S-Nitrosoglutathione Reductase Underlies the Dysfunctional Relaxation to Nitric Oxide in Preterm Labor

Tocolytics show limited efficacy to prevent preterm delivery. In uterine smooth muscle cGMP accumulation following addition of nitric oxide (NO) has little effect on relaxation suggesting a role for protein S-nitrosation. In human myometrial tissues from women in labor at term (TL), or spontaneously in labor preterm (sPTL), direct stimulation of soluble guanylyl cyclase (sGC) fails to relax myometrium, while the same treatment relaxes vascular smooth muscle completely. Unlike term myometrium, effects of NO are not only blunted in sPTL, but global protein S-nitrosation is also diminished, suggesting a dysfunctional response to NO-mediated protein S-nitrosation. Examination of the enzymatic regulator of endogenous S-nitrosoglutathione availability, S-nitrosoglutathione reductase, reveals increased expression of the reductase in preterm myometrium associated with decreased total protein S-nitrosation. Blockade of S-nitrosoglutathione reductase relaxes sPTL tissue. Addition of NO donor to the actin motility assay attenuates force. Failure of sGC activation to mediate relaxation in sPTL tissues, together with the ability of NO to relax TL, but not sPTL myometrium, suggests a unique pathway for NO-mediated relaxation in myometrium. Our results suggest that examining the action of S-nitrosation on critical contraction associated proteins central to the regulation of uterine smooth muscle contraction can reveal new tocolytic targets.


Results
Activation SGC Fails to Relax Human Myometrium. In order to examine the role of cyclic GMP in uterine smooth muscle relaxation in the absence of haem-dependent activation of soluble guanylyl cyclase by NO, we have assessed the effect of direct stimulation of myometrial tissue by the haem-independent agonist BAY58-2667 (BAY58, cinaciguat) on uterine relaxation. Cinaciguat has been evaluated for haemodynamic benefit in patients with acute decompensated heart failure (ADHF) 24 . Cinaciguat is well known to stimulate cyclic GMP accumulation 25 , but fails to relax human term myometrium despite the use of concentrations from 0.1 to 10 µM administered in an increasing cumulative dose at 10 min intervals after OT stimulation (8 nM, Fig. 1a). Treatment of rat aorta with cinaciguat undertaken as a comparative control, resulted in immediate relaxation at 1 µM, consistent with the known actions of cyclic GMP in vascular smooth muscle (Fig. 1b). The EC 50 for cinaciguat activation of the sGC is 6.4 nM and its effect to relax vascular smooth muscle is sub-μM 26 . Experiments repeated with term myometrium (Fig. 1c) from multiple human (n = 4) and guinea pig (n = 3) donors (Fig. 1d) confirmed the failure of cinaciguat to relax the tissue (one-way ANOVA: human p = 0.29; guinea pig p = 0.92).

NO-Mediated Relaxation is Blunted in Preterm Human Myometrium. Patients who enter labor
spontaneously preterm without infection have a blunted relaxation response to NO (Fig. 2), suggesting that the mechanism of NO action may be involved in the pathophysiology of preterm labor. Addition of NO donor in increasing concentrations to term tissues relaxed both OT-treated (Fig. 2a) and spontaneously active tissues (Fig. 2b). Treatment of sPTL tissues in an identical fashion revealed that the NO-donor cysteine-NO (CysNO) could not relax OT-treated tissues (Fig. 2a) and the relaxation of spontaneous contractions was blunted (Fig. 2b). This is the first study to measure the ability of NO to relax spontaneous preterm vs. term pregnant human myometrium.

Expression of S-Nitrosoglutathione Reductase (GSNOR) in Myometrium. Blunted relaxation to
the NO donor Cys-NO suggested the possibility that NO availability to S-nitrosate contraction associated proteins in sPTL tissue was somehow limited. Glutathione is the major thiol in mammalian cells (up to 10 mM) 27 . S-nitrosoglutathione (GSNO) is the likely form in which NO resides in the cell to trans-nitrosate proteins 28 . The metabolism of GSNO is accomplished by the Class-III alcohol dehydrogenase (ADH5) 23,29 . The principal substrate for ADH5 is GSNO. The enzyme, renamed S-nitrosoglutathione reductase (GSNOR), utilizes NADH to SCIeNtIFIC REPORTS | (2018) 8:5614 | DOI: 10.1038/s41598-018-23371-w carry out a 2 e− reduction of GSNO to generate glutathione sulfinamide 30 . Using a Wes ® Simple Western ™ assay we determined that GSNOR protein expression increased in sPTL myometrium compared to term laboring tissue (p = 0.04) and term non-laboring (p = 0.02), and it also increased in term laboring women compared to those not in labor (p = 0.007), as determined by a Dunnett's Multiple Comparisons test (Fig. 3a). In order to address the possibility that differences seen in sPTL were the result of gestational length timing rather than a pathological feature of sPTL, we explored GSNOR expression in guinea pig pregnancy. The pattern of GSNOR expression during gestation cannot be investigated over time in an individual pregnant patient. Because guinea pigs, like women and unlike mice, experience cyclic GMP independence of NO relaxation 19 and do not undergo progesterone withdrawal 31 , we examined the changes in GSNOR expression during normal guinea pig gestation. GSNOR expression in guinea pigs is biphasic, dropping during gestation and returning toward pre-pregnancy levels at term (Fig. 3b), and was significantly lower than the non-pregnant (NP) guinea pig by gestational days 40-45 (p = 0.0005), 50-55 (p = 0.002), and 60-65 (p = 0.005), as determined by one-way ANOVA followed by Dunnett's multiple comparisons test. Immunohistochemical examination of GSNOR expression in myometrial cells (PHUSMC) in culture, as well as myometrial tissue from term (TL) and preterm (sPTL) laboring patients (Fig. 3c), is consistent with measurements of protein. Decreased expression of GSNOR is expected to result in increased GSNO levels to promote critical S-nitrosations that are consistent with quiescence, while increased GSNOR expression, as with sPTL, would serve to lower the availability of GSNO and thus lower critical S-nitrosations that govern uterine quiescence. No response was seen to addition of BAY58-2667. Addition of the NO-donor at the end of the experiment confirmed the ability of the tissue to relax (not shown). (b) Rat aorta was hung in tissue baths with Krebs buffer at 37 °C with continuous oxygenation and allowed to equilibrate for 60 min in the absence of drug. Addition of phenylephrine (1 µM) produced immediate contractions (black ----) that could be relaxed (blue ) by addition of BAY58-2667 (10 µM). (c) The failure of tissues to relax to BAY58-2667 was repeatable in both human ( n = 4) and guinea pig ( n = 3) where control addition of GSNO at 300 μM relaxed spontaneous contractions. Data in (c) and (d) are quantified as AUC over 10 min.
Blockade of GSNOR Activity Relaxes Term Non-Laboring (TNL) Myometrium. In order to determine if GSNOR actively contributes to myometrial quiescence, we inhibited the enzyme with N6022, a potent and selective inhibitor of GSNOR. Ex vivo organ bath experiments using TNL myometrium revealed that the addition of N6022 relaxes the term non-laboring myometrium (Fig. 5a) whether quantified as peak force or area under the curve (AUC) that represents force over time. The effect of GSNOR inhibition is dose-dependent (Fig. 5b). While the effect of N6022 is modest (~15-20% relaxation), it is notable that GSNOR inhibition affects force, and that force reduction may be consistent with S-nitrosations lowering the threshold for contractions until term. Reduction in both peak (p = 0.005) force and area under the curve (p = 0.013) is consistent with S-nitrosation differences that favor relaxation.

The Effect of GSNO on Protein S-Nitrosation in Human Myometrium. Total protein S-nitrosations
were measured in GSNO treated term and sPTL myometrium using LC/MS/MS. S-nitrosated peptides were quantified and expression levels of 110 S-nitrosated proteins were quantified using normalized spectral counts and AUC measures of extracted ion chromatograms. We employed both quantification measures because spectral In tissues from women in labor spontaneously preterm Cys-NO relaxation was insignificant ( ) compared to relaxation in term tissue (p = 0.6). Cys-NO relaxed term laboring myometrium contracting spontaneously ( ) while the relaxation seen in tissues from women in labor spontaneously preterm was blunted ( , 26% vs. 84% relaxation) and the apparent IC50 was right-shifted 10 fold. Data are mean ± SEM of triplicate determinations in 5 patients in each pregnancy state.
SCIeNtIFIC REPORTS | (2018) 8:5614 | DOI:10.1038/s41598-018-23371-w counting, while using dynamic exclusion, limits the quantitative capability to a relative measurement between states and we wanted to verify that the number of MS2 events per peptide were indicative of the amount of peptide that was measured by AUC of the MS1 chromatograms. ANOVA demonstrated that proteins exhibited statistically significant differences between TL and sPTL tissues (Fig. 6) specified by an F statistic P value of P < 0.05 (Fig. 6). These proteins had log 2 fold changes of at least ±1 in preterm laboring patients compared with term laboring patients. Stringent controls were performed to avoid false positive identification of non-S-nitrosated cysteines that could have been mislabeled during the experimental procedures. These controls are standard when performing the biotin switch procedure and include removal of GSNO and/or ascorbate during the biotin switch. A small number of proteins were shown to be constitutively S-nitrosated and were labeled without the addition of GSNO. This is a common and expected result. Removal of ascorbate during the biotin switch removed any signal that was seen when ascorbate was present. Streptavidin purification of biotin switched proteins removes any signal from naturally biotinylated proteins, and these are therefore not present in our analysis. Streptavidin purification and LC/MS/MS analysis of ascorbate negative samples were shown to only contain the contaminating keratin proteins, trypsin and traces of serum albumin. Therefore, our ascorbate-positive sample identification contains almost no nonspecific binding proteins.

GSNO Alters Actin-Myosin Motility.
In order to examine the functional effect of protein S-nitrosation in human myometrium, myosin-dependent actin dynamics were measured as translocation, or sliding, of actin filaments over smooth muscle myosin bound to a coverslip. The assay permits determination of the velocity of the interaction as a measure of cross-bridge cycling. Addition of GSNO to the motility assay results in a reduction in velocity consistent with an effect of GSNO to S-nitrosate one or more constituents in the assay, specifically smooth muscle myosin, and/or actin, both of which contain numerous cysteines (Fig. 7). At myosin densities of 25, 50, 100, 200, and 400 (µg/ml), the addition of 300 µM GSNO significantly decreased actin velocity P < 0.05. The result is consistent with an effect of GSNO to relax the smooth muscle and suggests a mechanism for S-nitrosation of contraction associated proteins underling the non-cyclic GMP action of NO.

Discussion
Despite decades of research, tocolytics do not reliably extend pregnancy beyond 48 hours. Approaches to manage sPTL are employed without clear evidence of benefit for acute or maintenance tocolysis 34,35 , and no tocolytic currently in use is without potential adverse effects 36 . It is imperative that new approaches to prevent preterm  contractions of the uterus be found. An intriguing observation in uterine smooth muscle is its ability to relax independently of sGC-mediated cyclic GMP accumulation when exposed to NO 12,19,20,37 . Here we address three questions: (1) How does sPTL myometrium respond to NO; (2) what alternative pathways might NO act upon to mediate relaxation in the myometrium; and (3), is it possible that the dysregulation of GSNOR, an important modulator of S-nitrosations in the myometrium, is responsible for the decreased level of protein S-nitrosation measured in sPTL tissues, and does GSNOR serve as a contributing factor to the blunted relaxation response to NO in spontaneous preterm labor?
Our lab has previously determined that the inhibition of sGC by agents that prevent cyclic GMP accumulation do not prevent NO-mediated relaxation of the human term myometrium 15 . Soluble GC converts GTP to cyclic GMP to activate its cognate kinase, PKG, whose targets include myosin phosphatase, which dephosphorylates the regulatory light chain 38 . We do not posit that cyclic GMP has no role in myometrium, indeed, we have shown that accumulation of cyclic GMP following activation of the particulate guanylyl cyclase does relax the uterus 17 . These results suggest compartmentation of second messenger signaling in myometrium, a finding first established over three decades ago in cardiac myocytes 39 . This signaling exception in myometrium represents a unique opportunity to discover a new approach to tocolytic development. The failure of NO-induced sGC-mediated cyclic GMP accumulation to relax myometrial smooth muscle was unexpected. The assumption that cyclic GMP  measurements are sufficient to make the case is refuted by evidence establishing that blockade of cyclic GMP accumulation does not prevent the relaxation. Direct evidence for the failure of cyclic GMP to signal myometrial quiescence is further established by our finding that the activation of sGC by the haem-independent agonist cinaciguat does not promote relaxation of the myometrium (Fig. 1a,c,d), despite its robust capability to do so in other smooth muscles (Fig. 1b). We propose that NO drives an alternative pathway in myometrial smooth muscle integral to contraction/relaxation cycling.
One such pathway that may impact smooth muscle relaxation is protein S-nitrosation. Not only is this posttranslational modification important to cellular homeostasis, its dysregulation can drive disease states 23 and smooth muscle inflammation 40,41 . It is generally accepted that pregnancy is, to some degree, an inflammatory condition, whether due to maternal infection or not 42,43 , and we suggest that dysregulation of S-nitrosation of critical contractile proteins underlies sPTL both because S-nitrosation impacts contractile protein regulation as we show, and because S-nitrosation is a principal redox regulator 44 that may limit inflammatory mechanisms until term.
We have shown that many proteins critical to the uterine contraction/relaxation cycle are differentially S-nitrosated in in preterm laboring myometrium as compared to term laboring, and term non-laboring, tissue 45 . We investigated the functional relevance of protein S-nitrosation by examining the effects of GSNO addition on ATP-ase activity and acto/myosin binding dynamics (Fig. 7). The presence of GSNO in the actin motility assay depresses ATP-ase activity, and by extension, cross-bridge cycling. Furthermore, as suggested by a decrease in the slope of actin velocity at low myosin concentrations, we can infer that the acto-myosin "latch phase" time is likely lengthened. The notion that S-nitrosation can impact contractile mechanisms has been suggested by studies in skeletal muscle 46,47 . Both of these contributions were made examining skeletal muscle, which differs markedly from uterine smooth muscle. The motility assay data presented here are compelling because they employ purified smooth muscle myosin, and because they support an effect of GSNO to promote relaxation through S-nitrosation in the absence of cyclic GMP elevation.
Protein S-nitrosation is an important regulator to human health and disease 48 , and the level of S-nitrosation in the cell is highly dependent on the availability of intracellular GSNO 49 . As GSNO concentrations increase, so do levels of total protein S-nitrosation. Because GSNOR ( −/− ) mice have increased cellular levels of GSNO and SNO-proteins 50 , it is likely that GSNO and some S-NO-proteins are in equilibrium governed by Cys-to-Cys trans-nitrosation, and GSNOR de-nitrosation. We have found that women who undergo sPTL show decreased protein S-nitrosation (Fig. 6), and exhibit higher cytosolic levels and activity of GSNOR (Figs 3, 4), an enzyme that metabolizes GSNO to GSSG (Fig. 4b). Taken together with our finding that women who undergo sPTL present a blunted response to NO-mediated relaxation (Fig. 2), we conclude that GSNOR may be responsible for atypical myometrial activity in some women who undergo sPTL. We do not posit that the dysfunction of sPTL tissues to NO is a matter of availability per se. Indeed, no concentration of NO donor overcomes the dysfunction we demonstrate. What is likely is that some specific S-nitrosation differences are the result of regulation by GSNO metabolism. This conclusion is supported by or demonstration of both up and down S-nitrosation difference seen in sPTL tissues 45 .
The knowledge that GSNOR regulates GSNO availability, and that GSNOR is upregulated in sPTL myometrium, makes it an attractive therapeutic target. N6022, a potent and selective inhibitor of GSNOR, is well tolerated in humans and has already been tested in clinical trials as an airway smooth muscle relaxing agent in asthmatics, and as a treatment for cystic fibrosis 51 . Here we demonstrate its efficacy, and entertain its use as a tocolytic (Fig. 5). In organ bath experiments N6022 significantly decreased the peak force of contraction and AUC of term myometrium. Due to the drug's modest bioavailability (~10%) 52 , its potential as a marketable tocolytic remains in question. However, numerous derivatives of N6022 have been identified 53 as well as other compounds targeting GSNOR 40 that may prove useful as tocolytic agents. Beyond GSNOR, there are other NO-mediators in the cell such as the thioredoxin system 54 and carbonyl reductase 27 that may also prove attractive as therapeutic targets to increase the availability of critical protein S-nitrosations in the cell.
Ultimately, the underlying cause(s) of sPTL remain unknown. Here we provide a novel mechanism that may provide insight into the unique relaxation pathway in the myometrium. Our finding that GSNOR, and by extension protein S-nitrosation, are dysregulated in the myometrium of women undergoing sPTL affords an opportunity to investigate a new class of drugs as tocolytics.

Tissue collection. Human tissue collection and research was approved by the University of Nevada Biomedical
Review Committee for the protection of human subjects. Human uterine biopsies were obtained with written informed-consent from mothers with singleton pregnancies undergoing Cesarean section without known infection or rupture of membranes. All experiments were performed in accordance with the NIH guidance on the use of human tissues in research. The absence of maternal infection was determined by the obstetrical team at the time of surgery based on the appearance of placenta and membranes. The possibility of subclinical infection in patients delivering preterm is not ruled out. Exclusion criteria include age < 18 years, any history of drug abuse, co-morbid diagnoses such as HIV infection or AIDS, hepatitis C infection, uncontrolled diabetes, renal disease, preeclampsia, IUGR, placenta previa and any use of steroids other than betamethasone including topical use during pregnancy. Tissues were transported to the laboratory immediately in cold Krebs buffer containing: NaCl (118 mM), KCl (4.75 mM), CaCl 2 (2.5 mM), KH 2 PO 4 (1.2 mM), NaHCO 3 (25 mM), MgCl 2 (1.2 mM), dextrose (20 mM), and adjusted to pH 7.4. Tissues were microdissected under magnification to isolate smooth muscle, employed in contractile experiments or snap frozen in liquid nitrogen, and stored at -150 °C. The average age for patients in the pregnant laboring group was 28.9 ± 5.6 yr. and in the preterm laboring group 30 Animal studies were approved by the University Institutional Animal Care and Use Committee. All experiments were conducted in accordance with the NIH guidelines for the use of vertebrate animals in research. Dunkin-Hartley Guinea pigs (Elm Hill, Chelmsford, MA) were purchased as either virgin juveniles (300-350 g) and bred on site, or as timed-pregnancies (30-35d). Non-pregnant guinea pigs were estrogen primed (3 mg/kg β-estradiol) 48-hours prior to tissue collection to ensure alignment of estrous cycles. Virgin female guinea pigs, and timed-pregnant animals, were sacrificed under isoflurane anesthesia. Uterine tissue was dissected and used immediately as previously described 55 . Contractile studies. Strips of myometrium (~0.5 × 15 mM) were clip-mounted by silk thread, attached to a force transducer, and isometrically stretched to an initial tension of 1.2 × tissue length in an organ bath (WPI, Sarasota, FL) containing Krebs buffer. Tissues were maintained at 37 °C and gently bubbled with balanced oxygen (95% O 2 , 5% CO 2 ). Tissues were then challenged with KCL (60 mM replacing NaCl) for 3 min, followed by washout, then allowed to equilibrate for 1 hr. during which time regular spontaneous contractions were seen. Only tissues that responded to KCL-challenge were employed in experiments. Under some conditions, tissues were further challenged with oxytocin (OT, 8 or 100 nM), followed by washout. Tissues served as their own controls based on tension prior to addition of drug. Both NO donors Cysteine-NO (100 µM) or GSNO (300 µM) were made daily. Data were analyzed with LabScribe (version 3.015800, Mac OS 10.11, iWorx systems Inc., Dover, NH). Aorta was collected from 3-month-old Sprague-Dawley rats, cut into 2 mm rings, and hung by stainless steel triangles that were passed through the lumen of each ring. Enzyme Activity Assay. The GSNOR activity assay was performed as previously described 29 using total protein lysate from human uterine smooth muscle tissue taken from the superior portion of the transverse incision. The lysate was prepared to a final concentration of 1 mg/ml in oxygen-purged experimental buffer containing: Tris-HCl pH 8.0 (20 mM), EDTA (0.5 mM), NP-40 (0.1%) and 1 mM phenylmethylsulphonyl fluoride (PMSF). Lysate was equilibrated at r.t. for 10 min in the presence of NADH (300 µM) prior to addition of GSNO (200 µM). Absorbance at 340 nm (A340) (SmartSpec TM Plus: Bio-Rad Laboratories, Inc., Hercules, CA) was recorded at t = 0, 5, 10 min to ensure stability of the NADH pool prior to the addition of GSNOR and/or inhibitors. Following equilibration, GSNO was added to the lysate mixture and A340 recording were collected at time = 0, 5, 10 min. N6022 (8 nM) (S77589: Selleck Chemicals, Houston, TX), a GSNOR inhibitor, was used to verify negligible NADH conversion to NAD + in the presence of GSNO. For control experiments N6022 was added concurrently with NADH to the protein lysate and equilibrated for 10 min prior to the addition of GSNO. Absorbance data are presented as 1/A340 to represent GSNOR-dependent NADH consumption.

Wes Protein
Confocal Microscopy. Myometrial tissues were sliced into 10μm sections using a cryostat at −30 °C and placed on coated slides (Surgipath, Buffalo Grove, Illinois). Samples were fixed in 4% paraformaldehyde then permeabilized with 0.5% Triton X-100. GSNOR protein was labeled with goat anti-rabbit ADH5 (1:250) primary antibody (ab59134: Abcam, Cambridge, MA), followed by either TRITC-donkey anti-rabbit secondary (Santa Cruz Biotech) for tissue sections, or FITC-donkey anti-rabbit secondary (Santa Cruz Biotech) for cultured cells, then mounted in Vectashield plus DAPI (VectorLabs, Burlingame, California). Images were acquired on an Olympus IX81 Fluoview confocal microscope system at 40 × magnification and analyzed with bundled software FV10-ASW (version 04.02, Windows 7 professional, Olympus America, Inc., Melville, NY). Brightness and contrast were the only modifications to the images, and they were adjusted globally using identical values for each image to ensure consistency (brightness + 256, contrast + 53, Photoshop CC 2017.1.0, Adobe Systems Inc., San Jose, CA).
Phosphorylated smooth muscle myosin (pSMM) preparation. SMM was isolated from frozen chicken gizzard (ID: 43018-2: Pel-Freez Biologicals, Rogers, AR) as previously described 56 . The resulting SMM product was dialyzed twice for 8 hours each in 2-liters of DTT-free HMM buffer, described below, using a 3 ml, 3.5 kDa dialysis cassette (66330: Thermo Fisher Scientific Inc., Waltham, MA). Phosphorylation of the regulatory light chain (MYL9) was also performed as previously described 57  Actin Motility Assay. The actin motility assay was performed as previously described 58 with minor modifications to minimize the presence of DTT in the flow chamber. Flow cells were prepared by flushing the following reagents through the flow chamber and incubating at each step for one min prior to the next addition: i) myosin SCIeNtIFIC REPORTS | (2018) 8:5614 | DOI:10.1038/s41598-018-23371-w added in the appropriate experimental concentration (5,10,25,50,100,200, 300, 400 µg/ml), ii) BSA (5 mg/mL), iii) TRITC-labeled actin (10 nM), iv) two washes of DTT-free actin buffer (with or without 300 µM GSNO), and v) two washes of DTT-free motility buffer containing 1 mM ATP (with or without 300 µM GSNO). Motility assays were implemented using a Nikon TE2000 epifluorescence microscope with fluorescent images digitally acquired with an Andor iXon Ultra (Belfast, N. Ireland) camera. Each flow cell was imaged for 30 sec from three distinct fields to obtain a single sample (n = 1), and performed in triplicate. Data were analyzed using Simple PCI tracking software (Compix, Sewickley, PA) to obtain actin-sliding velocities. Objects were defined by applying an exclusionary area threshold to minimize background noise. Velocities too slow to be accurately measured by the PCI tracking software were hand-calculated in ImageJ (version 1.50i, Mac OS 10.11) by recording the linear velocity of (3) filaments per recording (9 velocities per n = 1). The action of GSNO was controlled by addition of GSH which had no effect.
Biotin Switch and Streptavidin Pulldown. Samples from each patient in each group was independently isolated by biotin switch and streptavidin pulldown and then pooled for tandem mass spectrometry (MS/MS) analysis (i.e., sPTL1, 4 unique patients; sPTL2, 4 unique patients; sPTL3, 4 unique patients; for a total of 12 unique patients split into 3 biological replicates to help control for human diversity). Pooling patients allows experimental economy while maintaining replication. Protein isolates (1.8 ml 0.8 mg/ml in HEN buffer) were incubated with GSNO (300 μM) for 20 min at r.t. At this concentration, GSNO will produce ~5μM reactive NO over 15-20 min without accumulation 59 . This reactive species concentration matches the IC 50 concentration for relaxation of isolated myometrium 37 . Neither biotin-HPDP nor a maleimide dye lead to false positives because the amines or tyrosines are not be labeled even if nitrosated. SDS (0.2 ml of 25% SDS) was added along with 30 mM NEM. Samples were incubated at 50 °C in the dark for 20 min and proteins precipitated in −20 °C acetone for 1 hr. and collected by centrifugation at 3,000 g for 10 min. The clear supernatant was aspirated, and the protein pellet was gently washed with 70% acetone (4 × 5 ml). After resuspension in 0.24 ml HEN buffer with 1% SDS (HENS), the material was transferred to a fresh 1.7-ml microfuge tube containing 30 μl biotin-HPDP (2.5 mg/ml). The labeling reaction was initiated by addition of 30 μl of 200 mM sodium ascorbate (final 20 mM ascorbate) for 1 hr. at r.t. in the dark. Four volumes of −20 °C acetone were added to the labeled samples and incubated at −20 °C for 20 min to remove biotin-HPDP. The samples were centrifuged at 3,000 g for 10 min at 4 °C, and the supernatant discarded. The pellet was resuspended in 140 μl of HENS buffer. Neutralization buffer (HEPES (20 mM) pH 7.7, NaCl (100 mM), EDTA (1 mM), and Triton X-100 (0.5%)), was added (280 μl) along with 42 μl of streptavidin-agarose. Proteins were incubated for 1 hr. at r.t. and washed five times with 1.5 ml of neutralization buffer with 600 mM NaCl. Beads were incubated with 100 μl elution buffer (neutralization buffer with 600 mM NaCl plus 100 mM β-mercaptoethanol) to recover the bound proteins. This step releases the protein from the streptavidin bead leaving the biotin-HPDP tag bound to the bead as well as natively biotinylated proteins still bound to the bead. Four volumes of −20 °C acetone were added to re-precipitate proteins. Samples were centrifuged at 3,000 g for 10 min at 4 °C, the supernatant was discarded, and the pellet was washed and dried for proteomic analysis.
Mass Spectrometry. The Nevada Proteomics Center analyzed proteins by trypsin digestion and liquid chromatography (LC)/MS/MS analysis. Acetone-precipitated pellets were washed twice with 25 mM ammonium bicarbonate and 100% acetonitrile, reduced, and alkylated using 10 mM dithiothreitol and 100 mM iodoacetamide and incubated with 75 ng sequencing grade modified porcine trypsin (Promega, Fitchburg WI) in 25 mM ammonium bicarbonate overnight at 37 °C. Peptides were first separated by Michrom Paradigm Multi-Dimensional Liquid Chromatography (MDLC) instrument [Magic C18AQ 3μ 200 Å (0.2 × 50 mM) column (Michrom Bioresources, Auburn, CA) with an Agilent ZORBAX 300SB-C18 5μ (5 × 0.3 mM) trap (Agilent Technologies, Santa Clara, CA)] using a 0.1% formic acid/1% formic acid in acetonitrile gradient. Eluted peptides were analyzed using a Thermo Finnigan LTQOrbitrap using Xcalibur v 2.0.7. MS spectra (m/z 300-2,000) were acquired in the positive ion mode with resolution of 60,000 in profile mode. The top 4 data-dependent signals were analyzed by MS/MS with CID activation, minimum signal of 50,000, isolation width of 3.0, and normalized collision energy of 35.0 with a targeted reject list 45 . Dynamic exclusion settings were used with a repeat count of two, repeat duration of 10 s, exclusion list size of 500, and exclusion duration of 30 s. Criteria for S-Nitrosation Identification. PROTEOIQ (V2.6, www.nusep.com) was used to validate MS/ MS-based peptide and protein identifications. Peptides were parsed before analysis with a minimum Xcorr value of 1.5 and a minimum length of six amino acids. There were no matches to the concatenated decoy database, and therefore, a false discovery value is not applicable or "0. " Peptide identifications were accepted if they could be established at > 95.0% probability as specified by the Peptide Prophet algorithm 60 . Protein identifications were accepted if they could be established at > 95.0% probability and contained at least two identified peptides with five spectra per peptide. Protein probabilities were assigned by the Protein Prophet algorithm 61 . Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Data availability. The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.