Studies of interaction between insulin and glutathione using electrospray ionization mass spectrometry

Article abstract of DOI:10.1002/rcm.6248

Rationale The interaction of glutathione (GSH) with insulin plays an important role in the degradation or regulation of insulin. The characterization of the reaction products of GSH and insulin is very important for a proper understanding of the mechanism of insulin regulation of GSH. Methods Solutions of insulin and glutathione were incubated under different experimental conditions in vitro. The reaction products were determined by electrospray ionization (ESI) ion trap mass spectrometry combined with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. Results: The multi-reaction products were identified, including insulin A chain with two intrachain disulfides, insulin B chain with one intrachain disulfides, GSH-modified insulin, GSH-modified A chain, GSH-modified B chain, aggregates of A chain and B chain, and reduced A chain and B chain. The binding site of the B chain with insulin was determined directly by tandem mass spectrometry (MS/MS) without enzyme digestion. It was found that the reaction between GSH and insulin was pH-, O₂- and temperature-dependent. CONCLUSIONS The Results: provide insight into the interaction between GSH and insulin. It has also been demonstrated that ESI-MS combined with high-resolution FTICRMS and MS/MS provides a powerful tool for screening the reactions of proteins and small molecules.

Full text of DOI:10.1002/rcm.6248

Research Article
Received: 11 December 2011
Revised: 26 March 2012
Accepted: 6 April 2012
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2012, 26, 1519–1526
(wileyonlinelibrary.com) DOI: 10.1002/rcm.6248
Studies of interaction between insulin and glutathione using
electrospray ionization mass spectrometry
1,2
1,2
1
1
1
*
Yonggang Du , Ningbo Zhang , Meng Cui , Zhiqiang Liu and Shuying Liu
1Changchun Center of Mass Spectrometry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625
Renmin Street, Changchun 130022, P.R. China
2Graduate School of the Chinese Academy of Sciences, Beijing 100039, P.R. China
RATIONALE: The interaction of glutathione (GSH) with insulin plays an important role in the degradation or regulation
of insulin. The characterization of the reaction products of GSH and insulin is very important for a proper understanding
of the mechanism of insulin regulation of GSH.
METHODS: Solutions of insulin and glutathione were incubated under different experimental conditions in vitro. The
reaction products were determined by electrospray ionization (ESI) ion trap mass spectrometry combined with Fourier
transform ion cyclotron resonance (FTICR) mass spectrometry.
RESULTS: The multi-reaction products were identified, including insulin A chain with two intrachain disulfides, insulin
B chain with one intrachain disulfides, GSH-modified insulin, GSH-modified A chain, GSH-modified B chain, aggregates
of A chain and B chain, and reduced A chain and B chain. The binding site of the B chain with insulin was determined
directly by tandem mass spectrometry (MS/MS) without enzyme digestion. It was found that the reaction between GSH
and insulin was pH-, O2- and temperature-dependent.
CONCLUSIONS: The results provide insight into the interaction between GSH and insulin. It has also been
demonstrated that ESI-MS combined with high-resolution FTICRMS and MS/MS provides a powerful tool for screening
the reactions of proteins and small molecules. Copyright © 2012 John Wiley & Sons, Ltd.
Glutathione (L-g-glutamyl-L-cysteinyl-glycine, GSH) is widely
distributed in most plants, bacteria and all mammalian cells at
concentrations ranging from 0.5 to 10 mM.[1] The majority of
intracellular GSH is in the reduced form, with less than 5% of
its total amount as the mixed disulfide (mainly GS-S-protein),
the disulfide (GSSG), and thioethers in the cell.[1] As the most
abundant free non-protein thiol, GSH is involved in a wide
variety of biological functions, such as a free radical scavenger,[2]
maintenance of protein thiol groups in the reduced states, and
detoxification of xeno- and endo-biotic electrophiles.[3,4] Among
them, the interactions of GSH and proteins play significant
biological roles in cells including the formation of the
mitotic apparatus during cell division[5,6] and regulation of
protein functions and protection of irreversible oxidation of
proteins.[7–9] Therefore, many proteins have been investigated
to react with GSH in vitro[10–20,23] for the better understanding
of mechanisms of interactions of GSH and proteins, for exam-
ple, serum albumin,[12–14] bovine pancreatic ribonuclease,[15,16]
eye-lens protein g-crystallin[18,19] and cytochrome c.[20]
and fat tissue to take up glucose from blood, storing it as
glycogen in the liver and muscle.[21] It has been postulated
that GSH plays an important role in degradation or secretion
of insulin, which has been extensively studied in vivo or
in vitro.[22,24,25] Direct in vitro interactions between insulin
and GSH have been reported that GSH can break insulin into
A and B chains by reduction of the disulfide bridges.[10,12,24,25]
Hird investigated the enzymic reduction of insulin by GSH
and found that this reaction was slow when insulin was in
the native state and speeded up after insulin was partly
digested by proteolytic enzymes.[12] Rafter found that an
aggregate of insulin was formed when it was incubated
with GSH[24] and Taniguchi and co-workers reported that
insulin aggregation can be promoted by protein disulfide
isomerase.[26] Alai and Fenselau demonstrated that insulin-
glutathione mixed disulfides were formed when insulin was
incubated with GSH.[27] Extending the incubation time
resulted in increased production of A and B chains of insulin.
Characterization of reaction products of GSH and insulin is
very important for proper understanding of the mechanism
of GSH regulation of insulin in vitro.
Insulin is a small globular protein to regulate energy
storage and glucose metabolism in the body. It is synthesized
in the pancreatic b-cells and causes cells in the liver, muscle
Electrospray ionization (ESI) and matrix-assisted laser
desorption ionization (MALDI) mass spectrometry (MS) have
become very powerful tools to investigate proteins,[28,29]
DNAs,[30] protein-ligands,[31–33] and other biomolecules.[34,35]
Deng reported that the reaction of cytochrome c with
glutathione generated multiple products characterized by
MALDI-TOF-MS and LC/ESI-MS combined with enzymatic
digestion.[20] Harding and co-workers identified a reaction site
* Correspondence to: M. Cui, Changchun Center of Mass Spec-
trometry, Changchun Institute of Applied Chemistry, Chinese
Academy of Sciences, 5625 Renmin Street, Changchun
130022, P.R. China.
E-mail: cuimeng@ciac.jl.cn
Rapid Commun. Mass Spectrom. 2012, 26, 1519–1526
Copyright © 2012 John Wiley & Sons, Ltd.

Y. G. Du et al.
of glutathione mixed disulfide formation on gamma crystallin S
in human lens by MALDI-TOFMS with trypsin digestion.[19]
However, unless specific enzymes or high-resolution mass
spectrometry (HRMS) are used, MS-based approaches still need
additional information to unambiguously identify proteolysis
products or modification of proteins, because many proteolysis
products or modified and unmodified proteins could have very
close masses hard to been distinguished without HRMS. With
the advent of Fourier transform ion cyclotron resonance mass
spectrometry (FTICRMS), it is proving to be one of the most
valuable techniques for the characterization of biomolecules
for its high resolution and high mass accuracy.[36] To the best
of our knowledge, the reaction products of GSH and insulin
have not been well characterized until now.
Herein, we studied in detail the interaction between insulin
and glutathione under different experimental conditions
without the presence of any enzyme using ESI-MS combined
with FTICRMS and tandem mass spectrometry (MS/MS).
Multiple reaction products at different mole ratios have
been well characterized without time-consuming column
separation. The bonding site of GSH to the insulin B chain
was directly assigned speedily by MS/MS without the need
for complex enzyme digestion. The effects of oxygen, pH and
temperature on the reaction between insulin and GSH were
also investigated.
Reactions of insulin with GSH or DTT
The stock solutions of insulin, GSH and DTT were prepared
at 200 mM, 10 mM and 10 mM in 25 mM NH4HCO3 (pH
9.0), respectively. The other buffer solutions (0.2% formic acid
aqueous solution (pH ~2.5), 25 mM NH4HCO3 (pH 8.25)
and 0.002% H2O2 in 25 mM NH4HCO3 (pH 9.0)) were
also prepared.
The mixtures of GSH and insulin at differenꢀt mole ratios
ꢀ
(1:1, 6:1 and 200:1) were incubated at 37 C, 50 C and room
temperature for certain times, respectively.
The reaction solution ofꢀDTT with insulin at mole ratio of
200:1 was incubated at 55 C for 30 min.
The reaction products with proper dilution (final concen-
tration of insulin 2 mM) with 50% aqueous methanol /0.1%
aqueous formic acid (1:1, v/v) were analyzed by MS.
Analysis of reaction products by mass spectrometry
All the experiments were performed on a LTQ linear ion trap
mass spectrometer (Thermo, San Jose, CA, USA), equipped
with a conventional ESI source. The spray voltage was 4 kV
and the capillary voltage was 32 V. The capillary temperature
was kept at 260 ꢀC. Sheath gas was set at 20 (arbitrary units).
Samples were introduced directly into the mass spectrometer
at a flow rate of 5 mL/min. For CID experiments, 28%
collision energy and an activation time of 30 ms were
typically applied. ESI spectra were acquired using Xcalibur
software (Thermo) in the positive ion mode.
EXPERIMENTAL
HRMS data of reaction products were obtained by a FTICR
mass spectrometer (IonSpec, Irvine, CA, USA), equipped
with a 7.0 T actively shielded superconducting magnet.
Materials and reagents
Insulin and GSH (shown in Scheme 1) were purchased from
Sigma-Aldrich and used without further purification. Dithio-
threitol (DTT) was purchased from Promega (Madison, WI,
USA). 30% H2O2 (analytical grade) was gained from Beijing
Chemical Factory (Beijing, China). Ammonium hydrogen
carbonate (analytical grade) was acquired from Fluka (Buchs,
Switzerland). Methanol (HPLC grade) was obtained from Fisher
Chemical Company (Fair Lawn, NJ, USA). Formic acid (HPLC
grade) was bought from TEDIA Co. (Fairfield, OH, USA).
Ultrapure water (specific conductivity, 18.2 MΩ/cm) was
produced by a MilliQ device (Millipore, Milford, MA, USA).
ꢀ
Source temperature was 100 C and probe temperature was
80 ꢀC. The ions were accumulated for 3.0 s in a hexapole ion
guide. All acquisitions were performed on a 1024 K data set
and 1 scan. The broad band analysis mode was used. The
samples were introduced by direct infusion at a flow rate of
3 mL/min.
RESULTS AND DISCUSSION
Identification of reaction products
Figure 1(a) shows the representative ESI ion trap mass
spectrum of reaction products of GSH with insulin at mole
ꢀ
ratio 1:1 in 25 mM NH4HCO3 (pH 9.0) at 37 C for 6 h. The
dominant peaks at m/z 1147, 1433 and 1911 corresponded to
multiply charged molecular ions of insulin, [insulin+5H]5+
,
[insulin+4H]4+ and [insulin+3H]3+, respectively. There were
also a protonated reduced GSH ion at m/z 308 and a
protonated oxidized glutathione (GSSG) ion at m/z 613.
Based on the masses, the other reaction adducts were
assigned to separated A chain ([A+2H]2+) and B chain
([B+4H]4+, [B+3H]3+ and [B+2H]2+), the complexes of chain
A or chain B with insulin ([M+A+5H]5+, [M+B+7H]7+
,
[M+B+6H]6+ and [M+B+5H]5+), and dimmer of B chain
([2B+7H]7+ and [2B+6H]6+). Because the ion trap mass
spectrometer has unit mass resolution throughout the
mass range, it is not easy for it to unambiguously differ-
entiate the oxidized and reduced forms of the reaction
products of insulin with GSH. FTICRMS was employed
Scheme 1. Amino acid sequence of insulin (top) and structure
of GSH (bottom).
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ESI-MS studies of insulin and glutathione interactions
Figure 1. (a) ESI ion trap mass spectrum of reaction products of GSH with
insulin (inserted are the experimental (top) and theoretical isotopic patterns
(bottom) of the [A+2H]2+ ion of chain A and the [B+4H]4+ ion of chain B
detected by FTICRMS), CID mass spectra of m/z 1168 ([A+2H]2+) ion of chain
A (b) and m/z 1133 ([B+3H]3+) ion of chain B (c) , and ESI FT-ICR mass spectrum
of reaction products of DTTat 55ꢀC for 2h with the solution after the incubation
of GSH with insulin (d) at mole ratio 1:1 in 25 mM NH4HCO3 (pH 9.0) at 37 ꢀC
for 6 h. Letters A, B and M represent chain A, chain B and insulin, respectively.
to identify them. Taking the m/z 850 ion and m/z 1168 ion
observed between Cys6 and Cys20, which suggested that
the two cysteine residues Cys7 and Cys20 formed a disulfide
linkage in chain A besides the Cys6–Cys11 intradisulfide
bond. Figure 1(c) shows the tandem mass spectrum of the
m/z 1133 ([B+3H]3+) ion. The b- and y-type fragment ions
observed was indicative of an intrachain disulfide bond
formed between Cys7 and Cys19 of chain B. No MS/MS data
could be obtained for complexes of A and B chains with
insulin and oligomers of B chains because of low signal
intensities. The intrachain disulfide linkages of chain A and
B were further confirmed by treatment with excess DTT
as examples, the inserted experimental FTICRMS mass
spectra of the m/z 850 ion and m/z 1168 ion matched very
well with the theoretical isotopic patterns of oxidized A
and B chains in Fig. 1(a). So the m/z 850 ion and m/z
1168 ion can be clearly assigned as the [B+4H]4+ ion of
the oxidized B chain and the [A+2H]2+ ion of the oxidized
A
chain, respectively, which suggested that the new
intradisulfides may be formed in chain A and chain B.
Similarly, based on the HRMS data, all cysteine residues
in complexes of A and B chains with insulin and dimmer
of B chains were in the oxidized form.
ꢀ
(200:1) at 55 C over 2 h after the incubation of insulin with
For confirmation of intradisulfide bonds formed in chain A
and B, collision-induced dissociation (CID) experiments
were applied. Figure 1(b) shows tandem mass spectrum of
the m/z 1168 ([A+2H]2+) ion. For clarity, the symbolism
proposed by Cooks and co-workers[37] for the representation
of the scan mode was used. There were no other fragments
GSH at 37 ꢀC for 6 h. Figure 1(d) shows the ESI-MS spectrum
of the reaction mixture of GSH and insulin treated with DTT.
The aggregates of chain A or B and complexes of chain A or B
with insulin were not observed. The dominant peaks in the
spectrum corresponded to chain A and chain B in reductive
forms, based on their FTICRMS data.
Rapid Commun. Mass Spectrom. 2012, 26, 1519–1526
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Y. G. Du et al.
Figure 1. (Continued)
According to the above data, it was evident that during the
incubation of insulin with GSH at the mole ratio 1:1 in
NH4HCO3 buffer, GSH can break insulin into chain A with
two intradisulfide bonds and chain B with one intradisulfide
bond in vitro by reduction of two interchain disulfide bridges
of insulin. Meanwhile, complexes of chain A or chain B with
insulin and aggregates of chain B by disulfide bonds were
also formed.
Additionally, usually the A chain of insulin is not
as easily observed as the B chain by ESI-MS, because the
B chain has five potential protonated sites including the
N-termini, arginine22, lysine29, histidine5 and histidine10
and chain A only has an N-terminus. It is interesting
that chain A can be strongly observed under our experi-
mental conditions.
Fig. 2(b)). It suggested that the reaction was pH-dependent,
which was similar to the reaction of cytochrome c with
GSH.[20] The SH group of GSH releases a proton only
under alkaline conditions, then S– can react with the
disufide bond of insulin. Though decreasing the pH values
may lead to the denaturing of insulin to enhance the
reaction of insulin with GSH, thiol/disulfide exchanges
were quenched at low pH due to protonation of the
thiolate species.[4]
Effects of temperatures on the reaction of insulin with GSH
Figure 2(c) shows the ESI-MS spectrum of the reaction
products of insulin and GSH in 25 mM NH4HCO3 (pH 9.0)
at room temperature for 6 h. The GSH and its oxidized
species (GSSG) were observed and multiply charged insulin
molecular ions, [insulin+5H]5+, [insulin+4H]4+ and [insulin
+3H]3+, were still dominant in the spectrum. The multiply
charged ions of insulin B chain were also observed.
Compared with Fig. 1(a), the extent of the reaction of insulin
with GSH at room temperature was much slower than that at
37 ꢀC. Increasing the temperature to 50 ꢀC, this reaction was
accelerated and the relative intensities of products were
increased as well (Fig. 2(d)). This suggested that the reaction
was temperature-dependent.
Effects of pH on the reaction of insulin with GSH
Figure 2(a) shows the ESI-MS spectrum of reaction
products of insulin with GSH in 0.2% formic acid aqueous
solution (pH ~2.5). The GSH at the low pH values mainly
remained in its reductive form. Insulin was hardly reduced
by GSH in 0.2% formic acid aqueous solution and only
multiply charged insulin molecular ions appeared in the
spectrum. Increasing the pH to ~8.25, the extent of
this reaction was still lower than that at pH 9 (shown in
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ESI-MS studies of insulin and glutathione interactions
Figure 2. ESI mass spectra of reacꢀtion products of GSH with insulin at mole
ratio 1:1 in 0.2% formic acid at 37 C (pH ~2.5) (a), in 25 mM NH4HCO3 (pH
~8.25) (b), in 25 mM NH4HCO3 (pH ~9.0) at room temperature (c), in 25 mM
NH4HCO3 (pH ~9.0) at 50 ꢀC (d), without oxygen (e) and in 25 mM NH4HCO3
ꢀ
(pH ~9.0) with 0.002% H2O2 at 37 C (f) for 6 h. Letters A, B and M represent
chain A, chain B and insulin, respectively.
Effects of oxygen and hydrogen peroxide on the reaction of
insulin with GSH
10% and the [B+3H]3+ ion of insulin B chain became the
base peak. The inserted FTICRMS data of chain A indicated
that there are two forms of chain A (one is chain A with
two intradisulfide bonds, the other is chain A with one
intradisulfide bond and two -SH groups). The inserted
FTICRMS spectrum of chain B showed that some of chain
B are in reductive form (containing two –SH groups) and
the others are in oxidized form (containing one intradisu-
fide bond). The relative abundances of complexes of chain
A and B with insulin and dimmer of B chain were
improved greatly. The GSH-modified A chain and B chain
were also observed.
Oxygen or hydrogen peroxide (H2O2) commonly exists in the
human body. Effects of oxygen or H2O2 on the reaction of
insulin with GSH were studied. All the samples were
prepared in a very short time, and then sealed to minimize
exposure to excess air. There is only the dissolved oxygen in
the sample solution. It was reported that the air-exposed
sample solution can contain oxygen ~ 3.2 Â 10–4 mol/L at
room temperature and 1 atm ambient pressure.[38] The
reaction products of insulin and GSH were less in sealed
solution than those in air (shown in Fig. 2(e)). Extending the
reaction time did not lead to any dramatic changes in reaction
products. Therefore, it was reasonable to believe that this
reaction was O2-dependent.
(2) Mole ratio of GSH and insulin at 200:1
To test the influence of H2O2 on this reaction, 0.002%
H2O2 (v/v) was added to the insulin solution in NH4HCO3
buffer prior to mixing with GSH. This reaction was much
slower in the presence of H2O2 than that without addition
of H2O2 (shown in Fig. 2(f)). The ions of oxidized GSH
were observed, which indicated that GSH was firstly oxi-
dized by H2O2 after addition of H2O2 in the mixture of
GSH and insulin.
Figure 3(b) showed the ESI mass spectrum of the reaction of
GSH with insulin at the high mole ratio 200:1 in 25 mM
NH4HCO3 buffer (pH 9.0) at 37 ꢀC for 6 h. The ions of
protonated GSH and its oligmers were dominant in the mass
spectrum. Based on the FTICRMS data, the B chain was
reduced completely and only one disulfide bond of chain A
was in the reductive form and the other intradisulfide bond
remained unbroken. Increasing the reaction time did not lead
to reduction of the intradisulfide bond of the A chain.
The GSH-modified B chain was observed. The reduced
B-chain has two free cysteines (Cys7, Cys19) which are
possible binding sites to GSH. To identify the bonding sites
of GSH, both the unmodified and GSH-modified B-chain ions
were fragmented by tandem mass spectrometry (shown in
Fig. 4). To simplify the spectrum, the neutral losses (H2O,
NH3) of b- and y-type fragment ions were not annotated.
The GSH-modified b- and y-type fragment ions indicated
GSH binding to Cys19 of the B chain. The GSH-modified A
chain was also observed but its CID data could not be
obtained due to its low sensitivity.
Effects of mole ratios of GSH to insulin on the reaction
(1) Mole ratio of GSH and insulin at 6:1
Figure 3(a) shows the ESI mass spectrum of the reaction
products of GSH with insulin at mole ratio 6:1 in 25 mM
NH4HCO3 (pH 9.0) at 37 ꢀC for 6 h. Compared with
Fig. 1(a), increasing the mole amount of GSH led to a
speeding up of the reaction of GSH and insulin. The abun-
dance of intact insulin molecular ions decreased to less than
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Y. G. Du et al.
Figure 3. ESI ion trap mass spectra of reaction products of GSH with insulin
ꢀ
at mole ratios 6:1 (a) and 200:1 (b) in 25 mM NH4HCO3 (pH 9.0) at 37 C for
6 h. Inserted are the experimental isotopic patterns of the [A+2H]2+ ion of chain
A, the [B+4H]4+ ion of chain B and the [B+GSH+3H]3+ ion of GSH-modified
chain B detected by FTICRMS. Letters A, B and M represent chain A, chain B
and insulin, respectively.
It is obvious that reaction products of GSH and insulin
depend on the mole ratios of GSH and insulin. At low
mole ratio, the insulin can be partly broken to oxidized
A and B chains by GSH. Formation of insulin-glutathione
mixed disulfides (GSH-modified insulin, A chain and B
chain and aggregates of A-chain and B-chain) was also
observed. With an increase in GSH, some of the B chain
was in its reductive form. When GSH was in great excess,
the B chain was in reductive species completely, while
there was an interchain disulfide bond in chain A. No
oligomers of A and B chains and complexes of A and B
chains with insulin were formed.
A and B chains were formed and insulin B chain and A
chain were reduced partially (Fig. 5(b)). When the reaction
time was increased to 1 h, the adduct of GSH-insulin was
not observed (Fig. 5(c)). The extent of the reaction was
increased with increase in the reaction time (Fig. 5(d)).
Extending the reaction time to 24 h, the intradisulfide
bond formed between Cys6-Cys11 of the insulin A chain
was still conserved and the insulin B chain was completely
reduced. However, all three disulfide bonds of insulin can
be reduced by DTT completely. Because the conformation
of chain A is kept much more compact in 25 mM
NH4HCO3 buffer and the Cys6–Cys11 disulfide bond is
not readily accessible, DTT is smaller than GSH and easily
attacks the Cys6–Cys11 disulfide bond avoiding the effect
of steric hindrance.
Time-course of the reaction of insulin and GSH
The time-course of the reaction of insulin and GSH at high
mole ratio 200:1 was monitored and the corresponding
mass spectra are shown in Fig. 5. After 10 min incubation,
the adduct of GSH-insulin was observed (Fig. 5(a)).
Extending the incubation time to 30 min, GSH-modified
Based on the above data, it was inferred that the GSH
firstly attacked one of the interdisulfide bonds of insulin to
form a mixed disulfide bond between insulin and GSH. Then
the GSH opened another interdisulfide bond of insulin to
generate the GSH-modified A chain and the GSH-modified
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ESI-MS studies of insulin and glutathione interactions
Figure 4. CID mass spectra of unmodified B chain ions at m/z 1134 (a) and GSH-modified
B chain ions at m/z 1236 (b).
Figure 5. ESI ion trap mass spectra of reaction products of GSH with insulin at
mole ratio 200:1 in 25 mM NH4HCO3 (pH 9.0) at 37 ꢀC for 10 min (a), 30 min (b),
1 h (c), and 3 h (d), respectively. Letters A, B and M represent chain A, chain B
and insulin, respectively.
B chain with a mixed disulfide bond with reduced A and B
chains. The GSH-modified A and B chains were further
reduced to GSSG, reduced A and B chains by GSH.
products indicate that insulin is metabolized by a far more
complex mechanism. These results provide an increased
understanding of the role of GSH in the metabolism and
processing of insulin.
CONCLUSIONS
Acknowledgements
This work was financially supported by the National
Natural Science Foundation of China (No. 21103160) and the
Science and Technology Development Project of Jilin Province
(No. 201105032).
It has been demonstrated that ESI-MS combined with high-
resolution FTICRMS and tandem mass spectrometry plays
an important role in unambiguous identification of multiple
reaction products of insulin with GSH. The multiple reaction
Rapid Commun. Mass Spectrom. 2012, 26, 1519–1526
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Y. G. Du et al.
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[23] W. J. Nickerson, G. Falcone. Enzymatic reduction of disul-
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