Detection and Proteomic Identification of S‐Nitrosated Proteins in Human Hepatocytes

C H A P T E R

S E V E N T E E N

Detection and Proteomic Identification of S-Nitrosated Proteins in Human Hepatocytes Laura M. Lo´pez-Sa´nchez,* Fernando J. Corrales,† Manuel De La Mata,* Jordi Muntane´,* and Antonio Rodrı´guez-Ariza* Contents 1. Introduction 2. Preparation of CSNO 3. Preparation of Primary Human Hepatocytes and Cell Culture 4. Treatment of Hepatocytes and Sample Preparation 5. Biotin Switch Assay 6. Detection and Purification of Biotinylated Proteins 7. Final Considerations Acknowledgments References

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Abstract The S-nitrosation of protein thiols is a redox-based posttranslational modification that modulates protein function and cell phenotype. Although the detection of S-nitrosated proteins is problematical because of the lability of S-nitrosothiols, an increasing range of proteins has been shown to undergo S-nitrosation with the improvement of molecular tools. This chapter describes the methodology used to identify potential targets of S-nitrosation in cultured primary human hepatocytes using proteomic approaches. This methodology is based on the biotin switch method, which labels S-nitrosated proteins with an affinity tag, allowing their selective detection and proteomic identification.

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Liver Research Unit, Hospital Universitario Reina Sof ´ıa, Co´rdoba, Spain Hepatology and Gene Therapy Unit, Universidad de Navarra, Pamplona, Spain

Methods in Enzymology, Volume 440 ISSN 0076-6879, DOI: 10.1016/S0076-6879(07)00817-8

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2008 Elsevier Inc. All rights reserved.

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1. Introduction Under physiological aerobic conditions, nitric oxide (NO) reacts with O2 to yield NO2 and N2O3, powerful electrophiles that S-nitrosate cysteines to form S-nitrosothiols. The S-nitrosation of protein thiols, often referred to as ‘‘S-nitrosylation,’’ is a form of posttranslational modification that has been regarded as a mechanism by which NO can transmit signals both within and between cells and tissues (Stamler et al., 2001). S-Nitrosation is being advanced as a central biological signaling mechanism, and first attempts are underway to identify potential targets of S-nitrosation by using proteomic approaches. Jaffrey et al. (2001) developed an original approach to assess S-nitrosation in which S-nitrosoproteins are selectively tagged with biotin and hence the method is termed the ‘‘biotin switch’’ method (Fig. 17.1). The assay consists of three steps. In the first step, free thiols are blocked with the thiol-specific methylating agent methyl methanethiosulfonate (MMTS). The second step involves the selective reduction S-S-CH3 MMTS

Ascorbate

SNO

SH

SSR

SH

S-S-CH3 Biotin-HPDP

SSR

S-S-CH3

“BIOTIN SWITCH”

SNO

S-S- Biotin

SSR

SSR

S-S-CH3

Streptavidin capture

S-S- Biotin SSR

Inmunodetection

S-S-CH3 S-S- Biotin

S-S-CH3 SH

Anti-biotin

SSR HS- Biotin

SSR

Figure 17.1 During the biotin switch assay, S-nitrosoproteins are selectively tagged with biotin. In the first step, free thiols (-SH) are blocked with the thiol-specific methylating reagent MMTS. During the second step, and under conditions where disulfides (-SSR) are not reduced, S-nitrosothiols (-SNO) are selectively reduced by ascorbate. Inthe final step,these newly formedthiols are reactedwiththethiol-specificbiotinylating reagent biotin-HPDP. Biotinylated proteins can be inmunodetected or, alternatively, can be captured on streptavidin resins for their purification and proteomic identification.

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of protein nitrosothiols to thiols with ascorbate. In the final step, these newly formed thiols are reacted with the sulfhydryl-specific biotinylating reagent N-[6-(biotinamido)hexyl]-30 -(20 -pyridyldithio)propionamide (biotin-HPDP). This method offers significant advantages in that biotinylated proteins can be detected on Western blots following incubation with antibiotin antibodies or recognition via streptavidin. Moreover, biotinylated proteins can be captured on streptavidin matrices for identification by mass spectrometry (Fig. 17.1). Detection of S-nitrosated proteins is problematical due to the lability of S-nitrosothiols, which are unstable under certain conditions. It has been established that they are sensitive to both photolytic and transition metal ion-dependent breakdown and that samples must be kept in the dark and in the presence of transition metal chelators. Several issues have been raised regarding the sensitivity and specificity of the biotin switch method. The complete blocking of free thiols is imperative to minimize background biotinylation, and therefore to improve signal-tonoise ratio and sensitivity. Another worrisome issue is the possibility that the ascorbate treatment may reduce other cysteine oxidation-derived modifications such as S-glutathionylation or S-oxidations, including sulfenic, sulfinic, and sulfonic acids (Kettenhofen et al., 2007). Importantly, Forrester et al. (2007) showed that ascorbate does not directly reduce the S–NO bond, but rather undergoes transnitrosation by SNO to generate O-nitrosoascorbate as the intermediate, which homolyzes rapidly to yield the semidehydroascorbate radical and NO. This reaction with ascorbate is unique among cysteine oxidation products, thus conferring specificity to the biotin switch assay. As in other assays, the biotin switch method poses potential problems and limitations. However, once they are recognized, this method represents a useful tool for the study of S-nitrosothiol biology and may facilitate the isolation and analysis of the S-nitrosoproteome. We have used this technique to detect and identify S-nitrosated proteins during the alteration of SNO homeostasis in human hepatocytes (Lopez-Sanchez et al., 2007; Ranchal et al., 2006). We use S-nitroso-L-cysteine (CSNO), a nitrosothiol that is effective at relatively low concentrations and has very rapid effects increasing the intracellular pool of S-nitrosothiols. This effectiveness has been related to the direct transfer of this intact molecule across cellular membranes (Mallis and Thomas, 2000). CSNO can be taken up into cells via amino acid transport system L, whereas nitrosoglutathione (GSNO) is not transported directly, but requires the presence of cysteine and/or cystine (Zhang and Hogg, 2005) or cleavage by g-glutamyl transpeptidase to the membrane-permeable S-nitroso-cysteinyl glycine dipeptide (Gaston et al., 2006). Once inside the cell, transnitrosation to both protein thiols and glutathione occurs, but GSNO once formed is broken down rapidly by GSNO reductase (Liu et al., 2001).

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2. Preparation of CSNO We synthesize CSNO following a previously described method (Shah et al., 2003; Mallis and Thomas, 2000). The CSNO solution is freshly prepared immediately before use by mixing 220 ml of 220 mM L-cysteine and 220 ml of 220 mM NaNO2 with 25 ml of 4.0 N HCl. This solution is incubated in the dark at room temperature for 10 min and is then neutralized by adding 25 ml of 4.0 N NaOH and maintained on ice. The final concentration is calculated from absorbance at 334 nm using a molar absorption coefficient of 740 M1 cm1. Usually, the yield is between 60 and 70% with CSNO solutions in the range of 60 to 70 mM.

3. Preparation of Primary Human Hepatocytes and Cell Culture Liver resections are obtained after written consent from patients submitted to surgical intervention for a primary or secondary liver tumor. Cell isolation is carried through ex vivo collagenase perfusion. The following steps are carried out maintaining the piece of liver and solutions at 37 under strict sterile conditions. We first perfuse the piece of liver with nonrecirculating washing solution I (20 mM HEPES, 120 mM NaCl, 5 mM KCl, 0.5% glucose, 100 mM sorbitol, 100 mM manitol, 100 mM GSH, 100 U/ml penicillin, 100 mg/ml streptomycin, and 0.25 mg/ml amphotericin B), pH 7.4, at a flow of 75 ml/min in order to remove blood cells. Afterward, the liver is perfused with nonrecirculating chelating solution II (0.5 mM EGTA, 58.4 mM NaCl, 5.4 mM KCl, 0.44 mM KH2PO4, 0.34 mM NaHPO4, 25 mM N-tris[hydroxymethyl]methylglycine, 100 mM sorbitol, 100 mM manitol, 100 mM GSH, 100 U/ml penicillin, 100 mg/ml streptomycin, and 0.25 mg/ml amphotericin B), pH 7.4, at a flow of 75 ml/min. The liver is finally perfused with recirculating isolation solution III (0.050% collagenase, 20 mM HEPES, 120 mM NaCl, 5 mM KCl, 0.7 mM CaCl2, 0.5% glucose, 100 mM sorbitol, 100 mM manitol, 100 mM GSH, 100 U/ml penicillin, 100 mg/ml streptomycin, and 0.25 mg/ml amphotericin B), pH 7.4, at a flow of 75 ml/min. The obtained cell suspension is filtered through nylon mesh (250 mm) and washed three times at 50g 5 min 4 in supplemented culture medium. DEM:Ham-F12 and William’s E mediums (1:1) are supplemented with 26 mM NaHCO3, 15 mM HEPES, 0.292 g/liter glutamine, 50 mg/liter vitamin C, 0.04 mg/ liter dexamethasone, 2 mg/liter insulin, 200 mg/liter glucagon, 50 mg/liter transferrin, and 4 ng/liter ethanolamine). We usually obtain a consistent cell viability >85%, as determined by trypan blue exclusion. Hepatocytes

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(150,000 cells/cm2) are seeded in type I collagen-coated 100-mm diameter dishes and cultured in culture medium containing 5% fetal calf serum for 12 h. Afterward, the medium is removed and replaced by fresh culture medium without fetal bovine serum. Treatment of cells is initiated 48 h after seeding of the cells to allow stabilization of the culture.

4. Treatment of Hepatocytes and Sample Preparation After culture stabilization, the medium is refreshed, CSNO is added at different final concentrations (0.5, 2, and 5 mM ), and cells are incubated for 2 to 24 h at 37 . After washing hepatocytes with 0.1 M phosphate-buffered saline, 1 ml of nondenaturing lysis solution (50 mM Tris-HCl, pH 7.4, 300 mM NaCl, 5 mM EDTA, 0.1 mM neocuproine, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 5 mg/ml aprotinin, and 10 mg/ml leupeptin) is added to a 100-mm diameter dish, and cells are scraped, harvested, and incubated on ice for 15 min. From now on, it is imperative to protect samples from light to avoid artifacts and decomposition of Snitrosothiols. After centrifugation at 10,000g, 4 for 15 min, supernatants are collected and protein is quantified with the Bradford reagent. N-Ethylmaleimide is included at a final concentration of 5 mM in the lysis solution for blockade of thiols during homogenization to prevent ex vivo transnitrosation of protein thiols. However, any thiol-blocking agent must be excluded from the lysis solution if in vitro transnitrosation experiments are planned. For example, as a positive control, we suggest incubating cellular lysates with 1 mM GSNO for 1 h at room temperature before performing the biotin switch method.

5. Biotin Switch Assay Hepatocyte lysates are adjusted to 300–400 mg of protein per milliliter, and equal volumes are mixed with 3 volumes of blocking buffer [9 volumes of HEN buffer (250 mM HEPES, pH 7.7, 1 mM EDTA, and 0.1 mM neocuproine), 1 volume of SDS [25% (v/v) in H2O], and 0.1 volumes of 2 M MMTS (stock solution in N,N-dimethylformamide)]. Free thiols are then blocked at 50 for 1 h with agitation. The SDS and temperature of 50 are designed to ensure that buried thiols are alkylated. The protein must be washed to remove MMTS so that any remaining blocking agent is not able to then block the newly exposed thiol group following the ascorbate reduction of S-nitrosothiols. To remove MMTS, proteins are precipitated twice with 2 volumes of prechilled (20 ) acetone, incubated at 20 for

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15 min, and centrifuged at 2000g for 10 min followed by gentle rinsing of the pellet with 4  1 ml 70% acetone/H2O. In the following step, nitrosothiols are reduced and labeled at once. To this end, precipitates obtained as described earlier are resuspended in 60 ml HENS buffer (250 mM HEPES, pH 7.7, 1 mM EDTA, 0.1 mM neocuproine, and 1% SDS); following the addition of 2 ml of 50 mM ascorbic acid (from a freshly prepared 50 mM stock solution in deionized water) and 20 ml of 4 mM biotin-HPDP (prepared fresh by diluting a 50 mM stock solution in N,N-dimethylformamide), they are incubated for 1 h at room temperature. Proteins are acetone precipitated as described earlier and resuspended in 40 ml of HENS buffer. After this step, S-nitrosated proteins are biotin tagged and there is no need to protect samples from light.

6. Detection and Purification of Biotinylated Proteins The detection of biotinylated proteins can be carried out by Western blot with antibiotin antibodies or by using streptavidin conjugated to alkaline phosphatase or horseradish peroxidase. Because biotin-HPDP is cleavable under reducing conditions, prepared samples are loaded onto SDS-PAGE gels without reducing agents and, to prevent nonspecific reactions of biotin-HPDP, are not boiled before electrophoresis. We usually separate samples from the biotin switch assay on 10% SDS-PAGE gels; after transfer of proteins to nitrocellulose membranes, they are incubated with a primary monoclonal antibiotin antibody (Sigma-Aldrich, St. Louis, MO; 1/2500 dilution) for 1 h and a secondary antibody coupled to horseradish peroxidase (Santa Cruz Biotechnology Inc., Santa Cruz, CA). Inmunoreactive proteins are then visualized using the ECL Advance detection system (Amersham Biosciences, Uppsala, Sweden). Appropriate controls in which hepatocyte lysates are processed in the absence of HPDP-biotin should be included to distinguish endogenously biotinylated proteins. Biotinylated proteins can be alternatively purified for proteomic identification. In this case, the starting material for the biotin switch assay should be in the order of 3 to 4 mg protein. Samples are diluted in 2 volumes of neutralization buffer (20 mM HEPES, pH 7.7, 0.1 M NaCl, 1 mM EDTA, and 0.5% Triton X-100). Add 18 ml of streptavidin agarose/mg of protein used in the initial protein sample. We use EZview Red streptavidin-agarose (Sigma-Aldrich) washed previously in neutralization buffer. Use of a colored resin helps minimize accidental aspiration of the resin during the washing and discarding steps that follow. Biotinylated proteins are incubated with the resin for 3 h at room temperature with continuous gentle inversion agitation. Samples are then centrifuged at 8200g, 4 for 30 s, and

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supernatants containing unbound proteins are discarded. The resin is washed five times with washing buffer (20 mM HEPES, pH 7.7, 600 mM NaCl, 1 mM EDTA, and 0.5% Triton X-100) and centrifuging as described previously. To elute bound proteins, the resin is incubated with 1 volume of elution buffer (20 mM HEPES, pH 7.7, 0.1 M NaCl, 1 mM EDTA, and 0.1 M 2-mercaptoethanol) for 20 min at 37 with gentle inversion agitation. As the biotin is incorporated via a disulfide bond and elution is carried out in reducing conditions, endogenously biotinylated proteins are not purified. Supernatants are collected and purified proteins are concentrated with a Microcon YM-3 centrifugal filter unit (3 kDa NMWL, Millipore, Billerica, MA) and separated on 10% SDS-PAGE. In our experience, twodimensional electrophoresis results in a low yield for protein identification. Gels are stained with Sypro Ruby protein stain (Biorad) and protein bands excised, in-gel digested with trypsin and proteins identified by mass spectrometry (MS) using matrix-assisted laser desorption/ionization–time of flight (MALDI-TOF) and peptide mass fingerprinting. Gel bands are excised using a robotic workstation (Investigator Propic, Genomics Solutions, Ann Harbor, MI) and trypsin digested using a robotic digestion system (ProGest, Genomic Solutions). Tryptic digests are then analyzed on a MALDI-TOF/TOF 4700 proteomics analyzer (Applied Biosystems, Foster City, CA). Alternatively, eluted proteins can be precipitated using trichloroacetic acid/acetone, and trypsin digested for liquid chromatography-MS/MS sequencing of the resulting peptides. This alternative has the advantage over gel studies in that a high yield results in a higher number of identified proteins. An ESI-MS/MS analysis is performed using a microcapillary reversed-phase LC system (CapLC, Waters, Milford, MA) coupled online to a Q-TOF Micro (Waters) using a PicoTip nanospray ionization source (Waters). MS/MS data are collected in an automated data-dependent mode, and the three most intense ions in each survey scan are fragmented sequentially by collision-induced dissociation. Data processing is performed with MassLynx 4 and ProteinLynx Global Server 2 (Waters).

7. Final Considerations The levels of protein S-nitrosothiols generated in vivo are too low for a proteomic analysis with the current available methods. Therefore, most studies have relied on exogenous treatments to increase the intracellular pool of S-nitrosothiols. It is important to assess if there is enough S-nitrosothiol in the sample under study to make the biotin switch assay results meaningful. The total S-nitrosothiol content of our protein samples is measured by a displacement chemiluminescence technique using

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tri-iodide as the reductant (Lopez-Sanchez et al., 2007). S-Nitrosothiols decompose rapidly in the presence of mercuric ion Hg2þ, and HgCl2 has been used as a diagnostic marker for the presence of S-nitrosothiols in chemiluminescence techniques. However, it has been demonstrated that Hg2þ interferes with thiol labeling by biotin-HPDP (Zhang et al., 2005), and we do not recommend the pretreatment of samples with HgCl2 as a diagnostic indicator of S-nitrosothiols in the biotin switch assay. Variations on the methodology pioneered by Jaffrey et al. (2001) demonstrate the versatility of this method in the study of S-nitrosothiol biology. For example, biotinylated proteins can be trypsin digested before streptavidin purification of biotinylated peptides. Then, after MS analysis, cysteine residues in identified peptides will be putative cysteine S-nitrosation sites (Hao et al., 2006). Alternatively, if peptides bound to streptavidin are eluted with formic acid instead of with mercaptoethanol, biotin remains associated to them and MS analysis can identify biotinylation sites directly (Greco et al., 2006). However, radioactive probes can be used instead of biotin-HPDP with the advantage that the stoichiometry of S-nitrosation can be quantified and experiments of peptide mapping can be performed using this approach ( Jaffrey et al., 2002). A detailed description of some of these methodological modifications can be found in other chapters in this volume.

ACKNOWLEDGMENTS This work was supported by grants from the Programa de Promocio´n de la Investigacio´n en Salud del Ministerio de Sanidad y Consumo (PI04/1470) and Consejerı´a de Salud de la Junta de Andalucı´a (099/06).

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