Conversion of S-phenylsulfonylcysteine residues to mixed disulfides at pH 4.0: Utility in protein thiol blocking and in protein-S-nitrosothiol detection

Article abstract of DOI:10.1039/c4ob00995a

A three step protocol for protein S-nitrosothiol conversion to fluorescent mixed disulfides with purified proteins, referred to as the thiosulfonate switch, is explored which involves: (1) thiol blocking at pH 4.0 using S-phenylsulfonylcysteine (SPSC); (2) trapping of protein S-nitrosothiols as their S-phenylsulfonylcysteines employing sodium benzenesulfinate; and (3) tagging the protein thiosulfonate with a fluorescent rhodamine based probe bearing a reactive thiol (Rhod-SH), which forms a mixed disulfide between the probe and the formerly S-nitrosated cysteine residue. S-Nitrosated bovine serum albumin and the S-nitrosated C-terminally truncated form of AdhR-SH (alcohol dehydrogenase regulator) designated as AdhR-SNO were selectively labelled by the thiosulfonate switch both individually and in protein mixtures containing free thiols. This protocol features the facile reaction of thiols with S-phenylsulfonylcysteines forming mixed disulfides at mild acidic pH (pH = 4.0) in both the initial blocking step as well as in the conversion of protein-S-sulfonylcysteines to form stable fluorescent disulfides. Labelling was monitored by TOF-MS and gel electrophoresis. Proteolysis and peptide analysis of the resulting digest identified the cysteine residues containing mixed disulfides bearing the fluorescent probe, Rhod-SH.

Full text of DOI:10.1039/c4ob00995a

Volume 12 Number 40 28 October 2014 Pages 7867–8101
Organic &
Biomolecular
Chemistry
www.rsc.org/obc
ISSN 1477-0520
PAPER
P. A. Grieco et al.
Conversion of S-phenylsulfonylcysteine residues to mixed disulfides at
pH 4.0: utility in protein thiol blocking and in protein-S-nitrosothiol
detection

Organic &
Biomolecular Chemistry
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PAPER
Conversion of S-phenylsulfonylcysteine residues
to mixed disulfides at pH 4.0: utility in protein
thiol blocking and in protein-S-nitrosothiol
detection†
Cite this: Org. Biomol. Chem., 2014,
12, 7942
B. D. Reeves,a N. Joshi,a G. C. Campanello,b J. K. Hilmer,a L. Chetia,a J. A. Vance,a
J. N. Reinschmidt,a C. G. Miller,a D. P. Giedroc,b E. A. Dratz,a D. J. Singela and
P. A. Grieco*a
A three step protocol for protein S-nitrosothiol conversion to fluorescent mixed disulfides with purified
proteins, referred to as the thiosulfonate switch, is explored which involves: (1) thiol blocking at pH 4.0
using S-phenylsulfonylcysteine (SPSC); (2) trapping of protein S-nitrosothiols as their S-phenylsulfonyl-
cysteines employing sodium benzenesulfinate; and (3) tagging the protein thiosulfonate with a fluorescent
rhodamine based probe bearing a reactive thiol (Rhod-SH), which forms a mixed disulfide between the
probe and the formerly S-nitrosated cysteine residue. S-Nitrosated bovine serum albumin and the
S-nitrosated C-terminally truncated form of AdhR-SH (alcohol dehydrogenase regulator) designated as
AdhR*-SNO were selectively labelled by the thiosulfonate switch both individually and in protein mixtures
containing free thiols. This protocol features the facile reaction of thiols with S-phenylsulfonylcysteines
forming mixed disulfides at mild acidic pH (pH = 4.0) in both the initial blocking step as well as in the con-
version of protein-S-sulfonylcysteines to form stable fluorescent disulfides. Labelling was monitored by
TOF-MS and gel electrophoresis. Proteolysis and peptide analysis of the resulting digest identified the
cysteine residues containing mixed disulfides bearing the fluorescent probe, Rhod-SH.
Received 14th May 2014,
Accepted 24th June 2014
DOI: 10.1039/c4ob00995a
www.rsc.org/obc
The biological relevance of protein S-nitrosothiols has led
to intense interest in variations of the biotin switch tech-
Introduction
Protein S-nitrosothiols, defined as a covalent bond between a nique,20,21 which switches the S-nitrosothiol group into a thiol
cysteine thiol and an NO equivalent, have been identified as by reduction with ascorbate. The resulting thiol is labelled,
important posttranslational modifications in cell biology.1–3 provided that native free thiols are blocked before reduction.
S-Nitrosothiols function as a means toward nitric oxide cellu- This method has facilitated extensive proteomic studies by
lar signal transduction4,5 and play a role in both physiological use of gel electrophoresis,22–24 resin capture,25 microarray
as well as pathophysiological processes.6,7 A wide array of bio- based analysis,26 and proteolysis followed by amino acid
logical processes have been found to be effected by protein sequencing.27,28 Phenyl mercury compounds have also been
S-nitrosothiols including transcriptional regulation,8,9 blood employed on resins and with biotin tags for enrichment and
flow,10 neuronal function,11,12 and phosphorylation.13 Aber- comprehensive detection of protein S-nitrosothiols.29,30
rant S-nitrosation has been implicated in various cancers,14
Due to the intricate chemistry associated with S-nitrosothiol
cardiovascular disease,15 and neurodegenerative diseases16,17 detection, there is the likelihood that the field will require a
and may well contribute to the resistance of bacterial patho- variety of complementary approaches. Switchless protocols
gens to both host-generated and endogenous sources of nitric have been developed that utilize S-nitrosothiol ligation to triaryl-
oxide.18,19
phosphine derivatives31 to form sulfenamides,32 which can
be further manipulated to form disulfides,33 thioethers,34 or
dehydroalanines.35 These reactions are promising but are
limited by aqueous solubility issues and compatibility with
amines and carboxylates. Water soluble triphenylphosphine
probes have been developed that convert protein-S-nitroso-
thiols into S-alkylphosphonium salts detectable by mass
spectrometry.36 While these reactions exhibit advantages by
aDepartment of Chemistry and Biochemistry, Montana State University, PO Box
173400, Bozeman, MT 59717-3400, USA. E-mail: grieco@chemistry.montana.edu
bDepartment of Chemistry, Indiana University, 800 East Kirkwood Avenue,
Bloomington, Indiana 47405-7102, USA
†Electronic supplementary information (ESI) available: Fig. S1–S10 and 1H and
13C NMRs of new compounds. See DOI: 10.1039/c4ob00995a
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rhodamine based fluorophore incorporating a reactive thiol,
denoted Rhod-SH (1), reacts with protein S-phenylthio-
sulfonates, giving rise to a mixed disulfide between the probe
and the formerly S-nitrosated cysteine residue. Since PhSO2Na
does not react with thiols,38 the sequential addition of a thiol
derivatized fluorophore is performed without purification.
Results and discussion
Control reactions
(i) Stability of GSNO at different pH’s with and without
DTT. The thiosulfonate switch protocol employs a mild acidic
pH in order to convert S-nitrosothiols into their corresponding
S-phenylthiosulfonates.38 In order to assess the stability of
S-nitrosothiols at pH 4.0, the absorbance at 545 nm of a 10 mM
GSNO solution (ε = 15 M−1 cm−1, 545 nm, nN→π*)41 was moni-
tored (Fig. 1) over 50 min in pH 4.0 buffer. This was repeated
at pH 7.0 (100 mM HEPES, 1 mM EDTA) and minimal degra-
dation of GSNO was observed at either pH. Denitrosation reac-
tions were also monitored between GSNO and dithiothreitol
(DTT) (10 mM each) at varying pHs. When DTT undergoes
transnitrosation, an unstable S-nitrosothiol is formed which is
converted to a disulfide along with concomitant release of
nitroxyl (HNO).42–44 A 1% decrease in absorbance is observed
when DTT is added to GSNO at pH 4.0 over 50 min. This is in
stark contrast to the results obtained at pH 4.8 (100 mM
ammonium formate, 1 mM EDTA), 5.5 (100 mM MES, 1 mM
EDTA), and 7.0 (100 mM HEPES, 1 mM EDTA), where 17%,
47%, and 75% (respectively) of the absorbance is lost. This is
consistent with previously reported findings where the rate of
transnitrosation increases with increasing pH.45 This stability
trend of S-nitrosothiols demonstrated here (Fig. 1) suggests
that there are clear advantages to designing a proteomics-
Scheme 1 BSA-SNO is converted into BSA-SSO2Ph and PhSO2NHOH
upon reaction with PhSO2Na at pH 4.0.
reacting directly with S-nitrosothiols, the resulting S-alkylphos-
phonium salts are not stable to gel electrophoresis or tryptic
digest. This work has been expanded by incorporating triaryl-
phosphines with ester and thioester functionalities along with
water solubilizing groups.37 While low molecular weight
S-nitrosothiols such as S-nitrosoglutathione (GSNO) have been
detected through mass fragmentation, this method has not
been applied to protein-S-nitrosothiols.
We have recently published38 a detailed study of the reac-
tion between S-nitrosated bovine serum albumin (BSA-SNO)
and benzenesulfinic acid sodium salt (PhSO2Na). PhSO2Na
was shown to react with BSA-SNO forming the corresponding
S-phenylthiosulfonate, BSA-SSO2Ph, and N-hydroxybenzene-
sulfonamide in a 1 : 1 ratio (Scheme 1) within minutes.
N-Hydroxybenzenesulfonamide, also known as Piloty’s acid,39
is not an NO donor at neutral and acidic pH. It should be
noted that in the biotin switch technique, ascorbate reacts
with S-nitrosothiols giving rise to O-nitrosoascorbate, which
releases NO within minutes at neutral pH.40 PhSO2Na was
shown to be unreactive to the protein thiol and disulfides in
bovine serum albumin.
While PhSO2Na has been employed as an S-nitrosothiol
probe,38 the reporter group linkage, i.e. the S-phenylsulfonyl-
cysteine functional group, is susceptible to hydrolysis at
neutral pH resulting in diminished signal. An expanded
methodology that displaces S-phenylthiosulfonates with a
thiol containing fluorescent probe resulting in
a stable
disulfide linkage has the potential of addressing this short-
coming and is the focus of the work presented here.
Herein, we explore a new protocol, denoted as the thiosulfo-
nate switch, that traps protein S-nitrosothiols as mixed disul-
fides bearing a fluorescent probe at pH 4.0. This protocol
involves initial blocking of protein thiols by S-phenylsulfonyl-
cysteine (SPSC),41 which forms cysteine bearing mixed disul-
fides. It should be noted that the byproduct of the initial
blocking step is benzenesulfinate itself. Subsequent addition
of PhSO2Na converts protein S-nitrosothiols into protein S-phenyl-
thiosulfonates. Addition of a highly water soluble zwitterionic
Fig. 1 % GSNO over time (min) taken from absorbance readings at
545 nm at different pH’s with and without equimolar DTT.
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Scheme 3 Reactions of Rhod-SH (1) with GSSG, (GlyCys)2, SPSC,
MMTS, NEM, and IAM at pH 4.0.
tions with disulfides GSSG and (Gly-Cys)2 were also observed
in order to assess the reactivity of Rhod-SH (1) with disulfides
over extended time. For both disulfides, no product formation
was observed after 1 h. For thiol blocking reactions, SPSC
reacted with Rhod-SH (1) the fastest with approximately full
conversion in 1 min. MMTS reacted the second fastest with
22% conversion over 1 min and 97% conversion after 30 min.
Therefore, both SPSC and MMTS are efficient thiol blockers at
pH 4.0. The reaction with NEM was slow at pH 4.0 with only
48% conversion over 1 h while IAM showed no product for-
mation in 1 h. All products were confirmed by collecting the
corresponding HPLC elution peak followed by HRMS analysis
(see Fig. S2† for HPLC traces).
(iv) SPSC control reactions. While SPSC reacts readily with
Rhod-SH (1), a series of thiols with varying pKa’s were blocked
with SPSC in order to validate that SPSC reacts with thiols and
not thiolates (Scheme 4). A solution of thiol (2 mM) and SPSC
(1.8 mM) in pH 4.0 buffer was analyzed by reverse phase HPLC
in tandem with UV-vis detection. Reactions were monitored
by measuring absorbance at 265 nm (λmax of SPSC and
PhSO2Na)38 at Rt = 10 min (PhSO2Na) and Rt = 12 min (SPSC)
(retention times were determined by running authentic
samples). Conversion of SPSC to PhSO2Na went to completion
after 2 min for penicillamine and glutathione and 87% com-
Scheme 2 Reactions of PhSO2Na and Rhod-SH (1) on GSNO, GSH and
GSSG.
based detection strategy at mild acidic pHs provided that
protein solubility issues can be overcome.
(ii) Reactions of PhSO2Na and Rhod-SH (1) with GSNO,
GSH and GSSG. First, GSNO was employed as the substrate to
undergo two of the essential steps in the thiosulfonate switch:
S-nitrosothiol conversion to an S-phenylthiosulfonate and dis-
placement with a thiol probe forming a mixed disulfide. Con-
version of GSNO to GSSO2Ph was performed by adding excess
benzenesulfinic acid sodium salt (250 eq.) at pH 4.0 (100 mM
ammonium formate, 1 mM EDTA) (Scheme 2). After 30 min,
the fluorescent rhodamine based probe bearing a reactive
thiol, Rhod-SH (1) (see Experimental for synthesis and optical
characterization), was added at 1 eq. and after 10 min, the
product Rhod-SSG, was observed at 95% conversion based on
the reverse phase HPLC trace (see Fig. S1†) with detection at
λmax of Rhod-SH (1) (562 nm). When this experiment was
repeated replacing GSNO with either GSSG or GSH, only Rhod-
SH (1) was observed, confirming the selectivity of the reaction
towards S-nitrosothiols. These reactions also confirm that the
thiol probe, Rhod-SH (1), does not readily undergo disulfide
exchange at pH 4.0 but does exchange with S-phenylsulfonyl-
glutathione in the presence of excess PhSO2Na.
(iii) Rhod-SH (1) control reactions with thiol blockers and
disulfides. The reactivity of SPSC was compared to well-known
commercially available thiol blockers such as MMTS, NEM,
and IAM by reacting each with Rhod-SH (1) at pH 4.0
(Scheme 3). While SPSC has been shown to react with thiols at
basic pH (0.1 M sodium bicarbonate),46 the reaction at mild
acidic pH’s is unreported to our knowledge. In addition, reac-
Scheme 4 Reactions of SPSC with a series of thiols of varying pKa.
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Fig. 2 % Conversion of SPSC to PhSO2Na over 1 h as measured from
absorbance detection at 265 nm from RP HPLC analysis.
pletion for captopril. Therefore, SPSC undergoes facile reac-
tions with thiols at pH 4.0 and does not require the deprotona-
tion of thiols to form thiolates for conversion.
Fig. 3 Deconvoluted mass spectra of either (red, top): BSA (100 µM) or
(blue, middle): BSA-SNO (44% SNO content, 100 µM total protein) after
addition of PhSO2Na and Rhod-SH (1) for 30 min. (green, bottom):
BSA-SNO (22% SNO content, 28% free thiol, 100 µM total protein) after
sequential addition of SPSC (1 h), PhSO2Na (30 min), and Rhod-SH (1)
(10 min).
The hydrolysis of a 2 mM solution of SPSC at different pH’s
was also measured using the same HPLC method as above
over 1 h (Fig. 2). Hydrolysis at pH 4.0, 4.8, 5.5, and 6.0 was
detected at 2%, 5%, 9%, and 31%, respectively. At pH 7.0 the
hydrolysis was almost complete (96%). Minimal detection of
benzenesulfonic acid (Rt = 5.5 min) was observed. This con-
firms that S-phenylsulfonylcysteines are unstable at neutral
pH. Since common proteomic methodology requires neutral to
slightly basic pH, such as polyacrylamide-SDS gel electro-
phoresis and trypsinolysis, S-phenylsulfonylcysteines are an
unlikely choice as a reporter adduct for protein-S-nitrosothiols.
free thiol content of BSA was found to be 49% employing the
DTDP method.38,51 S-Nitrosation of BSA with 5 eq. Cys-SNO in
HEN buffer (100 mM HEPES, 1 mM EDTA, 0.1 mM neo-
cuproine, pH 8.0) for 1 h in the dark was 90% complete (44%
overall SNO) based on a Griess/Saville assay as modified by
Basu et al.52 BSA-SNO was purified and the buffer was
exchanged to pH 4.0 (100 mM ammonium formate, 1 mM
EDTA) by centrifugation through a Sephadex G25 column.
When PhSO2Na (250 eq.) followed by Rhod-SH (1) (2 eq.)
was added to BSA-SNO (44% SNO content, 100 µM total
protein, 100 µL) at pH 4.0, the mixed disulfide BSA-SS-Rhod
(67 376 amu) was detected as the major product within 30 min
(Fig. 3, blue, middle). The glycosylated adduct, BSA(Glc)-
SS-Rhod (67 538 amu), was also detected as a minor product.
In a control reaction with commercially available BSA (Sigma)
employing PhSO2Na and Rhod-SH (1), no fluorescent adducts
were detected (Fig. 3, red, top). Other peaks were observed
corresponding to unidentified BSA variants. After labelling of
BSA-SNO, these variants show a corresponding mixed disulfide
peak (+946 amu, +SRhod).
As shown in Fig. 3 (red, top) commercial BSA and the con-
taminant BSA-SSCys upon exposure to PhSO2Na and Rhod-SH
(1) does not give rise to BSA-SS-Rhod. However, protein thiols
can react with protein S-phenylsulfonylcysteines resulting in
diminished signal. A thiol blocking step before SNO labelling
addresses this concern. To demonstrate the blocking of free
thiols at pH 4.0, BSA was partially nitrosated (22% SNO, 28%
free thiol) by addition of GSNO (5 eq., 1 h, HEN pH 7.0) in the
dark followed by buffer exchange to pH 4.0. The addition of
Conversion with
a thiol probe readily generates mixed
disulfides, which are the linkage utilized in the biotin switch
technique used in protein-S-nitrosothiol proteomics.20
Labeling of individual protein-S-nitrosothiols with
Rhod-SH (1)
(i) TOF analysis of Rhod-SH (1) labelled BSA-SNO. S-Nitro-
sated bovine serum albumin (BSA-SNO) was used to probe the
reaction between an S-nitrosated protein and PhSO2Na and
Rhod-SH (1). Serum albumin has been implicated as an NO
reservoir in plasma47 and exerts NO bioactivity by transnitrosa-
tion to other proteins such as cell surface Protein Disulfide
Isomerase (csPDI).48 The detection of BSA-SNO by the biotin
switch assay is hindered by a slow reduction rate with ascor-
bate.49 BSA contains a single reactive thiol (Cys34) and 17
protein disulfides which allows for evaluation of selectivity of
S-nitrosothiol labeling employing PhSO2Na and Rhod-SH (1)
in the presence of disulfides.
Initial TOF-MS analysis of commercial BSA (Sigma-Aldrich)
shows that in addition to the free thiol (66 430 amu), covalent
adducts were detected including the mixed disulfide
BSA-SSCys (66 549 amu), and glycosylation adduct BSA(Glc)
(66 592 amu) which have been observed previously.38,50 The
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S-phenylsulfonylcysteine (SPSC, 1 eq., 1 h) to the mixture of was also identified as a derivative of RhodSSRhod lacking an
BSA-SNO and BSA (100 µM, 10 nmol total protein) resulted in ethyl group (Table S1†).
blocking of the free thiol group (Fig. 3, green, bottom) as seen
The same protocol was used on BSA as a control (Fig. 4,
by the increase in the BSA-SSCys peak (66 549 amu) as well as red), which exhibited low absorbance overall, demonstrating
a new peak corresponding to BSA(Glc)SSCys (66 711 amu). the selectivity of the technique. The major peaks in the control
Sequential addition of PhSO2Na (250 eq., 30 min) and Rhod- experiment correspond to RhodSSMe and RhodSSRhod (Fig. 4,
SH (1) (3 eq., 10 min) formed BSA-SS-Rhod (67 376 amu).
(ii) Trypsinolysis of BSA-SS-Rhod. To demonstrate the
red).
(iii) 1D gel electrophoresis on BSA-SS-Rhod. One dimen-
compatibility of the mixed disulfide reporter group with sional SDS-PAGE nonreducing gels were run on the samples of
trypsinolysis and confirm the location of the tagged cysteine BSA-SS-Rhod prepared from a BSA-SNO dilution series contain-
residue, BSA-SS-Rhod was subjected to trypsin digestion. ing the same amount of total protein (6.6 μg) with varying
Toward this end, BSA-SNO (44% SNO, 100 µM total protein) amounts of BSA-SNO (1.8, 1.2, 0.6, 0.3, 0.15, and 0.0 μg). In
was exposed to PhSO2Na (250 eq.) and Rhod-SH (1) (2 eq.) at each dilution series, BSA-SNO was subjected to the sequential
pH 4.0 for 30 min. MMTS was added to trap the excess dye addition of SPSC (0.8 nmol, 1 h), PhSO2Na (2.5 µmol, 30 min)
and safeguard against disulfide scrambling between residual and Rhod-SH (1) (2 nmol, 10 min) at pH 4.0. Before addition
Rhod-SH (1) and protein disulfides after buffer exchange to pH of non-reducing sample buffer and gel electrophoresis (pH
7.5. Trypsinolysis was performed (10 h, 37 °C) on BSA-SS-Rhod 8.0), an excess of MMTS (80 nmol) was added to block free
and the peptides obtained were separated by reverse phase Rhod-SH (1). After electrophoresis, the gels were scanned at
HPLC and analyzed by UV-vis in tandem with mass spectro- 532 nm with a 30 nm band pass filter at 580 nm. A linear
metry. As seen in Fig. 4 (blue), the BSA-SS-Rhod proteolysis response was observed between spot volume and µg BSA-SNO
produced two major peptides that exhibited strong absorbance over the dilution range as seen in Fig. 5.
at 565 nm. The isotopic pattern (Table S1†) of the major peak
(iv) AdhR*SNO synthesis and labelling by the thiosulfonate
(3.85 min) corresponds to the fifth fully digested tryptic switch. In order to evaluate the general utility of the thiosulfo-
peptide from the C-terminus [GLVLIAFSQYLQQC(SSRhod)- nate switch to react with biologically important S-nitrosothiols,
PFDEHVK)] wherein Cys34 is a mixed disulfide derived from we investigated the ability of two homologous (46% identity)
Rhod-SH (1) (T5SSRhod) while the first peak (3.7 min) in the bacterial transcriptional activators, NmlR (novel MerR-like reg-
HPLC trace corresponds to a tryptic peptide with two misclea- ulator) from Streptococcus pneumoniae and AdhR (alcohol
vages [FKDLGEEHFKG LVLIAFSQYLQQC(SSRhod)PFDEHVK] dehydrogenase regulator) from Bacillus subtilis, to undergo
wherein Cys34 is a mixed disulfide derived from Rhod-SH (1) S-nitrosation. NmlR protects S. pneumoniae from nitric oxide
(T3-5SSRhod) (Fig. S4, Table S1†). The S-methyl capped dye
species RhodSSMe (4.35 min), as well as the dimer RhodSSR-
hod (4.68 min), were also observed. The small peak at 4.5 min
Fig. 5 Spot volume vs. µg BSA-SS-Rhod from 1D SDS-PAGE nonredu-
cing gels. BSA-SNO (1.8, 1.2, 0.6, 0.3, 0.15, and 0.0 μg) was subjected to
the sequential addition of SPSC (1 h), PhSO2Na (30 min), Rhod-SH (1)
Fig. 4 Blue: Chromatogram for a tryptic digest of BSA-SS-Rhod (λ =
565 nm). Red: Chromatogram for a tryptic digest of BSA subjected to
the same reaction and digestion conditions.
(10 min), and MMTS (5 min). The error bars represent the standard devi-
ation. The image above is the fluorescent image of the gel and coomas-
sie stain.
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stress required for survival of this human pathogen in the
bloodstream,53 while AdhR protects B. subtilis from naturally
occurring carbon electrophiles such as methylglyoxal and form-
aldehyde. AdhR activates transcription of Fdh and cysteine
proteinase YraA, both of which protect against aldehyde toxi-
city.54 Recent studies also suggest that NmlR may be capable
of sensing carbon electrophiles in the cell.55 Both NmlR and
AdhR possess a single cysteine residue, Cys52, derivatization
of which is linked to transcriptional activation via an unknown
mechanism.
Purified recombinant and reduced NmlR (13 463 amu) and
AdhR*-SH (14 438 amu), a C-terminally truncated and func-
tional (Fig. S5†) variant of AdhR-SH (see Experimental section
for expression and purification details), each readily react with
GNSO to form NmlR-SNO and AdhR*-SNO, unambiguously
established by their characteristic absorption spectra
(Fig. S6†). We reasoned that AdhR*-SNO may be difficult to
reduce with ascorbate since S-nitrosation of Cys52 is regulat-
Fig. 7 Deconvoluted mass spectra after labelling by the thiosulfonate
switch of (red, top): AdhR*-SNO (53% SNO, 47% free thiol, 25 µM total
protein); (blue, bottom): AdhR*-SH (25 µM). thiosulfonate switch = SPSC
(1 h) + PhSO2Na (30 min) + Rhod-SH (1) (10 min).
ory, converting AdhR*-SH from a weak repressor to a potent AdhR*-SNO and ascorbate much like the reaction between
activator of transcription.54 To test this, AdhR*-SH was nitro- BSA-SNO and ascorbate.49
sated with GSNO (10 eq., 25 mM HEPES, 400 mM NaCl, 1 mM
A series of mixing experiments were next carried out in
EDTA, 0.1 mM bathocuproine disulfonate, pH = 7.0, 1 h) in the order to establish the specificity of the thiosulfonate switch to
dark and the SNO content was determined to be 53% by uniquely detect S-nitrosated cysteines in the presence of
UV-Vis (AdhR*-SH: ε280 nm = 12 950 cm−1 M−1 and AdhR*- underivatized cysteines. Initially, the thiosulfonate switch was
SNO: ε340 nm = 1000 cm−1
M
−1, Fig. S6†). Fig. 6 (dotted line) carried out on a mixture of AdhR*-SNO (53%) and AdhR*-SH
shows the deconvoluted mass spectrum of AdhR*-SNO (14 467 (47%) (25 µM total protein concentration, 0.2 nmol). SPSC was
amu) and AdhR*-SH (14 438 amu) mixture. When exposed to used to block the free thiol over 1 h to form AdhR*-SSCys
ascorbate (20 mM) at pH 7.5 (HEN buffer) under ambient con- (14 557 amu) while subsequent addition of PhSO2Na (30 min)
ditions for 1 h, we observed only a modest 10% increase in and Rhod-SH (1) (10 min) gave rise to AdhR*-SS-Rhod (15 384
free thiol relative to the starting mixture (Fig. 6, solid line). amu) (Fig. 7, red, top). When subjecting underivatized AdhR*-
While denaturing agents, heat, and increased ascorbate con- SH (25 µM) to the identical protocol, only the blocked AdhR*-
centration would likely increase the conversion of S-nitroso- SSCys was observed (Fig. 7, blue, bottom).
thiol to thiol as employed in the biotin switch technique,20
Protein-S-nitrosothiol labelling of mixtures of BSA-SNO/AdhR*-
SH and BSA/AdhR*-SNO
this initial experiment demonstrates a slow reaction between
(i) Thiosulfonate switch labelling of BSA-SNO/AdhR*-
SH. To demonstrate the selectivity of the thiosulfonate switch
protocol toward S-nitrosothiols in the presence of a second
protein bearing a free thiol, a mixture of BSA-SNO (52% SNO,
negligible free thiol content) and underivatized AdhR*-SH
(50 µM, 5 nmol total protein each) at pH 4.0 was subjected to
the thiosulfonate switch. SPSC was added to block the free
thiol of AdhR*-SH for 1 h followed by the sequential addition
of PhSO2Na (30 min) and Rhod-SH (1) (10 min). Fig. 8A shows
the BSA-SS-Rhod adduct (67 376 amu) is dominant 10 min
after addition of Rhod-SH (1) while AdhR*-SH was completely
converted to AdhR*-SSCys (14 557 amu). Scrambling of the
reporter group was not observed, as noted by the arrow point-
ing to the mass of the undetected Rhod-SH (1) adduct, AdhR*-
SS-Rhod. The labelled protein mixture was precipitated in
acetone, dissolved in 20 mM Tris, 1% deoxycholate (pH 7.7)
and proteolyzed with trypsin for 24 h at 37 °C. The peptides
were analysed by reverse phase HPLC/MS with absorbance
detection at 565 nm. Two absorbance peaks were observed
Fig. 6 Deconvoluted mass spectra of (dotted line): AdhR*-SNO; (solid
line): AdhR*-SNO+ ascorbate (20 mM), 1 h. Buffer = 100 mM HEPES,
corresponding to labelled peptides QQC(SSRhod)PF and LQQC
(SSRhod)PF from the BSA sequence (Fig. S8, Table S1†). 1D
1 mM EDTA, 0.1 mM neocuproine (HEN), pH 7.5. AdhR*-SNO = 53%
SNO, 47% free thiol, 25 µM total protein.
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Fig. 8 Deconvoluted spectrum of thiosulfonate switch labelled (A)
BSA-SNO# and AdhR*-SH (50 µM each) and (B) Spot volume of
BSA-SNO# and AdhR*-SH (5 µM each) labelled by the thiosulfonate
switch and run on a 1D SDS-PAGE nonreducing gel. The image shows
the actual gel spots with their corresponding molecular weights.
BSA-SNO# = 52% SNO, 0% free thiol.
Fig. 9 (A) Deconvoluted spectrum of thiosulfonate switch labeled BSA
and AdhR*-SNO (50 µM each). (B) Spot volume of BSA and AdhR*-SNO
(5 µM each) labelled by TST and run on a 1D SDS-PAGE nonreducing
gel. The image shows the actual gel spots with their corresponding
molecular weights. Buffer = 100 mM ammonium formate, 1 mM EDTA,
pH 4.0. AdhR*-SNO = 53% SNO, 47% free thiol.
SDS-PAGE nonreducing gel electrophoresis was performed on
BSA-SNO (52% SNO, negligible free thiol content) and underi-
vatized AdhR*-SH (5 µM, 0.25 nmol total protein each) after
thiosulfonate switch labelling. MMTS (4 µL, 20 mM,) was
added to quench the excess fluorophore. As shown in Fig. 8B,
labelling was selective for the BSA-SNO with the background
labelling of AdhR*-SH minimal (<1%). The image shows the
actual gel spots with their corresponding molecular weights
(Fig. 8B). Coomassie staining of the gel is shown in Fig. S9.†
(ii) Thiosulfonate switch labelling of BSA/AdhR*-SNO. In
the reciprocal experiment, a mixture of AdhR*-SNO (53% SNO
content, 47% free thiol) and underivatized BSA (50 µM, 5 nmol
total protein each) was subjected to the thiosulfonate switch
and the deconvoluted spectra are shown in Fig. 9A. Both
AdhR*-SSCys (14 557 amu) and AdhR*-SS-Rhod (15 384 amu)
were detected as anticipated. BSA-SSCys was also formed and
BSA was not labelled by Rhod-SH (1) as evidenced by the
absence of BSA-SS-Rhod. The mixture was precipitated in
acetone, dissolved in 20 mM Tris, 1% deoxycholate (pH 7.7)
and proteolyzed with trypsin for 24 h at 37 °C. The absorbance
peaks observed correspond to the labelled tryptic peptide
C(SSRhod)MR and overdigested peptide C(SSRhod)M from the
AdhR sequence (Fig. S8, Table S1†). 1D SDS-PAGE nonreducing
gel electrophoresis of a BSA and AdhR*-SNO (53% SNO
content, 47% free thiol) mixture (5 µM, 0.25 nmol total protein
each) labeled by the thiosulfonate switch shows that labeling
was selective for AdhR*-SNO and background labeling of BSA
was minimal (∼1%) (Fig. 9B). The image shows the actual gel
spots with their corresponding molecular weights (Fig. 9B).
Coomassie staining of the gel is shown in Fig. S9.†
To further confirm the identity of the AdhR*-SNO adduct,
AdhR*-SS-Rhod was proteolyzed by Glu-C, which allows the
detection of a larger labelled peptide. AdhR*-SNO (53% SNO,
100 μM) was subjected to labelling by the thiosulfonate switch
and the protein was precipitated in acetone and dissolved in
100 mM ammonium bicarbonate (pH 8.0), deoxycholate (1%),
and Glu-C (8 µg mL−1) and heated to 37 °C for 16 h. The
peptide mixture was analyzed by reverse phase HPLC and
detected the labeled peptide FIKC(SSRhod)MRNAGLSIE from
the AdhR*-SH sequence (Table S1†).
Conclusions
A new method designated as the thiosulfonate switch for
protein-S-nitrosothiol conversion to mixed disulfides in mild
acidic aqueous solution has been developed. This method uti-
lizes the conversion of S-nitrosothiols to S-phenylthiosulfo-
nates mediated by benzenesulfinate. Both BSA-SNO and
AdhR*-SNO are readily converted within minutes into their
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respective S-phenylsulfonylcysteines through the addition of
benzenesulfinic acid sodium salt. The thiol-modified rhod-
amine fluorescent probe, Rhod-SH (1), upon addition to the
protein S-phenylthiosulfonates, provides the corresponding
fluorescent mixed disulfides. This robust labelling protocol is
compatible with 1D non-reducing SDS PAGE and tryptic proteo-
lysis followed by peptide analysis. The thiosulfonate switch
is unique to other S-nitrosothiol detection strategies such as
the biotin-switch technique in that it is not reliant upon the
reduction of the S-nitrosothiol functionality to a thiol. There-
fore, incomplete thiol blocking does not generate false posi-
tives, which is a major obstacle to overcome using reductive
techniques. Thiols have been shown, however, to react readily
with S-phenylsulfonylcysteines and therefore have the poten-
tial of diminishing signal. This discovery has been exploited as
both a thiol blocking strategy and as a means to convert the
thiosulfonate to a mixed disulfide, a more robust reporter
group for proteolysis/peptide analysis and gel electrophoresis.
The thiol blocking reagent, S-phenyl sulfonylcysteine (SPSC),
that efficiently blocks free thiols at pH 4.0 in the presence of
S-nitrosothiols, is used to block thiols that possess reactivity
and proximity to displace protein-S-phenylsulfonylcysteines.
Residual SPSC does not interfere with the subsequent reac-
tions and therefore purification steps are not required. While
this study has examined labelling of purified proteins
BSA-SNO and AdhR*-SNO, future work will explore the label-
ling of the S-nitroso proteome of biological systems, including
human and bacterial cells, in lysate form. Also, cross reactivity
with other cysteine posttranslational modifications such as
sulfenic acids and persulfides will be evaluated by global
modifications of lysates. On the whole, this study has allowed
for new observations in thiol and S-nitrosothiol chemistry,
which will aid in developing prospective S-nitrosothiol detec-
tion strategies.
Scheme 5 Synthesis of S-phenylsulfonylcysteine (SPSC).
Phenomenex Synergi 4μ Polar RP 80A HPLC column (250 ×
10.00 mm) using Shimadzu Lab Solutions software. A gradient
of A (0.1% TFA, H2O) and B (0.1% TFA, 20 : 1 CH3CN, H2O)
was used in all cases. NMR spectroscopy was performed on
Bruker Avance DPX 300 or Bruker Avance DRX 500 NMR
spectrometer.
(ii) Synthetic procedures
S-Phenylsulfonylcysteine (SPSC). The synthesis of SPSC was
performed by modifying a literature procedure (Scheme 5).41
To a stirred solution of L-cysteine (240 mg, 2.0 mmol) in 2 N HCl
(2 mL) was slowly added a solution of NaNO2 (138 mg, 2.0 mmol)
in water (0.5 mL). After 40 min at 5 °C the deep red solution was
treated with a solution of PhSO2Na (650 mg, 4.0 mmol) in
water (4 mL) and stirred for 1 h at 5 °C and 4 h at rt. The pre-
cipitate was filtered and washed with water (2 × 4 mL), acetone
(2 × 3 mL) and ether (2 × 3 mL). Drying by lyophilization pro-
vided 332 mg of SPSC as a white solid (66% yield): 1H NMR
(300 MHz, d-DMSO): δ 3.30–3.15 (m, 2H), 3.35–3.41 (m, 1H),
7.66–7.80 (m, 3H), 7.91 (d, 2H, J = 7.7 Hz); 13C NMR (125 MHz,
CD3OD):
δ 36.36, 53.91, 128.50, 131.08, 135.90, 169.79;
HRMS-EI (m/z): calcd for C9H11NO4S2 [M + H]+ 262.0202,
found 262.0227.
N-(9-(2-(4-(5-Carboxypentanoyl)piperazine-1-carbonyl)phenyl)-
6-(diethylamino)-3H-xanthen-3-ylidene)-N-ethylethanaminium
2,2,2-trifluoroacetate (3). The synthesis of N-(6-(diethylamino)-
9-(2-(piperazine-1-carbonyl) phenyl)-3H-xanthen-3-ylidene)-N-
ethylethanaminium 2,2,2-trifluoroacetate (2) was performed by
following a literature procedure (Scheme 6).56 To a solution of 2
(2.39 g, 4.37 mmol), adipic anhydride (700.5 mg, 5.47 mmol),
and 4,4-dimethylaminopyridine (659 mg, 5.39 mmol) in dry
methylene chloride (15 mL) was added dropwise freshly distilled
triethylamine (0.65 mL, 4.66 mmol). The solution was stirred for
2 h under Argon at which time the reaction was complete by TLC
analysis (Rf = 0.8, 2 : 1 CH2Cl2–MeOH). The resulting solution
was partitioned between 100 mL EtOAc and 150 mL 1 M
K2CO3 and washed with 100 mL × 3 EtOAc to remove starting
material. NaCl (15 g) was added to the aqueous layer which
was then extracted with 2 : 1 isopropanol–methylene chloride
until clear. The organic layer was dried with Na2SO4 and the
solvent was removed in vacuo. The resulting red solid was dis-
solved in 50 mL of chloroform and filtered through a 0.45 mm
filter (Whatman, Spartan 13). The solvent was removed
in vacuo and the resulting red solid was purified by reverse phase
HPLC (B 40% to 65%, 0–12.5 min, 65% to 95%, 12.5–25 min,
95% to 50%, 25–30 min, flow = 25 mL min−1, λ = 525 nm, Rt =
14 min, multiple runs) and after lyophilization provided 2.48 g
Experimental
Synthesis and characterization
(i) Materials. Boc-Cys(Trt)-OSu was purchased from
Advanced Chemtech. Adipic anhydride was purchased from
Wako Chemicals USA. All other reagents were purchased from
Aldrich. Methylene chloride, triethylamine and N-methyl-
morpholine were distilled over CaH2. Reversed phase HPLC puri-
fication was performed with a Waters 2487 Dual l Absorbance
detector, 600 controller and pump, and a Phenomenex Synergi
4μ Polar RP 80A HPLC column (250 × 21.2 mm,) using Waters
Empower 3 software. Compound 3 was purified by reverse
phase HPLC using a Shimadzu SPD-10AV UV-vis detector, a
Shimadzu LC-8A pump, a Phenomenex Synergi 4µ Polar RP
80A HPLC column (250 × 30 mm), a Shimadzu SIL-10AP auto-
sampler, and a Shimadzu FRC-10A fraction collector using Shi-
madzu Lab Solutions software. Compound 9 was purified and
control reactions were monitored by reversed phase HPLC
using a Shimadzu SPD-M20A prominence diode array detector,
an LC-20AB prominence liquid chromatography pump, and a
1
of 3 as a red powder (84% yield): H NMR (300 MHz, CD3OD):
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Scheme 7 Synthesis of cysteic acid side chain 6. (i) Boc-Cys(Trt)-OSu,
Na2CO3, aq. dioxane, rt, 2 d, 88%, (ii) Et3SiH, TFA, CH2Cl2, rt, 1 h, (iii)
HCO2H, H2O2, 0 °C, 1 h, 81%, 2 steps.
157.17, 157.35, 159.42, 160.27, 160.77, 161.28, 169.72, 170.30,
171.93, 173.79; HRMS-EI (m/z): calcd for C42H50N5O7 [M]+
+
736.3705, found 736.3719.
5-((R)-2-((N-Boc)-amino)-(3-tritylthio)-propionamido)-pentanoic
acid (5) (Scheme 7). A mixture of 5-aminovaleric acid hydrochlo-
ride (1.40 g, 9.11 mmol), Boc-Cys(Trt)-OSu (5.0 g, 8.92 mmol) and
Na2CO3 (1.89 g, 17.8 mmol) in H2O (52.5 mL) and 1,4-dioxane
(52.5 mL) was stirred at ambient temperature for 2 d before
the solvents were removed in vacuo. The residue was dissolved
in H2O (1 L) and acidified to pH 2 using 1 M HCl, at which
point a white precipitate fell out of solution. The precipitate
was filtered and washed with H2O, followed by drying under
vacuum providing 4.40 g of a white solid (88%): FTIR (CH2Cl2):
700 (s), 742 (s), 1166 (s), 1490 (s), 1527 (s), 1708 (s), 2341 (s),
Scheme 6 Synthesis of compounds 2–4.
1
2359 (s), 2931 (s), 2975 (s) 3304 (br); H NMR (500 MHz, D2O):
δ 1.31 (t, 12H, J = 7.0), 1.59 (m, 4H), 2.30 (m, 2H), 2.37 (m, 2H),
3.39 (m, 8H), 3.69 (q, 8H, J = 7.1 Hz), 6.97 (m, 2H), 7.08 (m,
2H), 7.28 (m, 2H), 7.52 (m, 1H), 7.74 (m, 3H); 13C NMR
(125 MHz, CD3OD): δ 12.97, 25.71, 25.87, 33.68, 34.66, 42.88,
47.04, 97.50, 115.00, 115.54, 129.06, 131.44, 131.91, 132.43,
133.31, 136.65, 157.15, 157.35, 159.41, 161.41, 169.69, 174.01,
δ 1.40 (s, 9H), 1.49–1.62 (m, 4H), 2.28 (t, 2H, J = 7.0 Hz),
2.51–2.59 (m, 2H), 3.15–3.22 (m, 2H), 4.08 (m, 1H), 6.08 (d,
1H, J = 8.0 Hz), 7.23 (t, 3H, J = 7.2 Hz), 7.35 (t, 6H, J = 7.5 Hz,),
7.40 (d, 6H, J = 7.5 Hz,); 13C NMR (125 MHz, CDCl3): δ 22.05,
28.44, 28.86, 33.59, 34.01, 39.25, 53.82, 67.36, 80.67, 127.06,
128.23, 129.75, 144.59, 155.74, 170.87, 177.82; HRMS-EI (m/z):
calcd for C32H38N2O5S [M + Na]+ 585.2399, found 585.2393.
+
177.22; HRMS-EI (m/z): calcd for C38H47N4O5 [M]+ 639.3541,
found 639.3528.
5-((R)-2-Amino-sulfono-propionamido)-pentanoic
acid
(6).
N-(6-(Diethylamino)-9-(2-(4-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-
oxohexanoyl)piperazine-1-carbonyl)phenyl)-3H-xanthen-3-ylidene)- Triethylsilane (4.58 mL, 28.6 mmol) and TFA (40.4 mL)
N-ethylethanaminium 2,2,2-trifluoroacetate (4). To an Argon
flushed flask cooled to °C containing (141 mg,
were added to a solution of acid 5 (8.075 g, 14.3 mmol) in
anhydrous DCM (40.4 mL) stirred under Argon at ambient
temperature for 1.25 h before the solvents were removed
in vacuo. The white residue was partitioned between Et2O and
H2O. The aqueous phase was removed in vacuo providing a
yellow oil. A solution of 30% H2O2 (22.6 mL) and formic acid
(203.4 mL) were allowed to stand together at ambient tempera-
ture for 1 h. The solution was used to dissolve the yellow oil, at
which point the mixture was stirred at ambient temperature
for 1.5 h. The solvent was removed in vacuo, and the white
solid was dissolved in water. The water was removed in vacuo
and the white solid was dried by lyophilization providing
3.38 g of 6 (81%): 1H NMR (500 MHz, D2O): δ 1.49–1.58 (m,
4H), 2.35 (t, 2H, J = 7.0 Hz), 3.21 (m, 2H), 3.31 (m, 1H), 3.39
(m, 1H), 4.30 (dd, 1H. J = 8.7, 4.1 Hz); 13C NMR (125 MHz,
D2O): δ 21.48, 27.42, 33.18, 39.29, 49.95, 50.26, 167.15, 178.57;
HRMS-EI (m/z): calcd for C8H16N2O6S [M + Na]+ 291.0627,
found 291.0609.
0
3
0.19 mm) and N-hydroxysuccinimide (430 mg, 3.74 mmol, 20
eq.) was added dry DCM (12 mL) followed by dicyclohexyl-
carbodiamide (386 mg, 1.87 mm, 10 eq.). The reaction was com-
plete by TLC analysis (Rf (product) = 0.7, Rf(3) = 0.4, 9 : 1 CH2Cl2–
MeOH) in 2.5 h at which time the solvent was removed
in vacuo. The resulting red solid was purified by reversed
phase HPLC (B 40% to 70% over 30 min, flow = 20 mL min−1
,
λ = 530 nm, Rt = 24 min) and after lyophilization provided
134 mg of 4 as a red powder (84%): 1H NMR (500 MHz,
CD3OD): δ 1.31 (t, 12H, J = 7.0), 1.67 (m, 2H), 1.74 (m, 2H),
2.40 (t, 2H, J = 7.1 Hz), 2.65 (m, 2H), 2.82 (s, 4H), 3.39 (m, 7H),
3.49 (m, 1H), 3.69 (q, 8H, J = 7.0 Hz), 6.97 (m, 2H), 7.08 (m,
2H), 7.29 (m, 2H), 7.53 (m, 1H), 7.74 (m, 1H), 7.78 (m, 2H); 13
NMR (125 MHz, CD3OD): δ 12.96, 25.37, 25.47, 26.19, 26.63,
31.40, 33.49, 34.85, 42.89, 46.12, 47.04, 97.50, 115.00, 115.38,
115.54, 119.20, 129.06, 131.43, 131.91, 132.44, 133.31, 136.65,
C
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(R)-3-((4-Carboxybutyl)amino)-2-(6-(4-(2-(6-(diethylamino)-3-
(diethyliminio)-3H-xanthen-9-yl)benzoyl)piperazin-1-yl)-6-oxohexan-
amido)-3-oxopropane-1-sulfonate (7). To an Argon flushed
flask containing 6 (424 mg, 1.58 mm) was added dry DMF
(15 mL) followed by NMM (0.34 mL, 3.16 mmol). A solution of
4 (134 mg, 0.158 mmol) in dry DCM (5 mL) was added drop-
wise to the solution and stirred. The reaction was complete by
TLC analysis (Rf (product) = 0.0, Rf (4) = 0.7, 9 : 1 CH2Cl2–MeOH)
after 23 h. Removal of the solvent by lyophilization gave rise to
a red solid which was purified by reverse phase HPLC (B 40%
to 70% over 30 min, flow = 20 mL min−1, λ = 530 nm, Rt =
10 min) and after lyophilization afforded 87 mg of 7 as a red
powder (62% yield): 1H NMR (300 MHz, CD3OD): δ 1.31 (t,
12H, J = 7.0 Hz), 1.58 (m, 8H), 2.31 (m, 6H), 3.17 (m, 4H), 3.38
(m, 7H), 3.54 (m, 1H), 3.69 (q, 8H, J = 7.0 Hz), 4.60 (m, 1H),
6.97 (s, 2H), 7.09 (m, 2H), 7.30 (d, 2H, J = 9.5 Hz), 7.51 (m, 1H),
7.76 (m, 3H); 13C NMR (125 MHz, CD3OD): δ 13.02, 23.40,
25.95, 26.11, 29.84, 33.60, 33.90, 34.60, 36.51, 40.27, 42.86,
43.33, 47.06, 52.74, 52.92, 97.47, 115.00, 115.71, 129.15,
131.39, 131.47, 131.87, 132.34, 133.35, 136.72, 157.34, 159.40,
169.67, 172.84, 174.24, 175.78, 177.26; HRMS-EI (m/z): calcd
for C46H60N6O10S [M + Na]+ 911.3984, found 911.3972.
(R)-2-(6-(4-(2-(6-(Diethylamino)-3-(diethyliminio)-3H-xanthen-9-
yl)benzoyl)piperazin-1-yl)-6-oxohexanamido)-3-((5-((2,5-dioxo-
pyrrolidin-1-yl)oxy)-5-oxopentyl)amino)-3-oxopropane-1-sulfonate
(8) (Scheme 8). To an Argon flushed flask containing 7 (38 mg,
43 mmol) and N-hydroxysuccinimide (104 mg, 0.9 mmol)
was added dry DMF (2 mL) followed by DCC (93 mg,
0.45 mmol, 10 eq.) and stirred. The reaction was complete by TLC
analysis (Rf(product) = 0.7, Rf(8) = 0.4, 3 : 1 CH2Cl2–MeOH) after 17 h
at which time the solvent was removed by lyophilization. The
resulting red solid was purified by reverse phase HPLC (B 40% to
70% over 30 min, flow = 4 mL min−1, λ = 530 nm, Rt = 12 min) and
after lyophilization afforded 38 mg of 8 as a red powder (91%
yield): 1H-NMR (300 MHz, CD3OD) δ 1.31 (t, 12H, J = 7.0 Hz), 1.60
(m, 6H), 1.71 (m, 2H), 2.27 (m, 2H), 2.35 (m, 2H), 2.64 (t, 2H, J =
7.0 Hz), 2.81 (s, 4H), 3.19 (m, 4H), 3.39 (m, 7H), 3.54 (m, 1H),
3.69 (m, 8H), 4.60 (m, 1H), 6.96 (s, 2H), 7.13 (m, 2H), 7.30 (d,
2H, J = 9.4 Hz), 7.51 (m, 1H), 7.77 (m, 3H); 13C-NMR (125 MHz,
CD3OD): δ 13.01, 23.09, 25.90, 26.12, 26.65, 29.46, 31.33, 33.64,
33.90, 34.85, 36.47, 39.93, 42.21, 42.91, 43.31, 46.22, 47.06,
52.69, 52.87, 97.46, 115.01, 115.72, 129.16, 131.48, 131.88,
132.34, 132.56, 133.36, 136.74, 157.35, 159.42, 160.20, 169.66,
170.33, 171.93, 172.93, 174.25, 175.78; HRMS-EI (m/z): calcd
C50H63N7O12S [M + 2Na]+2 515.7020, found 515.7041.
Scheme 8 Synthesis of Rhod-SH (1). (i) 6, NMM, DMF, 23 h, 62%, (ii)
DCC, NHS, DMF, 17 h, 91%, (iii) cysteamine hydrochloride, NMM, DMF,
22 h, 61%.
19 min) and after lyophilization provided 11 mg of 1 (61%
1
yield) as a red powder: H NMR (500 MHz, CD3OD): δ 1.31 (t,
12H, J = 6.8 Hz), 1.51 (m, 2H), 1.61 (m, 6H), 2.19 (m, 2H), 2.27
(m, 2H), 2.37 (m, 2H), 2.58 (m, 2H), 3.19 (m, 4H), 3.40 (m, 8H),
3.53 (m, 2H), 3.69 (m, 8H), 4.61 (m, 1H), 6.96 (s, 2H), 7.08 (m,
1H), 7.13 (m, 1H), 7.29 (d, 2H, J = 9.4 Hz), 7.52 (m, 1H), 7.73
(m, 1H), 7.78 (m, 2H); 13C NMR (125 MHz, CD3OD): δ 13.02,
24.35, 24.62, 25.93, 26.14, 29.79, 33.64, 33.90, 36.57, 36.72,
40.18, 42.23, 42.92, 43.35, 44.04, 46.22, 47.07, 52.78, 97.49,
115.03, 115.62, 115.73, 129.17, 131.39, 131.49, 131.88, 132.55,
133.37, 136.76, 157.39, 159.43, 169.71, 172.85, 174.26, 175.76,
176.24; HRMS-EI (m/z): calcd C48H65N7O9S2 [M + H]+ 948.4358,
found 948.4363.
(R)-2-(6-(4-(2-(6-(Diethylamino)-3-(diethyliminio)-3H-xanthen-9-
yl)benzoyl)piperazin-1-yl)-6-oxohexanamido)-3-((5-((2-mercap-
toethyl)amino)-5-oxopentyl)amino)-3-oxopropane-1-sulfonate
(Rhod-SH, 1). Argon was bubbled through dry DMF (1 mL) for
(iii) Optical characterization of Rhod-SH (1). Absorbance
20 min at which time NHS ester 8 (19 mg, 0.019 mmol) was and emission profiles for Rhod-SH (1), were determined using
added followed by cysteamine hydrochloride (3.2 mg, both a Varian Cary 6000i UV-VIS NIR Spectrophotometer and a
0.028 mmol). Then NMM (6 μL, 0.127 mmol) was added and Varian Cary Eclipse Fluorescence Spectrophotometer. Ethanol
the solution was stirred for 22 h under Argon at which time was used as the spectroscopic solvent, utilizing 1 cm cuvettes
the solvent was removed by lyophilization and the resulting for both absorbance and emission readings. Molecular extinc-
red solid was purified via reverse phase HPLC (30% B to 60% tion coefficient values were determined by finding the slope of
B over 30 min, 20 mL min−1 flow, λ = 530 nm, product Rt = the concentration versus absorbance at λmax (562 nm, 86 000
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M−1 cm−1). The quantum yield was determined in triplicate by The BSA-SS-Rhod solution was subjected to trypsinolysis (see
comparing 1 to Rhodamine B (510 nm excitation, QY = 70%).57 below).
Dilution series’ were made with a maximum absorbance of 0.1
(ii) Thiosulfonate switch labeling of BSA-SNO. To an
at 510 nm. The quantum yield was determined to be 48
amber vial containing a solution of BSA-SNO (22% SNO
0.1%. Fig. S10† shows the absorbance and emission profiles content, 28% free thiol, 100 µM total protein, pH 4.0 buffer,
for 1.
100 µL, 10 nmol) was added S-phenylthiosulfonylcysteine
(SPSC) (10 µL, 1 mM, pH 4.0 buffer, 10 nmol). After 1 h,
PhSO2Na (5 µL, 0.5 M, pH 4.0 buffer, 2.5 µmol, 250 eq.) was
added. After 30 min, Rhod-SH (1) (3 µL, 10 mM, pH 4.0 buffer,
30 nmol, 3 eq.) was added and after 10 min, TOF MS analysis
was performed (Fig. 3, green, bottom).
(iii) TOF MS analysis. Intact-protein mass spectra were col-
lected via HPLC-ESI on a Bruker microTOF. A Phenomenex
size-exclusion column (Phenomenex S2000, 300 × 4.6 mm) was
used in denaturing conditions to separate the proteins from
buffer and small molecules. The solvent consisted of 50%
water, 50% acetonitrile, with 0.1% formic acid, at a flow rate of
1.0 mL min−1: the protein eluted in a wide band at approxi-
mately 5 minutes.
(iv) Trypsinolysis and peptide analysis on BSA-SS-Rhod.
The BSA-SS-Rhod solution prepared and analyzed in Fig. 3
(blue, middle) was buffer exchanged with Sephadex G25 to
HEN (pH 7.5). To 80 µL of BSA-SS-Rhod (pH 7.5) in an amber
vial was added MMTS (2 µL, 20 mM in DMF, 40 nmol). Trypsin
(Trypsin Gold, Promega, 2 µL, 0.1 mg mL−1 in water) was
added and the solution was heated to 37 °C for 10 h in the
dark. The solution was centrifuged and the supernatant was
analyzed by reverse phase HPLC in tandem with MS and UV/
vis detection (Fig. 4). Before peptide analysis, TOF MS analysis
was performed using a Biosep column to ensure protein
digestion. Digested samples were eluted through an Agilent
Zorbax reverse phase column with 1.8 µm particles and
dimensions of 50 mm length and 2.1 mm diameter with an
Agilent 1290 HPLC system and detected by UV-Vis and mass
spectrometry. The column was maintained at 50 °C with a flow
rate of 500 µL min−1. Chromatography was as follows: solvent
consisted of H2O with 0.1% (v/v) formic acid for channel “A”
and acetonitrile with 0.1% formic acid for channel “B”.
Following column equilibration at 5% B, the sample was
injected via autosampler, and the column was flushed for
1.0 min to waste. From 1.0 min to the end of the run, the
column eluant was directed to the MS source. From 1.0 min to
7.0 min, the gradient was linearly ramped from 5% to 95%
B. From 7.0 to 9.0 min, the gradient was held at 95%, and
from 9.0 to 10.0 minutes the gradient was held at 5% for re-
equilibration. A UV detector was used in-line following the
column and prior to the mass spectrometer. The mass spectro-
Control reactions
(i) Reactions of PhSO2Na and Rhod-SH (1) on GSNO, GSH
and GSSG. GSNO was synthesized and characterized using a
previously developed method.38,41 All solutions were in pH 4.0
buffer (100 mM ammonium formate, 1 mM EDTA). GSNO
(20 μL, 1 mM) was added to PhSO2Na (50 μL, 0.1 M) and incu-
bated for 30 min at which time Rhod-SH (1) (4 μL, 5 mM) was
added. After 10 min, an aliquot was taken and analyzed by
reversed phase HPLC with absorbance detection at λmax of
Rhod-SH (1) (B 5% to 70% 0–20 min, 70% to 100%,
20–30 min, flow = 4 mL min−1, λ = 562 nm). Rhod-SH (1)
exhibited a retention time of 18.9 min. The reaction with
GSNO results in a new peak with a retention time of 17.3 min
(95%) while the peak at 18.9 min is drastically diminished.
HRMS analysis of the 17.3 min peak confirmed the product to
be the mixed disulfide Rhod-SSG (HRMS-EI (m/z): calcd
C58H81N10O15S3+ [M]+ 1254.5040 found 1254.5031.
(ii) Rhod-SH (1) control reactions with thiol blockers and
disulfides. Thiol blocking reactions were performed at pH 4.0
(100 mM HCO2NH4, 1 mM EDTA) between 0.27 mM Rhod-SH
(1) and 0.27 mM thiol blocker (SPTC, MMTS, NEM, IAM,
GSSG, or (Gly-Cys)2). The reactions were monitored by taking
aliquots at given time points and analysing by reverse phase
HPLC (B 5% to 70% 0–20 min, 70% to 100%, 20–30 min, flow
= 4 mL min−1, λ = 562 nm). The products were further con-
firmed by HRMS: Rhod-SSCys: Rt = 17.7 min; HRMS (m/z): calc
for C51H70N8O11S3 [M + H]+ 1067.4399, found 1067.4375;
Rhod-SSMe: Rt = 20.2 min; HRMS (m/z): calc for C49H67N7O9S3
[M + H]+ 994.4235, found 994.4200; Rhod-S-NEM: Rt
=
19.5 min; HRMS (m/z): calc for C54H72N8O11S2 [M + H]+
1073.4835, found 1073.4808.
(iii) SPSC control reactions. SPSC (2 mM) in the following
buffers were analyzed by reverse phase HPLC (B 5% to 70%
0–20 min, 70% to 100%, 20–30 min, flow = 4 mL min−1, λ =
265 nm) after 1 h: pH 4.0 and 4.8: 100 mM ammonium
formate, 1 mM EDTA; pH 5.5, 6.0: 100 mM MES, 1 mM EDTA;
pH 7.0: 100 mM HEPES, 1 mM EDTA.
Protein analysis
(i) Reactions of BSA-SNO and PhSO2Na and Rhod-SH meter used was a Bruker microTOF with ESI source: resolution
(1). To an amber vial containing a solution of BSA-SNO (44% is approximately 10 000 and accuracy is 10 ppm (Table S1†).
SNO content, 100 µM total protein, 100 µL, 10 nmol, pH 4.0 Source parameters were as follows: drying gas 5.5 L min−1
,
buffer) was added PhSO2Na (5 µL, 0.5 M, 2.5 µmol, nebulizer 4.0 bar, capillary voltage 4500 V, capillary exit
pH 4.0 buffer, 250 eq.) followed by Rhod-SH (1) (2 µL, 10 mM, 100 V. Spectra were collected in positive mode from 50 to
pH 4.0 buffer, 20 nmol, 2 eq.). After 30 min, TOF MS analysis 1600 m/z at a rate of 1 Hz. The compounds of interest were
was performed (Fig. 3, blue, middle). A control reaction detected using a combination of UV detection at 565 nm
was performed under identical conditions employing and MS ion intensities. Compound identification was
commercial BSA and analyzed by TOF MS (Fig. 3, red, top). confirmed via accurate-mass isotope distribution matches
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Paper
compared to calculated values based upon theoretical for- Hepes, 200 mM NaCl, 2 mM EDTA, 1 mM DTT, pH 7.0), col-
mulas (Table S1†). lecting 1 mL fractions. Fractions exhibiting >95% purity as
(v) 1D SDS-PAGE nonreducing gels on BSA-SS-Rhod. A judged by SDS-PAGE were combined, concentrated, and stored
solution of BSA-SNO (36% SNO content, pH 4.0 buffer) was in Buffer G at −80 °C. Molecular masses determined by ESI
diluted with a solution of BSA to generate a dilution series MS or MALDI MS for NmlR and AdhR were 13 462 D (13 463.4
with identical amounts of total protein. To each vial was
D expected; residues 1–117) and 14 438 D (16 415.8 D
added SPSC (8 μL, 100 μM) and after 1 h, PhSO2Na (5 µL, 0.5 expected), respectively. Tandem MS/MS of purified AdhR
M, pH 4.0 buffer, 2.5 µmol) was added. After 30 min, Rhod-SH reveals a C-terminal tryptic peptide R|ENEAKL123 giving an
(1) (4 µL, 0.5 mM, pH 4.0 buffer, 2 nmol) was added and after expected molecular mass of 14 438.6 D (14 438 D observed;
10 min, MMTS (4 µL, 20 mM, pH 4.0 buffer, 80 nmol) was residues 1–123). We designate this C-terminally truncated
added. 15 μL of the final reaction solutions were added to 5 μL form of AdhR as AdhR*-SH. This C-terminal tail is not found
non-reducing sample buffer. For each lane, 15 μL were added in Spn NmlR (117 residues) and both reduced AdhR*-SH and
and the gel was run with Tris-HEPES-SDS buffer pH 8.0 at 100 NmlR bind to a 30-base pair nml operator promoter-containing
V through a 4–20% Precise Protein Gel from Thermo Scientific DNA (NmlO) with high affinity (Fig. S5†). NmlR and AdhR*-SH
using a Biorad Power Pac HC and mini protean tetracell. Pre- are 46% identical over 117 residues and both contain a single
cision plus all blue standards from Biorad were used as mole- Cys, Cys52. Following gel filtration, NmlR and AdhR*-SH
cular weight markers. Gels were washed for 1 h in distilled stocks were incubated with 5 mM TCEP, and then dialyzed
water and fluorescence images were acquired on a Typhoon anaerobically into a degassed buffer containing 10 mM Hepes,
9210 (GE Healthcare) scanning at 400 PMT, 100 micron, with 200 mM NaCl, pH 7.0. The reduced thiol content (1 expected)
excitation and emission filters at 532/580 nm. Image J was was verified using a DTNB assay carried out as previously
used to determine relative fluorescence intensities. After described60 and was found to be greater than 80% in all
imaging, the gel was stained with coomassie.
preparations.
(vi) Bacterial expression and purification of S. pneumoniae
(vii) NmlR and AdhR*-SH C52 reactivity experiments.
D39 NmlR.53 and B. subtilis AdhR.54 The nmlR gene was GSNO was either purchased (Axxora) or synthesized using a
amplified from S. pneumoniae D39 genomic DNA (locus tag procedure also used to prepare S-nitrosylated cysteine (CysNO).
SPD_1637) using primers 5′AGGTC
̲A̲T̲A̲T̲G̲AATATTAAAT- Briefly, a 1.1 : 1 ratio of sodium nitrite to cysteine or gluta-
CTGCCAGTG-3′ and 5′-CCG̲G̲A̲T̲C̲C̲CATTTAAAATTTTCCTTC-3′ thione was mixed in 1 M HCl in darkness and under
containing NdeI and BamHI restriction sites (underlined). The argon.41,61 Following a 15 min reaction, a 10-fold volumetric
adhR gene was amplified from B. subtilis str. 168 genomic DNA excess of ice cold acetone was added to the solution to precipi-
using primers 5′GGGC
̲A̲T̲A̲T̲G̲AATATTGCTCAGGTGGCAAAG-3′ tate CysNO or GSNO. The precipitate was filtered and then
and GGGGGG CTACCTTCT-TTGCTCCACTGATCC also washed with water, acetone, and finally anhydrous diethyl
̲G̲A̲T̲C̲C̲
containing NdeI and BamHI restriction sites. Following double ether. The resulting pink powder was allowed to dry overnight
digestion and ligation into pET-3a, all plasmids were con- in darkness. The powder was then resuspended in either 20%
firmed to contain the desired gene by DNA sequencing. Bac- DMSO or reaction buffer and stored at −80 °C until use. GSNO
terial expression of NmlR and AdhR proteins was carried out and CysNO stock concentrations were estimated using the ε340
in E.coli BL21(DE3)/pLysS essentially as described previously.58 nm value for GSNO of 1000 cm−1 M−1 61
GSNO and CysNO
.
Briefly, cell pellets were resuspended in a lysis buffer contain- stocks were aliquoted into 100–200 μL aliquots into either 20%
ing 25 mM Tris, 1 M NaCl, 2 mM DTT, 2 mM EDTA, pH 8.0, DMSO or reaction buffer and stored at −80 °C until used. C52
lysed via sonication, nucleic acids precipitated by polyethylene- reactivity experiments were conducted by adding a 10-fold
imine (PEI) addition, followed by protein precipitation of (GSNO) or 15-fold (CysSNO) molar excess of NO donor to
the PEI supernatant by ammonium sulfate essentially as 1 mM (AdhR*-SH) or 0.1 mM (NmlR) in 0.4 mL (AdhR*-SH) or
described previously.59 Following ammonium sulfate precipi- 4 mL (NmlR), respectively, in Buffer R (25 mM Hepes, 400 mM
tation, the pellet was resuspended and dialyzed into Buffer A NaCl, 1 mM EDTA) containing 0.1 mM BCS, the latter used to
(25 mM Tris-HCl, 2 mM EDTA, 1 mM DTT, pH 8.0) sup- chelate any Cu(I) formed during the reaction.61 Reactions were
plemented with 50 mM NaCl, loaded onto an in-house packed allowed to proceed for 1 h at room temperature anaerobically
Q (quaternary ethylamine)-column equilibrated with the same with rotational mixing on a LabQuake. A HiPrep 26/10 desalt-
buffer and eluted by running a linear gradient in Buffer A plus ing column was run in Buffer R and the absorbance at
750 mM NaCl (4 mL fractions were collected). The highest 280 nm, 338 nm, and 575 nm monitored in darkness
purity fractions as assessed by SDS-PAGE were pooled, concen- (Fig. S6A†). At the conclusion of the run, NmlR-SNO and
trated, and dialyzed again into Buffer A/50 mM NaCl, loaded AdhR*-SNO fractions were concentrated, and the UV-Vis absor-
onto a MonoQ column equilibrated with the same buffer, and bance spectrum measured (Fig. S6B†). The percentage of S-NO
eluted with Buffer A/750 mM NaCl over a 25–30 column reacted thiol was estimated by the 280 nm/340 nm ratio (see
volume gradient, collecting 2 mL fractions. Highest purity frac- main text). An NmlR prep reacted with GSNO was additionally
tions were again pooled, concentrated, and further purified by injected onto a μRPC C2/C18 column (GE Life Sciences)
gel filtration chromatography in either a G75 or G200 column reverse phase column with buffers of 0.1% formic acid (Buffer
16/60 (GE Life Sciences), equilibrated with Buffer G (25 mM A) and 0.1% formic acid, 60% acetonitrile (Buffer B), with
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reduced, S-glutathionylated and S-nitrosylated NmlRs held at 20% for re-equilibration. The higher percentage of “B”
(NmlR-SNO) eluted with a very shallow gradient (Fig. S7†). All in this experiment was tailored for the specific retention time
C52 adducts of NmlR and AdhR*-SH were routinely analyzed of dye-labeled peptides. A UV detector was used in-line follow-
by ESI/MS equipped with either a C4 or C8 column with ing the column and prior to the mass spectrometer. The mass
buffers of 0.1% formic acid (Buffer A) and 90% acetonitrile, spectrometer used was an Agilent 6538 QToF with ESI source:
0.1% formic acid (Buffer B), running either a 7 minute or resolution is approximately 20 000 and accuracy is 1 ppm.
20 minute gradient from 10% to 90% Buffer B.
Source parameters were as follows: drying gas 8.0 L min−1
,
(viii) AdhR*-SNO reduction with ascorbate. To an amber nebulizer 55 psi, capillary voltage 3500 V, and capillary exit
vial containing AdhR*-SNO (53% SNO, 47% free thiol, 25 µM 120 V. Spectra were collected in positive mode from 50 to 1700
total protein, HEN, pH 7.5, 100 µL, 3 nmol) was added ascor- m/z at a rate of 1 Hz. The compounds of interest were detected
bate (1 M, 2 µL, 2 µmol). After 1 h, the solution was analyzed using a combination of UV detection at 565 nm and MS ion
by TOF MS (Fig. 6).
intensities. Compound identification was confirmed via accu-
(ix) Thiosulfonate switch labelling of AdhR*-SNO. To an rate-mass isotope distribution matches compared to calculated
amber vial containing AdhR*-SNO (53% SNO, 47% free thiol, values based upon theoretical formulas (Table S1†). In a paral-
25 µM total protein, pH 4.0 buffer, 8 µL, 0.2 nmol) was added lel experiment, a mixture of AdhR*-SNO (47% SNO, 53% SH)
SPSC (4 µL, 130 µM, pH 4.0 buffer, 0.5 nmol). After 1 h, and commercial BSA (50 µM total protein each, 100 µL total,
PhSO2Na (5 µL, 0.25 M, pH 4.0 buffer, 1.25 µmol) was added pH 4.0 buffer) was subjected to the entire protocol and
and after 30 min, Rhod-SH (1) (4 µL, 0.5 mM, pH 4.0 buffer, analysis.
2 nmol) was added. After 10 min, the solution was analyzed by
TOF MS (Fig. 7, red, top). AdhR*-SH was subjected to the same subjected to labeling by the thiosulfonate switch. To an
procedure and analysis (Fig. 7, blue, bottom). amber vial was added BSA-SNO (52% SNO, 25 µM total
(xi) 1D SDS-PAGE nonreducing gels on protein mixtures
(x) Thiosulfonate switch labelling of protein mixtures. protein, pH 4.0 buffer, 10 µL, 0.25 nmol) and AdhR*-SH
BSA-SNO (52% SNO, 0% free thiol) and AdhR*-SH were mixed (25 µM total protein, 10 µL, 0.25 nmol) followed by 15 µL
in an amber vial to 50 µM total protein each (100 µL total, pH pH 4.0 buffer. SPSC (4 µL, 0.1 mM, pH 4.0 buffer, 0.4 nmol)
4.0 buffer, 5 nmol each). To the protein mixture was added was added. After 1 h, PhSO2Na (5 µL, 0.5 M, pH 4.0 buffer,
SPSC (10 µL, 1 mM, pH 4.0 buffer, 10 nmol). After 1 h, 2.5 µmol) was added and after 30 min, Rhod-SH (1) (2 µL,
PhSO2Na (5 µL, 0.5 M, pH 4.0 buffer, 2.5 µmol) was added and 0.5 mM, pH 4.0 buffer, 1 nmol) was added. After 10 min,
after 30 min, Rhod-SH (1) (2 µL, 10 mM, pH 4.0 buffer, MMTS (4 µL, 20 mM, pH 4.0 buffer, 80 nmol) was added
20 nmol) was added. After 10 min, the solution was then ana- followed by non-reducing sample buffer. Image J was used to
lyzed by TOF MS (Fig. 8A). 80 µL of the protein mixture was determine relative fluorescence intensities (Fig. 8B). In a paral-
precipitated in cold acetone (240 µL) and kept at −20 °C for lel experiment, AdhR*-SNO (47% SNO, 53% SH) and commer-
1 h. After centrifugation, the acetone was removed and the cial BSA was subjected to the entire protocol (Fig. 9B).
resulting pellet was dissolved in 20 mM Tris (1% deoxycholate,
pH 7.7, 80 µL). Trypsin (5 µL, 1 mg mL−1, Sigma-Aldrich) was
added and the vial was heated to 37 °C for 24 h. After cooling
to room temperature, formic acid (1 M) was added until a pH
Acknowledgements
of 3.0 was reached and the mixture was centrifuged to separate P.A.G., D.J.S., and E.A.D. receive support from the National
the precipitated deoxycholate. The supernatant was analyzed Institutes of Health (CoBRE 5P20RR024237-04 and
by reverse phase HPLC in tandem with MS and UV/vis detec- P20GM104935-05). D.P.G. receives support from NIH grants
tion. Detection of peptide products for the mixing experiments GM042569 and GM097225. We would like to acknowledge
(Fig. S8†) was performed in the same manner as the BSA-SS-R- David Fouchard for synthetic support. The Mass Spectrometry,
hod digest, except with longer column and chromatography to Proteomics and Metabolomics core facility receives support
ensure accurate detection of multiple peptides. An Agilent from the Murdock Charitable Trust and INBRE MT Grant No.
Zorbax C18 column with 1.8 µm particles and dimensions of P20 RR-16455-08.
100 mm length and 2.1 mm diameter was used for separation,
with an Agilent 1290 HPLC system. The column was main-
tained at 50 °C with a flow rate of 600 µL min−1. Chromato-
graphy was as follows: solvent consisted of H2O with 0.1% (v/v)
Notes and references
formic acid for channel “A” and acetonitrile with 0.1% formic
acid for channel “B”. Following column equilibration at 20%
B, the sample was injected via autosampler, and the column
was flushed for 1.0 min to waste. From 1.0 min to the end of
the run, the column eluant was directed to the MS source.
From 1.0 min to 26.0 min, the gradient was linearly ramped
from 20% to 45% B. From 26.0 to 28.0 min, the gradient was
held at 95%, and from 28.0 to 30.0 minutes the gradient was
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