Journal of Proteomics 138 (2016) 40–47 Contents lists available at ScienceDirect Journal of Proteomics journal homepage: www.elsevier.com/locate/jprot Identification of novel S-nitrosation sites in soluble guanylyl cyclase, the nitric oxide receptor Annie Beuve a, Changgong Wu b, Chuanlong Cui b,TongLiub,MohitRajaJainb, Can Huang a, Lin Yan b, Vladyslav Kholodovych c,d,HongLib,⁎ a Department of Pharmacology, Physiology and Neuroscience, Rutgers University, New Jersey Medical School, Newark, NJ 07103, United States b Center for Advanced Proteomics Research, Department of Microbiology, Biochemistry and Molecular Genetics, Rutgers University, New Jersey Medical School Cancer Center, Newark, NJ 07103, United States c High Performance and Research Computing, OIRT, Rutgers University, New Brunswick, NJ 07103, United States d Department of Pharmacology, Rutgers University, Robert Wood Johnson Medical School, Piscataway, NJ article info abstract Article history: Soluble Guanylyl Cyclase (sGC) is the main receptor for nitric oxide (NO). NO activates sGC to synthesize cGMP, Received 2 December 2015 triggering a plethora of signals. Recently, we discovered that NO covalently modifies select sGC cysteines via a Received in revised form 29 January 2016 post-translational modification termed S-nitrosation or S-nitrosylation. Earlier characterization was conducted Accepted 12 February 2016 on a purified sGC treated with S-nitrosoglutathione, and identified three S-nitrosated cysteines (SNO-Cys). Available online 18 February 2016 Here we describe a more biologically relevant mapping of sGC SNO-Cys in cells to better understand the multi- Keywords: faceted interactions between SNO and sGC. Since SNO-Cys are labile during LC/MS/MS, MS analysis of nitrosation Soluble guanylyl cyclase typically occurs after a biotin switch reaction, in which a SNO-Cys is converted to a biotin-Cys. Here we report the S-nitrosation identification of ten sGC SNO-Cys in rat neonatal cardiomyocytes using an Orbitrap MS. A majority of the SNO-Cys Biotin switch identified is located at the solvent-exposed surface of the sGC, and half of them in the conserved catalytic domain, Tandem mass spectrometry suggesting biological significance. These findings provide a solid basis for future studies of the regulations and functions of diverse sGC S-nitrosation events in cells. © 2016 Elsevier B.V. All rights reserved. 1. Introduction described as a non-enzymatic process, but there is now solid evidence that some proteins can catalyze specific transnitrosation and/or Nitric oxide (NO) signaling is a key modulator of the functions of denitrosation of target proteins via protein–protein interactions [2,3]. different cells, particularly the ones involved in cardiovascular and neu- This emerging role of specific protein–protein interaction-driven ronal function. NO stimulates the activity of sGC to produce cGMP, transnitrosation is apparent in critical cellular events such as regulation which in turn supports synaptic plasticity, relaxes smooth muscle cells of cell death (transnitrosation from thioredoxin to caspase 3, CASP3), of (SMC) and protects from cardiac hypertrophy. Of note, sGC can produce ubiquitination (transnitrosation between CASP3 and XIAP) and nuclear cGMP only as an αβ heterodimer, as its catalytic domain is formed by translocation (transnitrosation between GAPDH and GOSPEL) [4,5]. the association of the C-terminal α and β subunits. sGC isoforms most Thus, dysfunction of protein–protein transnitrosation could lead to apo- commonly expressed are α1 and β1 subunits. NO also modulates the ptosis [6] and mitochondrial dysfunction in neurodegenerative diseases. signaling and redox environment via S-nitrosation, a post-translational We have previously shown that sGC, which contains more than modification (PTM) of cysteines (Cys) that modifies protein localiza- thirty conserved Cys in its α and β subunits, is readily, yet specifically, tion, activity and interactions [1]. S-nitrosation was previously S-nitrosated in vitro [7], and in vivo in an Angiotensin II-induced hyper- tensive model and during development of nitrate tolerance [8,9].After MALDI-TOF-TOF MS analysis of the chemical S-nitrosation of sGC puri- fied from bovine lungs and treated with S-nitrosoglutathione (GSNO), Abbreviations: Asc, ascorbate; BCA, the bicinchoninic acid assay; BST, biotin fi α α switch technique; CID, collision-induced dissociation; CSNO, S-nitrosocysteine; GSNO, S- we initially identi ed three sGC SNO-Cys sites, C243 and C516 in nitrosoglutathione; HCD, higher-energy collisional dissociation; MMTS, methyl the α subunit and βC122 in the β subunit. Using site-directed mutagen- methanethiosulfonate; NCM, neonatal cardiomyocytes; PTM, post-translational modifica- esis, these three Cys were shown to be involved in desensitization of tion; sGC, soluble guanylyl cyclase; SMC, smooth muscle cells. sGC activity to NO stimulation [7–9]. ⁎ Corresponding author at: Department of Microbiology, Biochemistry and Molecular Due to SNO-Cys instability, indirect biotin switch techniques (BST) Genetics, Rutgers University, NJMS Cancer Center, 205 S. Orange Ave. F1226, Newark, NJ 07103, United States. are traditionally used to identify nitrosated peptides [10]. BST indirectly E-mail address: [email protected] (H. Li). detects SNO-Cys through ascorbate (Asc) reduction of SNO-Cys into free http://dx.doi.org/10.1016/j.jprot.2016.02.009 1874-3919/© 2016 Elsevier B.V. All rights reserved. A. Beuve et al. / Journal of Proteomics 138 (2016) 40–47 41 thiols for subsequent biotinylation, avidin affinity enrichment and LC/ removed by cold acetone precipitation and the protein pellets were col- MS/MS detection. BST specificity is predicated on SNO Asc-reduction lected by centrifuging at 5000g for 10 min at 4 °C and washed 3× with specificity and multiple controls are necessary [11–13]. Herein, we de- ice cold acetone. The protein pellets were re-suspended and the SNO- scribe the more in-depth identification of nine novel SNO-Cys from rat Cys were biotinylated in 900 μl of the HENS2 buffer (25 mM HEPES, neonatal cardiomyocytes overexpressing sGC via adenoviral infection, pH 7.7, 1 mM EDTA and 1% SDS) plus 0.7 mM biotin-HPDP, 75 mM using a combination of the classic BST approach with the highly sensi- Asc and 7 μM copper (I) chloride at room temperature for 1 h in the tive Orbitrap tandem mass spectrometry. We also built a model of the dark. Excess biotin-HPDP was removed by cold acetone precipitation catalytic domain of sGC harboring SNO modified Cys. Newly discovered (Supplemental Fig. S1). Negative controls were samples without Asc SNO-Cys are conserved among the mammalian species and are mostly treatment. exposed at the surface of sGC, in particular in the catalytic domain. This observation suggests that SNO-Cys in the catalytic domain could 2.5. In-solution digestion modulate sGC activity hence the NO-cGMP pathway. Since sGC SNO- Cys are solvent exposed, they could be involved in specific protein– Protein pellets were dissolved in 300 μl of a buffer containing 8 M protein interactions and possibly transnitrosation reactions to modulate urea with 100 mM Tris, at pH 7.0. The protein concentrations were mea- other signaling pathways. Thus identifying sGC, the main NO receptor, sured with the BCA assay and adjusted to 2 μg/μl. Two hundred micro- to be itself extensively and specifically S-nitrosated in cells provides a grams of proteins were diluted 10-fold with 50 mM NH4HCO3 strong rationale for further biological studies to investigate the func- (pH 8.3) and digested at a protein/trypsin weight ratio of 30:1, at tions of SNO-Cys in sGC. 37 °C overnight. 2. Materials and methods 2.6. Enrichment of biotinylated peptides 2.1. Materials The resulting peptides were diluted with the AB Sciex ICAT Affinity Buffer-Load buffer. After slowly injecting the diluted sample onto the Phenylmethylsulfonyl fluoride (PMSF), methyl methanethiosul- avidin cartridge, the Affinity Buffer-Load and the Affinity Buffer-Wash fonate (MMTS), protease inhibitor cocktail, copper (I) chloride, and 1 buffers were sequentially injected onto the cartridge to remove non- formic acid were purchased from Sigma (St. Louis, MO). N-[6- biotinylated peptides, according to the manufacturer's instructions. (biotinamido)hexyl]-3′-(2′-pyridyldithio)propionamide (Biotin-HPDP), Subsequently, the Affinity Buffer-Wash 2 and HPLC grade water were EDTA, HEPES, C18 spin columns, trypsin and ammonium bicarbonate injected onto the cartridge to further remove the nonspecifically were purchased from Fisher Scientific (Fair Lawn, NJ). SDS was pur- bound peptides. The Affinity Buffer-Elute buffer was slowly injected to chased from BioRad (Hercules, CA). Acetonitrile (ACN) and water were elute the biotinylated peptides. The resulting biotinylated peptides purchased from J. T. Baker Inc., (Center Valley, PA). The ICAT avidin en- were desalted with Pierce C18 spin columns, completely dried in a richment kit was purchased from AB Sciex (Framingham, MA). SpeedVac and were re-suspended in 5 μl of mobile phase A (2% ACN and 0.1% formic acid in H2O) for LC/MS/MS analysis. 2.2. Cell culture 2.7. Identification of biotinylated-peptides by LC/MS/MS Neonatal cardiomyocytes (NCM) were isolated from 1 to 2 days old control Wistar rats (Harlan Laboratories, Somerville) by Percoll The enriched biotinylated-peptides were analyzed by LC/MS/MS on gradient centrifugation and plated overnight in cardiomyocyte culture an LTQ-Orbitrap Velos Pro mass spectrometer (Thermo Fisher Scientif- medium with 5% horse serum and 100 μM BrdU. Plates were coated ic) coupled with an Ultimate 3000 Chromatography System. First, the with gelatin. The medium was modified from Dulbecco's modified desalted peptides were trapped on a C18 pre-column (Pepmap C18, Eagle's medium (DMEM)/F-12, supplemented with sodium pyruvate, 5mm×300μm, Dionex) at 2% mobile phase B (mobile phase A, 2% glucose, L-ascorbic acid, bovine serum albumin, sodium selenite, sodium ACN and 0.1% formic acid; mobile phase B, 85% ACN and 0.1% formic bicarbonate, and antibiotics. Twelve hours after seeding, the cells were acid) at a flow rate of 30 μl/min.