ESI investigation of non-covalent complexes between phosphorylated daidzein derivatives and insulin

Article abstract of DOI:10.1080/10426500701761763

Since the end of the last century, ESI-MS has begun to be used to reveal the existence of non-covalent complexes, providing important stoichiometric information. Many researchers have reported the use of ESI-MS to determine dissociation constants of such

Full text of DOI:10.1080/10426500701761763

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Phosphorus, Sulfur, and Silicon
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ESI Investigation of Non-
Covalent Complexes between
Phosphorylated Daidzein
Derivatives and Insulin
Xiaolan Chen a , Xiaona Shi a , Lingbo Qu b c , Jinwei
Yuan a & Yufen Zhao a d
a Key Laboratory of Chemical Biology and Organic
Chemistry of Henan Province, Department of
Chemistry , Zhengzhou University , Zhengzhou,
Henan, China
b Henan University of Technology, Henan Province ,
Zhengzhou, P. R. China
c Department of Chemistry , Zhengzhou University ,
Zhengzhou, P. R. China
d The Key Laboratory for Bioorganic Phosphorus
Chemistry, Department of Chemistry, School of Life
Sciences and Engineering , Tsinghua University ,
Beijing, China
Published online: 21 Jun 2008.
To cite this article: Xiaolan Chen , Xiaona Shi , Lingbo Qu , Jinwei Yuan & Yufen Zhao
(2008) ESI Investigation of Non-Covalent Complexes between Phosphorylated Daidzein
Derivatives and Insulin, Phosphorus, Sulfur, and Silicon and the Related Elements,
183:2-3, 527-537, DOI: 10.1080/10426500701761763
To link to this article: http://dx.doi.org/10.1080/10426500701761763
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Phosphorus, Sulfur, and Silicon, 183:527–537, 2008
Copyright © Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426500701761763
ESI Investigation of Non-Covalent Complexes between
Phosphorylated Daidzein Derivatives and Insulin
Xiaolan Chen,1 Xiaona Shi,1 Lingbo Qu,2,3 Jinwei Yuan,1
and Yufen Zhao1,4
1Key Laboratory of Chemical Biology and Organic Chemistry of Henan
Province, Department of Chemistry, Zhengzhou University, Zhengzhou,
Henan, China
2Henan University of Technology, Henan Province, Zhengzhou,
P. R. China
3Department of Chemistry, Zhengzhou University, Zhengzhou,
P. R. China
4The Key Laboratory for Bioorganic Phosphorus Chemistry, Department
of Chemistry, School of Life Sciences and Engineering, Tsinghua
University, Beijing, China
Since the end of the last century, ESI-MS has begun to be used to reveal the existence
of non-covalent complexes, providing important stoichiometric information. Many
researchers have reported the use of ESI-MS to determine dissociation constants
of such complexes. Gas phase dissociation constants measured in this way have
been found to correlate well with those measured by solution based techniques.1,2
In this article, ESI results show that all the phosphorylated daidzein derivatives
can form non-covalent complexes with the protein insulin, while non-covalent com-
plexes were not detected in solutions of unphosphorylated daidzein and insulin. The
relative affinity of each non-covalent complex was obtained according to its differ-
ent decomposition orifice voltage. Compound e has the highest disappearing orifice
voltage and therefore the strongest binding affinity. The relative stability of the
non-covalent complexes was closely associated with the length of the hydrophobic
chain.
Keywords Daidzein; ESI-MS; insulin; non-covalent complex; phosphorylation
INTRODUCTION
Cellular functions are often triggered by weak non-covalent enzyme-
substrate, protein-ligand, protein-protein or antibody-antigen.3 In
The authors thank the National Natural Science Foundation of China (No. 20472076
and 20175026) and Henan Academic Foundation of Science and Technology for their
financial support.
Address correspondence to Chen Xiaolan, Department of Chemistry, Zhengzhou Uni-
versity, Zhengzhou, Henan 450052, China. E-mail: chenxl@zzu.edu.cn
527

528
X. Chen et al.
recent years, isoflavonoids have attracted increasing interest due
to their various beneficial pharmacological effects. Daidzein (7,
4ꢀ-dihydroxyisoflavone) is one of the most abundant isoflavonoids, found
in legumes, especially soybeans. It has been implicated in the pre-
vention of cardiovascular disease,4 lessening the symptoms of the
menopause5 and protection against osteoporosis.6 It was also shown
to inhibit proliferation of different cancer derived cell lines in cul-
ture and reduced the multiplicity, but not incidence, of mammary tu-
mors in rats.7-- 8 Our previous researches indicated that the phospho-
rylated flavonoid derivatives possess relatively strong affinities, form
non-covalent complexes with the proteins more easily than the non-
phosphorylated compounds.9-- 10 Preliminary biological activity screen-
ing tests also indicated that the phosphorylated flavone derivatives
indeed had stronger activity against Hela tumor cells than the cor-
responding flavones in vitro.11 Five solutions of the phosphorylated
daidzein derivatives were mixed with the solution of insulin, which
were then injected individually into an ion trap mass spectrometer. The
results show that the phosphorylated daidzein derivatives could form
non-covalent complexes with insulin. Through modulating the orifice
voltage the covalent complexes could be decomposed, different phospho-
rylated daidzein derivative has different decomposition orifice voltage
due to its different affinity. Therefore, the stability of different non-
covalent complex can be obtained by measuring their decomposition
orifice voltage.
EXPERIMENTAL
SCHEME 1
These five phosphorlated daidzein derivatives were synthesized by
our laboratory (Scheme 1), and their structures were determined by
X-ray, IR, NMR, and ESI-MS.

ESI Investigation of Non-Covalent Complexes
529
Mass spectrometric conditions: Solutions of the complexes were an-
alyzed on a Bruker-Esquire 3000 mass spectrometer fitted with an ion
spray source working in the positive ion mode. Nitrogen was used as
drying gas at a flow rate of 4 µ L/min. The nebulizer pressure was
110.3 kPa and the dry gas flow rate was 6.5 L/min. The capillary was
typically held at 4 kV. Nine spectra were averaged, and the rolling av-
erage was 20. The ICC was set at 40,000.
RESULTS AND DISCUSSION
The development of electrospray ionization and the discovery that
highly charged ions of proteins are readily formed have led to dra-
matic growth in the application of mass spectrometry to biomolecules.
Electrospray ionization is sufficiently gentle to allow the ionization
and detection of intact non-covalent complexes between proteins and
small molecules and of multiunit protein structures.12-- 13 ESI-MS has
served as a powerful tool in providing evidence in support of the exis-
tence of non-covalently associated macromolecular complexes in the gas
phase. For many years, ESI-MS has been used to reveal the existence
of non-covalent complexes, providing important stoichiometric infor-
mation. Some examples of non-covalent complexes studied by ESI-MS
are protein-ligand,14,15,16 protein-metal,17,18 protein-protein,19,20 and
protein-sugar21 complexes. An excellent review concerning the use of
mass spectrometry for analyzing non-covalent interactions has been
published by Loo.22 Many researchers have reported the use of ESI-MS
to determine dissociation constants of such complexes. Gas phase dis-
sociation constants measured in this way have been found to correlate
well with those measured by solution based techniques.1,2
Detection of non-covalent complexes by ESI requires that the com-
plex tolerate the interface conditions (i.e., heat, collisional activation
due to electrical voltage) during the desolvation/desorption processes.
In the work described in this article, the solution of different phosphory-
lated daidzein derivative and insulin was first injected in suitable orifice
voltages and capillary temperature, and then the orifice voltage was in-
creased gradually until the peak of the phosphorylated daidzein-insulin
complex disappeared. Relative affinity of every non-covalent complex
was obtained according to its different decomposition orifice voltage.
The stability of different no-covalent complex was therefore compared.
The mass spectra of phosphorylated daidzein derivative-insulin non-
covalent complexes are shown in Figure 2 and the decomposition orifice
voltages of the corresponding complexes in Table I.
Figure 1 shows an ESI mass spectrum obtained from a neutral so-
lution of insulin. The charge-state distribution comprises four charged

530
X. Chen et al.
FIGURE 1 Electrospray ionzation mass spectrum of insulin. The solution was
prepared by mixing equal volumes of methanol and 0.04 mmol · L−1 insulin.
The orifice voltage was set at 113.0 V. The source temperature was maintained
at 250◦C. means multiply charged ion peaks of insulin.
•
states ranging from 6+ to 3+, with 5+ the most intense. The average
molecular mass measured from these peaks is 5734 Da.
Figure 2a shows an ESI mass spectrum obtained from a mixed solu-
tion of insulin and compound a. Besides the expected mulitiply proto-
nated molecule ions, a mass spectrum revealed several groups of new
protonated ions, corresponding to several kinds of highly charged mul-
tiple adducts, respectively, for example, ions at m/z: 1220 and 1296,
corresponding to (insulin +a +5H)5+, (insulin +2a +5H)5+
.
Figure 2b shows an ESI mass spectrum obtained from a mixed so-
lution of insulin and compound b. The mass spectrum also revealed
several groups of new protonated ions, corresponding to several kinds
of highly charged multiple adducts, respectively, for example, there is
a set of ions at m/z 1226, 1304, 1382, 1460, 1532, corresponding to (in-
sulin +b +5H)5+, (insulin +2b +5H)5+, (insulin +3b +5H)5+, (insulin
+4b +5H)5+, (insulin +b +4H)4+
.
Figure 2c shows an ESI mass spectrum obtained from a mixed so-
lution of insulin and compound c. The mass spectrum also revealed
several groups of new protonated ions, corresponding to several kinds
of highly charged multiple adducts, respectively, for example, there is a
set of ions at m/z 1231, 1315, 1398, 1482, 1566, 1539, corresponding to

ESI Investigation of Non-Covalent Complexes
531
(insulin +c +5H)5+, (insulin +2c +5H)5+, (insulin +3c +5H)5+, (insulin
+4c +5H)5+, (insulin +5c +5H)5+, (insulin +c +4H)4+
.
Figure 2d shows an ESI mass spectrum obtained from a mixed so-
lution of insulin and compound d. The mass spectrum also revealed
several groups of new protonated ions, corresponding to several kinds
of highly charged multiple adducts, respectively, for example, there is
FIGURE 2 (a) ESI mass spectrum of insulin with Pa. The solution was pre-
pared by mixing equal volumes of 0.04 m mol · L−1 inlulin and 0.4 m mol ·
L−1 methanol solution of compound a. (b) ESI mass spectrum of insulin with
compound b. The solution was prepared by mixing equal volumes of 0.04 m
mol · L−1 insulin and 0.4 m mol · L−1 methanol solution of compound b. (c)
ESI mass spectrum of insulin with compound c. The solution was prepared by
mixing equal volumes of 0.04 m mol · L−1 insulin and 0.4 m mol · L−1 methanol
solution of compound c. (d) ESI mass spectrum of insulin with compound d.
The solution was prepared by mixing equal volumes of 0.04 m mol · L−1 insulin
and 0.4 m mol · L−1 methanol solution of compound d. (e) ESI mass spectrum
of insulin with compound e. The solution was prepared by mixing equal vol-
umes of 0.04 m mol · L−1 insulin and 0.4 m mol · L−1M methanol solution of
compound e. The orifice voltage was set at 113.0 V. The source temperature
was maintained at 250◦C. means multiply charged ion peaks of insulin.
•
•◦
means multiply charged ion peaks of insulin-phosphorylated isoflavonoid (PF)
complex; means multiply charged ion peaks of insulin-2PF complex;
•◦◦
•◦◦◦
means multiply charged ion peaks of insulin-3PF complex;
ply charged ion peaks of insulin-4PF complex,
ion peaks of insulin-5PF complex; and
means multi-
•◦◦◦◦
means multiply charged
means multiply charged ion
•◦◦◦◦◦
•◦◦◦◦◦◦
peaks of insulin-6PF complex. (Continued)

532
X. Chen et al.
FIGURE 2 (Continued).

ESI Investigation of Non-Covalent Complexes
533
FIGURE 2 (Continued).

534
X. Chen et al.
(a)
(b)
FIGURE 3 (a) ESI mass spectrum of insulin with Pb. The orifice voltage was
set at 105.0 V; (b) ESI mass spectrum of insulin with Pb. The orifice voltage was
set at 113.0 V; and (c) ESI mass spectrum of insulin with Pb. The orifice voltage
was set at 120.4 V.

ESI Investigation of Non-Covalent Complexes
535
(c)
FIGURE 3 (Continued).
a set of ions at m/z 1232, 1315, 1399, 1539, corresponding to (insulin
+d +5H)5+, (insulin +2d +5H)5+, (insulin +3d +5H)5+, (insulin +d
+4H)4+
.
Figure 2e shows an ESI mass spectrum obtained from a mixed so-
lution of insulin and compound e. The mass spectrum also revealed
several groups of new protonated ions, corresponding to several kinds
of highly charged multiple adducts, respectively, for example, there is
a set of ions at m/z 1237, 1326 and 1539, corresponding to (insulin +e
+5H)5+, (insulin +2e +5H)5+, (insulin +e +4H)4+
.
The experiment described above shows that phosphorylated daidzein
derivative could interact with the protein insulin in the gas phase.
Even though different parameters were tried, no corresponding non-
covalent protein-daidzein complexes were detected. The experiment de-
scribed herein suggests that the phosphorylated daidzein derivatives
could form non-covalent complexes with insulin more easily than the
unphosphorylated daidzein. The phosphate group of the phosphory-
lated daidzein played important role in the processes of phosphorylated
daidzein-protein interactions.10,23
From the Figure 3, it is clear that the intensity of the non-covalent
complexes was reducing when the orifice voltage was increased. The

536
X. Chen et al.
TABLE I The Orifice Voltage of the
Non-Covalent Complexes Disappearing
Compound
a
b
c
d
e
Orifice voltage/V
133.8 146.0 151.8 157.4 162.9
interaction between the phosphorylated daidzein derivatives and in-
sulin was destroyed when the orifice voltage was increased. If the non-
covalent complexes were disappeared at higher orifice voltage, the bind-
ing affinity is stronger. We found that different phosphorylated daidzein
derivative could tolerance different high orifice voltage. The relative
affinity of each non-covalent complex was obtained according to its dif-
ferent decomposition orifice voltage. From the Table I, it is clear that
compound e has the highest disappearing orifice voltage and therefore
the strongest binding affinity. The relative stability of the non-covalent
complexes was closely associated with the length of the hydrophobic
chain.
CONCLUSION
The phosphorylated daidzein derivatives could form non-covalent com-
plexes with the protein insulin. Different phosphorylated daidzein
derivative shows different binding affinity with the same protein. The
compound e shows stronger binding affinity to insulin than others. The
relative stability of the non-covalent complexes was closely associated
with the length of the hydrophobic chain.
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