 1998; accepted 1 December 1998
Abstract
Electrospray ionization mass spectrometry (ESIMS) was used for relative quantification of glycated Cu-Zn superoxide
dismutase (SOD-1) in human erythrocytes. SOD-1 samples were prepared from erythrocytes by removing hemoglobin using
hemoglobind gel followed by ethanol and chloroform extraction. The reproducibility in measurement of the relative
percentage of glycated protein was good, and the standard deviation of each measurement was 4.0%. From the mass spectral
analysis of a mixture of commercial SOD-1 and in vitro partially glycated SOD-1 in several ratios, it was found that free and
glycated SOD-1 have the same ionization efficiencies. The percentage of glycation on SOD-1 was measured in 30 individuals,
including patients with diabetes mellitus. The glycation levels ranged from 4.5% to below the detection limit. The SOD-1
sample extracted from erythrocytes was fractionated by Glyco-Gel B chromatography, and the separated fractions were
analyzed by MS. The mass spectra of absorbed fraction showed significant amounts of non-specific binding of non-glycated
proteins to Glyco-Gel B. ß 1999 Elsevier Science B.V. All rights reserved.
Keywords: SOD-1 glycation; Erythrocyte; Hemoglobind; Glyco-Gel B chromatography; Electrospray ionization mass spectrometry
1. Introduction
Under physiological conditions, various proteins,
such as hemoglobin, albumin, low- and high-density
proteins, lens crystalline and various forms of colla-
gen undergo modi¢cations with glucose [1^5]. The
glycation proceeds through the formation of a Schi¡
base between glucose and a free amino group of
protein followed by Amadori rearrangement to yield
a relatively stable ketoamine adduct [6,7]. Glycation
of proteins is known to have a signi¢cant e¡ect on
protein conformation and its biological activity [8,9].
It has also been suggested that the concomitant
structural and functional alterations in various gly-
cated proteins due to the formation of Maillard
products contribute to the development of cataract,
renal failure, neuropathy and vascular diseases in
both diabetes and aging [10^12]. The glycation of
Cu-Zn superoxide dismutase (SOD-1), like that of
other proteins, also causes enzyme inactivation and
self degradation initially at a speci¢c site (between
Pro
62
and His
63
), followed by random fragmentation
[13,14]. It has also been reported that the OH radi-
cals released during the fragmentation of glycated
SOD-1 damage the nuclear DNA [15].
Measurement of percentage of glycated hemoglo-
bin has been used as a marker in the treatment of
diabetes mellitus [16]. Mass spectrometry has been
0304-4165 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved.
PII: S0304-4165(98)00171-8
* Corresponding author. Fax : +81 (726) 846548 ;
E-mail: shimizu@poh.osaka-med.ac.jp
BBAGEN 24757 28-1-99
Biochimica et Biophysica Acta 1426 (1999) 483^490