(Received for publication, March 23, 1995; and in revised form, May 3, 1995)
From the
Recent studies have demonstrated the biological importance of
the interaction of nitric oxide (NO) with proteins. Protein-associated
targets of NO include heme, Cys, and Tyr. Electrospray ionization-mass
spectrometry was used to monitor the results of exposure of model
peptides and an enzyme to NO under different conditions and thus
addressed aspects of NO-protein interactions. The molecular mass of a
decapeptide containing a single Cys residue increased by 29 Da upon
treatment with NO under aerobic and acidic conditions, consistent with
the substitution of one NO moiety. The mass of reduced somatostatin, a
peptide containing two Cys residues, increased by 58 Da, consistent
with the substitution of two NO moieties. These substitutions were
prevented by pretreatment of the peptides with Nethylmaleimide. The strength of the nitrosothiol bond was
examined by varying the amount of energy applied to the peptide ions
and indicated a labile species. Cys residues were very rapidly
nitrosated, while other reactions were observed to occur at much slower
rates. These include the further oxidation of nitrosothiol to sulfonic
acid and nitration of Tyr. Peptides treated with NO at physiological pH
were observed to undergo dimerization as well as nitrosation. These
studies were extended to the enzyme p21
The interaction of NO
Nitrosothiol
formation is considered a likely outcome of NO-Cys interactions at
acidic pH(3, 7) . In fact, the function of several
proteins has been postulated to be modulated by NO through formation of
RSNO, including p21
Electrospray ionization-mass spectrometry (ESI-MS) is a very
accurate method of determining molecular mass(10) , and
conditions can be varied such that the protein of interest is subjected
to gentle perturbations during analysis in an effort to preserve labile
structures. We employed ESI-MS to monitor the results of exposure of
model peptides and an enzyme to NO under different conditions and thus
addressed aspects of NO-protein interactions.
In ESI-MS experiments, the degree of energy input
into the peptide and protein ions can be controlled by two independent
variables(12) . One variable is the temperature of the
capillary transfer tube. The other variable is the potential difference
between the capillary transfer tube and the tube lens of the mass
spectrometer (
Figure 1:
Determination of peptide nitrosothiols
using ESI-MS. A, peptide (Pep, 100 µM,
in 5 µl of 20 mM ammonium bicarbonate, pH 7.8) was mixed
with 20 µl of H
Figure 2:
Kinetics of reaction of peptide with NO. A, NO (100 µM) was added to peptide (Pep) for 2 h and then mixed with
H
Figure 3:
Effect of capillary transfer tube
temperature on nitrosothiols. NO (100 µM) was added to
peptide (Pep) for 5 min and immediately mixed with
H
Figure 4:
Effect of varying NO concentration on
nitrosothiol formation. Somatostatin was first treated with DTE
overnight and then with NO for 5 min (A, 1:5:10; B,
1:5:30, somatostatin:DTE:NO, mol/mol/mol) prior to mixing with
H
Figure 5:
ESI-MS of NO-treated p21. p21 (20
µM) was treated without (A) or with (B) NO (100
µM) for 5 min prior to mixing with
H
We have used ESI-MS to identify, quantify, and characterize
modifications of peptides and protein as a result of exposure to NO. In
a peptide containing one Cys residue, a nitrosothiol bond was
identified and its thermodynamic properties examined by altering the
temperature of the electrospray capillary transfer tube. The bond was
found to be labile, and therefore its detection requires the relatively
gentle conditions used in these studies. It should also be possible to
calculate the
The high accuracy of molecular mass determination using ESI-MS
(error
Additional chemical modifications were also
identified, which include nitration and sulfonic acid formation. These
latter modifications formed at a much slower rate than nitrosothiols,
which were detected in less than 5 min. Formation of SO
The number
of nitrosothiols formed on reduced somatostatin, a peptide hormone with
two Cys residues, was found to be dose-dependent and controlled by the
amount of reducing agent present. This suggests that in the cell, where
endogenous modulators of redox state such as glutathione exist,
formation of RSNO on proteins may be regulated. Furthermore,
p21
The regulation of some
heme-containing enzymes, such as guanylyl cyclase, by NO is known to
occur through binding of NO to the Fe
Current methods of
colorimetric detection of nitrosothiols require large amounts of sample (20) and require acidification of the sample. Other
spectroscopic methods exist including UV, IR, and NMR techniques. Again
sample amount, purity, and quantitation are serious difficulties
associated with these methods. ESI-MS can detect nitrosothiols in very
small amounts of sample and can determine the stoichiometry of
substitution. Furthermore, this method allows coupling to high pressure
liquid chromatography, which should enable on-line mass spectrometric
peptide mapping of sites of RSNO formation.
, whose
activity has been postulated to be modulated by nitrosothiol formation,
and revealed the formation of a single nitrosothiol on p21
upon NO treatment. These data suggest that electrospray
ionization-mass spectrometry allows for quantitation and
characterization of nitrosothiol bonds in peptides and proteins.
(
)with proteins is
known to play a critical role in regulating blood pressure, host
defense, and neurotransmission(1, 2) . Known targets
of NO on proteins are Cys and Tyr residues (3, 4) and
metals such as heme-Fe
(5, 6) .
Although much work has been performed characterizing the
NO-Fe
interaction(6) , there is considerably
less data on the result of NO-Cys interactions. One reason is the
labile nature of nitrosothiol (RSNO) bonds. Nitrosothiol bonds are
thought to occur via substitution by NO
, or other
nitrogen oxides, on free sulfhydryl groups(3) .
(8) . At
physiological pH, RSNO formation is thought to be less favorable,
although RSNO on proteins has been found in vivo(9) .
Furthermore, it has been suggested that transnitrosation reactions on
vicinal thiols can occur, leading to disulfide formation(3) .
Source of Peptides and
p21
A peptide (KNNLKECGLY, mass =
1181.4 Da) corresponding to the C terminus of the G protein
G was commercially synthesized. Somatostatin was from
Sigma (catalog no. S-9129). Purified bacterially expressed
p21
was kindly provided by Dr.
Daniel Manor, Department of Pharmacology, Cornell University (Ithaca,
NY).
Preparation of Nitric Oxide Solutions
NO solutions
were prepared as we previously described(11) . Briefly, a
solution of 20 mM ammonium bicarbonate solution, pH 8.0, in a
rubber-stoppered tube was sparged for 15 min with N and
then 15 min with NO gas (Matheson Gas, East Rutherford, NJ). This
resulted in a saturated solution of NO (1.25 mM). This
solution also contained higher oxides of NO that were not quantified.
Electrospray Ionization-Mass Spectrometry
The
electrospray ionization mass spectra were obtained on a Finnigan-MAT
TSQ-700 triple quadrupole instrument. Unless otherwise indicated,
peptides and protein samples were electrosprayed from acidified (acetic
acid) 50% methanolic solutions (pH 3.0), and the concentrations of the
analyte electrospray solutions were in the range of 10-20
µM. The measurements of pH were made with a PHM 95 pH
meter (Radiometer, Copenhagen) calibrated in aqueous solutions. No
corrections were applied for the pH measurements of solutions
containing methanol. The analyte solutions were infused into the mass
spectrometer source using a Harvard syringe pump (model
24000-001) at a rate of 3 µl/min through a 100-µm (inner
diameter) fused silica capillary. The positive ion spectra obtained
were an average of 16 scans and were acquired at a rate of 3 s/scan.
The ion signals were recorded by a Finnigan ICIS data system operated
on a DECstation 5000/120 system. The reconstructed molecular mass
profiles were obtained by using a deconvolution algorithm
(FinniganMAT).
V). The majority of mass spectra were
obtained at a mass resolution that did not resolve the individual
isotopic components. However, when more detailed information was
required, the mass resolution was increased sufficiently to resolve the
individual isotopic components.
Determination of Peptide Nitrosothiol Content Using
ESI-MS
The regulation of protein and enzyme function by NO
through nitrosation of critical Cys residues is emerging as an
important control switch in NO
signaling(3, 7, 8, 13) . The RSNO
bond is of a labile nature and thus difficult to study and quantify.
Therefore, we applied ESI-MS to characterize NO-peptide interactions
under conditions where RSNO formation is favored. A peptide
(KNNLKECGLY, mass = 1181.4 Da) corresponding to the C terminus
of the G protein G was either untreated (Fig. 1A) or treated with (Fig. 1B)
10-fold molar excess NO. Treatment with NO for 5 min at pH 3.0 resulted
in detection of a new species with a mass 29.0 ± 0.2 Da greater
than the starting peptide. The observed difference of 29 Da corresponds
to the molecular mass of NO (30 Da) minus the mass of the substituted
proton (1 Da). This mass difference of 29 Da shows unequivocally that
treatment of this peptide with NO at pH 3.0 resulted in a substitution
reaction. Pretreatment of this peptide with N-ethylmaleimide
(NEM), an irreversible thiol-binding reagent(14) , led to a new
species with a mass equal to that of peptide + NEM (Fig. 1C). Subsequent treatment of this complex with NO
did not yield a species with higher mass (Fig. 1D),
indicating that the NEM modification blocked reaction of NO with the
peptide. This demonstrates that NO substitution had originally occurred
on the Cys residue (Fig. 1B). Treatment of peptide with
NO at pH 7.8 also led to nitrosothiol formation but to a much lesser
extent. The major product at pH 7.8 was peptide dimer (Fig. 1E).
O:CH
OH:CH
COOH
(1:1:0.05, v/v/v, pH 3.0); B, NO (100 µM) was
added to peptide and immediately mixed with
H
O:CH
OH:CH
COOH (1:1:0.05, v/v/v, pH
3.0); C, NEM (1 mM) was added to peptide for 5 min at
22 °C and then mixed with
H
O:CH
OH:CH
COOH (1:1:0.05, v/v/v, pH
3.0); D, NO (100 µM) was added to the same
preparation as in C and immediately mixed with
H
O:CH
OH:CH
COOH (1:1:0.05, v/v/v, pH
3.0); E, same preparation as in B but mixed with
H
O:CH
OH (1:1, pH 7.8). Samples were left at 22
°C for 5 min prior to mass spectrometric analysis. T = 125 °C;
V = 80. Satellite peaks
arise from adventitious cation adduction.
Determination of Reaction Kinetics by
ESI-MS
Modification of proteins by NO to yield tyrosine
nitration (Tyr-NO) or sulfonic acid (SO
H) has
been suggested(15, 16) . We examined the peptide-NO
adducts formed after various times of exposure of the test peptide
(KNNLKECGLY) to NO using ESI-MS. After 5 min of exposure to NO,
nitrosothiol was the major product formed at pH 3.0 (Fig. 1B), which persisted to 45 min (data not shown).
However, after 2 h and more prominently at 7 h, two new species arose.
One had an increased mass of 49 Da greater than that of the starting
peptide, likely corresponding to a sulfonic acid derivative. The other
had an increased mass of 94 Da greater than that of the starting
peptide, likely corresponding to a peptide with both Tyr-NO
(45 Da) and SO
H (Fig. 2, A and B). After 24 h, very little starting peptide and nitrosothiol
remained (Fig. 2, C and D), and the majority
of the peptide had either SO
H or both Tyr-NO
and SO
H (Fig. 2, C and D).
Formation of SO
H was confirmed by pretreatment of the
peptide with NEM. Treatment of the peptide-NEM complex (mass =
1307 Da) for 70 h with NO yielded both the starting complex and a
peptide complex with a mass increased by 45 Da (mass = 1352 Da, Fig. 2E), indicating that only nitration occurred.
Neither sulfonic acid nor nitration and sulfonic acid derivatization
was seen (expected mass = 1356 and 1401 Da, respectively). A
number of low abundance reaction products was also observed. Thus,
ESI-MS detected nitrosation, nitration, and sulfonic acid formation on
a peptide upon exposure to NO. The order of reactivity was: RSNO
Tyr-NO
= SO
H.
O:CH
OH:CH
COOH (1:1:0.05, v/v/v, pH
3.0); B, same treatment as in A but for 7 h; C, 24 h; D, 70 h; E, NEM (1 mM) was
added to peptide for 5 min at 22 °C, and then NO (100
µM) was added and the sample left at 22 °C for 70 h
prior to mixing with H
O:CH
OH:CH
COOH
(1:1:0.05, v/v/v, pH 3.0). T = 125 °C;
V = 80.
Using ESI-MS to Study the Chemistry of
Nitrosothiols
We studied the nature of the peptide-RSNO bond by
altering the temperature of the metal capillary transfer tube. Our
indicated temperatures of the capillary transfer tube do not reflect
the temperatures of the peptide and protein ions but rather provide a
relative measure of energy input. We found that at 125 °C (Fig. 1B) or 150 °C (Fig. 3A), the
nitrosated peptide was readily detected. At 175 °C, approximately
50% of the nitrosothiol bonds was broken (Fig. 3B), and
at 200 °C nitrosothiols were undetectable (Fig. 3C). The observed mass difference between the
nitrosated peptide and the heat-induced decomposition product was 30.1
± 0.2 Da. This mass difference of 30 Da demonstrates that
homolytic decomposition of the RSNO bond occurred, yielding the thiyl
radical (i.e. R-S). These results indicate that ESI-MS
can also be used to determine the relative strength of RSNO bonds and
that gentle conditions are required for their detection.
O:CH
OH:CH
COOH (1:1:0.05, v/v/v, pH
3.0) and infused at the indicated capillary temperatures.
V = 80.
Concentration-dependent Nitrosation of Reduced
Somatostatin
Somatostatin is a 1637.7-Da peptide hormone
containing two Cys residues that are normally disulfide-linked. We
reduced somatostatin with dithioerythritol (DTE) overnight at room
temperature (somatostatin:DTE, 1:5). We then added NO in either a
1:5:10 (somatostatin:DTE:NO) ratio (Fig. 4A) or a
1:5:30 ratio (Fig. 4B). As seen in Fig. 4A, addition of a slight excess of NO yielded
oxidized somatostatin and species of increased mass consistent with one
or two RSNO bonds. When NO is added in greater excess, analysis by
ESI-MS detects oxidized somatostatin and somatostatin with two RSNO
bonds (Fig. 4B). Pretreatment of reduced somatostatin
with NEM yielded a species of 250-Da increased mass (arising from
modification of reduced somatostatin by 2 NEM molecules) and prevented
NO from altering the mass (data not shown), indicating that RSNO bonds
are responsible for the increased molecular mass observed in Fig. 4, A and B. NO had no effect on native
somatostatin after treatment for 10 min (data not shown). These data
demonstrate that nitrosothiols can form in a concentration-dependent
manner and that the stoichiometry of modification is easily determined
with ESI-MS.
O:CH
OH:CH
COOH (1:1:0.05, v/v/v, pH
3.0). T = 125 °C;
V = 80. Somat, somatostatin; ox., oxidized; red.,
reduced.
Nitrosothiol Formation on an Enzyme
We have
previously identified G proteins, and particularly
p21, as potential targets of
NO(8, 11, 17) . Therefore, we utilized ESI-MS
to determine if NO could modify p21
through
formation of nitrosothiol bonds. Purified bacterially expressed
p21
yielded three major species on ESI-MS (Fig. 5A). The species with a molecular mass of 21,296
± 2 Da corresponds to that of the native enzyme and the species
20,575 ± 2 Da to that of the enzyme from which 7 C-terminal
amino acids have been lost during purification. Treatment of our
p21
preparation for 5 min with 4-fold excess NO
resulted in an increase of both species by 30 ± 3 Da (Fig. 5B). These modified species are not stable at a
capillary temperature of 200 °C, indicating their labile character.
Bacterially expressed p21
has five reduced Cys
residues available for nitrosation. The present ESI-MS studies indicate
that only one Cys is nitrosated. Thus, this technique also allows for
quantitation of RSNO on proteins.
O:CH
OH:CH
COOH (1:1:0.05, v/v/v, pH
3.0). T = 125 °C;
V = 80. Ras
indicates truncated p21; Ras indicates
full-length p21; and X indicates an unidentified impurity,
whose mass does not shift upon NO
treatment.
G of this bond relative to other protein
modifications known to regulate protein function (e.g. phosphorylation). At physiological pH, nitrosothiol formation
occurred to a lesser extent than at acidic pH, and peptide dimer became
the predominant product. Thus, NO can facilitate transnitrosation
reactions as has previously been suggested(3, 18) .
0.02% or ±0.2 Da in our peptide of 1181.4 Da)
permitted us to determine that the NO-modified peptide differed from
that of the starting peptide by 29.0 ± 0.2 Da. Therefore, we
have clearly identified that an RSNO formed concurrent with removal of
the RSH proton.
H
and Tyr-NO
was first seen after 2 h of treatment and was
fully developed after 7 h. The order of reaction in our model peptide
was RSNO Tyr-NO
= SO
H. Therefore,
nitrosothiol formation would likely be a preferred regulator of protein
function as compared with other modifications due to NO.
, a 21-kDa enzyme whose regulation by NO has
been postulated to be controlled by nitrosation(8) , was
demonstrated to have one nitrosothiol formed upon treatment with NO.
This enzyme has five free Cys residues, and, therefore, the formation
of only one RSNO suggests the possibility that nitrosation of this
enzyme occurs at a specific site.
of
heme(6) . Recently, evidence has emerged that suggests that NO
may also regulate certain heme-containing enzymes, such as
cyclooxygenase-1 (prostaglandin H synthase-1), through
nitrosothiols(19) . We are currently exploring the interaction
of NO with heme-containing enzymes using ESI-MS.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.