S-nitrosoproteome in Endothelial Cells Revealed by a Modified Biotin

Aug 12, 2009 - Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan, The Genomic Research Center, Academia Sinica, Taipei 11529, ...
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S-nitrosoproteome in Endothelial Cells Revealed by a Modified Biotin Switch Approach Coupled with Western Blot-Based Two-Dimensional Gel Electrophoresis Bin Huang,† Chung Ling Liao,‡ Ya Ping Lin,† Shih Chung Chen,§,| and Danny Ling Wang*,† Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan, The Genomic Research Center, Academia Sinica, Taipei 11529, Taiwan, Division of Cardiology, Taipei Medical University, Wan Fang Hospital, Taipei 11696, Taiwan, and Division of Cardiology, Taipei County Hospital, Taipei County 22051, Taiwan Received June 30, 2009

NO-mediated S-nitrosation of cysteine residues has been recognized as a fundamental post-translational modification. S-Nitrosation of endothelial cell (EC) proteins can alter function and affect vascular homeostasis. Trace amounts of S-nitrosoproteins in endothelial cells (ECs) in vivo coupled with lability of the S-nitroso bond have hindered a comprehensive characterization. We demonstrate a convenient and reliable method, requiring minimal sample, for the screening and identification of S-nitrosoproteins. ECs treated with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) were subjected to the biotin switch method of labeling, then detected by analytical Western blot-based two-dimensional gel electrophoresis (2-DE). More than 89 SNAP-increased S-nitrosoproteins were detected and 28 of these were successfully excised from preparative 2-DE gel and identified by LC-MS/MS. Moreover, the nitrosocysteine residue for each protein (HSPA9/368, β-actin/16, TMP3/170, vimentin/328) was also determined, and the relative ratio of S-nitrosation/non-S-nitrosation for Cys328 of vimentin was estimated using MASIC software. By the combination of the biotin switch method with 2-DE and Western blot analysis, S-nitrosoproteins can be screened and characterized by MS, providing a basis for further study of the physiological significance of each S-nitrosoproteins. Keywords: S-nitrosation • biotin switch • mass spectrometry • MS • nitric oxide • Endothelial cell

Introduction Intracellular nitric oxide (NO) is generated from the conversion of L-arginine to L-citrulline by the enzymatic action of NO synthase (NOS).1,2 Endothelial cells (ECs) constantly release NO, due to the activation of endothelial NO synthase. Released NO is known to act on guanylate cyclase and increase cGMP levels in the cardiovascular system, consequently contributing to vasodilation, antithrombosis and antiproliferation.3-5 Recent studies have shown that NO can also nitronate cysteine residues (here represented as Cys-SH) at the thiol group, generating nitrosocysteine (Cys-S-NO). This leads to altered function and signaling properties of the protein.6-8 S-nitrosation has been suggested to play an important role in regulating cardiovascular function.9 A comprehensive study of the intracellular S-nitrosoproteome in cells10-12 is, however, hindered by the labile nature of the S-NO bond. The biotin switch method has been developed and routinely used to screen for intracellular S-nitrosoproteins (S-nitrosoproteins).13 This method * To whom correspondence should be addressed. Dr. Danny Ling Wang, Institute of Biomedical Sciences, Academia Sinica, 128 sec. 2 Academia Rd. NanKang, Taipei 11529, Taiwan. Tel, +886-2-23699132; fax, +886-2-27899143; e-mail, [email protected]. † Institute of Biomedical Sciences, Academia Sinica. ‡ The Genomic Research Center, Academia Sinica. § Taipei Medical University. | Taipei County Hospital. 10.1021/pr9005662 CCC: $40.75

 2009 American Chemical Society

consists of four steps, as depicted schematically in Figure 1A. Treated ECs were lysed immediately followed by blocking of free thiols (Cys-SH) with methyl methanethiosulfonate (MMTS) to form methylthiols (Cys-S-S-CH3). The nitrosothiols (Cys-SNO) are selectively reduced by ascorbate to generate free thiols (Cys-SH) and are then labeled with biotin, using a thiol specific biotinylating reagent (biotin-HPDP), to form (Cys-S-S-biotin); Finally, the resulting biotinylated proteins (i.e., S-nitrosoproteins) can then be isolated using neutravidin-agarose beads and subjected to proteomic analysis. However, the yield of Snitrosoproteins has been too low to allow comprehensive study.12-15 In an attempt to establish a convenient and reliable tool to study S-nitrosoproteins in cells, we have modified the traditional approach of biotin switch method. By omitting the avidin affinity step (for isolation of biotinylated proteins), we were able to evaluate S-nitrosation levels of proteins directly using Western blot-based 2-DE. More than 89 SNAP-increased S-nitrosoproteins were detected and 28 of which were successfully excised from preparative 2-DE gel. In addition to identifying S-nitrosoproteins and the site of S-nitrosation with LC-MS/ MS, the relative ratio of S-nitrosated to non-S-nitrosated cysteine was also determined. With this modified approach, only two Petri dishes of cultured ECs were sufficient for detection of S-nitrosation. Moreover, this approach of detecting S-nitrosated proteins is reproducible, then, provides a method for identification and Journal of Proteome Research 2009, 8, 4835–4843 4835 Published on Web 08/12/2009

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Figure 1. Biotin switch method applied to isolate S-nitrosoproteins in ECs. (A) Schematic diagram represents the four steps of biotin switch method.13 ECs cultured on a Petri dish treated with either SNAP (nitrosating agent) or NAP (inert control) were lysed following the blocking with MMTS, reduction with ascorbate and biotinylation with biotin-HPDP. Finally, the resulting biotinylated proteins (i.e., S-nitrosoproteins) can then be captured by neutravidin-agarose beads, and eluted with 2-ME. (B) The actual protein amounts recovered from each step of biotin switch as a Petri dish (10 cm) of cultured ECs treated with either SNAP or NAP were originally applied. (C) Silver stain was applied to achieve a maximum detecting sensitivity in the 2-DE gels with indicated protein amount applied: whole-cell lysate (50 µg), MMTS-treated lysate (50 µg), biotinylated lysate (50 µg), and 2-ME eluent, i.e., former S-nitrosoproteins (0.34 µg) from SNAP-treated ECs. Gels were developed for 10 s, except that of 2-ME eluant which was developed for 30 s (arrowhead indicates the overdeveloped protein marker). The protein spots detected on the silver-stained 2-DE gels were counted and the results are shown as mean ( SE from three independent experiments.

characterization of the S-nitrosation of proteins in cells, and could lead to further insight into the physiological significance of this process.

Materials and Methods Endothelial Cell Culture and NO Treatment. Endothelial cells (ECs) used were derived from the EAhy 926 cell line kindly 4836

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donated by Dr. Edgell (University of North Carolina, Chapel Hill, NC). ECs cells were cultured in Petri dishes (100 × 20 mm) with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 µg/mL streptomycin, 100 U/mL penicillin and incubated at 37 °C under 5% CO2. Prior to NO donor treatment, the medium was replaced with DMEM containing 2% fetal bovine serum and incubated

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overnight. S-Nitroso-N-acetylpenicillamine (SNAP, 1 mM) and L-cysteine (1 mM) were added into medium for 30 min in the dark. NAP (N-acetylpenicillamine, 1 mM), an inert NO donor obtained after exposing SNAP to UV overnight, was used as a negative control. Biotin Switch Method for Labeling and Isolating S-Nitrosated Proteins. The biotin switch method17 was used as described below. ECs were washed with 1× cord buffer (10 mM HEPES, pH 7.4, 0.14 M NaCl, 4 mM KCl, 11 mM glucose). Cell lysates were obtained using ultrasound and lysis buffer (250 mM HEPES, pH 7.7, 1 mM EDTA, 0.1 mM neocuproine, 0.4% (w/v) CHAPS). After a centrifuge step at 10 000 rpm for 5 min at 4 °C, free thiols in the supernatant that contains protein extracts (0.8 mg/mL) were methylated with blocking buffer (225 mM HEPES, pH 7.7, 0.9 mM EDTA, 0.09 mM neocuproine, 2.5% (w/v) SDS, 20 mM MMTS) at 50 °C for 20 min with agitation. To remove residual MMTS, the MMTS-treated lysate was precipitated with cold acetone and the resulting pellet was resuspended in HENS buffer (250 mM HEPES, pH 7.7, 1 mM EDTA, 0.1 mM neocuproine and 1% (w/v) SDS). This was followed by addition of one-third of the HENS suspension’s final volume of 4 mM N-[6-(biotinamido)hexyl]-3′-(2′-pyridyldithio) propionamide (biotin-HPDP/DMF; Thermo Fisher Scientific.) mixed with 1 mM ascorbate. The protein lysate/ biotin-HPDP mixture was incubated at room temperature for 1 h to allow biotinylation to occur. These mixtures were precipitated with cold acetone to remove excess biotin-HPDP and then resuspended in HENS buffer. The biotinylated proteins (i.e., the former S-nitrosoproteins) were recovered using neutravidin-agarose beads (15 µL/per mg of initiated protein input) in neutralization buffer (20 mM HEPES, pH 7.7, 100 mM NaCl, 1 mM EDTA and 0.5% (v/v) Triton X-100). The agarose beads were rinsed with washing buffer (20 mM HEPES, pH 7.7, 600 mM NaCl, 1 mM EDTA and 0.5% (v/v) Triton X-100) and then incubated with elution buffer (20 mM HEPES, pH 7.7, 100 mM NaCl, 1 mM EDTA, 100 mM 2-mercaptoethanol, 2-ME) for 20 min at 37 °C, with gentle agitation to release the avidinbound proteins. The concentrations of these proteins recovered from each step of biotin switch were determined using the Lowry assay.18 Two-Dimensional Gel Electrophoresis (2-DE). To reveal the qualitative difference of detected protein spots in whole-cell lysate, MMTS-treated lysate, biotinylated lysate and 2-ME eluant (S-nitrosoprotein), these lysates from SNAP-treated ECs were alternatively dissolved in 100 µL of a denaturing solution of 9 M urea, 2% (w/v) CHAPS, and 2% IPG buffer pH 4-7 (GE Healthcare, Piscataway, NJ) at room temperature for 30 min (Figure 1C). The solution was made up to a final volume of 340 µL by adding 240 µL of a rehydration solution of 8 M urea, 0.5% IPG buffer pH 4-7, and 2% (w/v) CHAPS. The solution was soaked into an 18 cm Immobiline DryStrip pH 4-7, (GE Healthcare) for up to 12 h on an isoelectric focusing system (Ettan IPGphor IEF, GE Healthcare) with the accumulated voltage at 40 kVh. After electrophoresis, stripped gels were equilibrated with a denaturing Tris-buffer (6 M urea, 30% (v/ v) glycerol, 2% (w/v) SDS, 50 mM Tris-HCl, pH 8.8) for 40 min. The equilibrated isoelectric focusing strip was laid on top of a vertical SDS-PAGE system. Only trace (∼0.3 µg) of S-nitrosoprotein from SNAP-treated ECs could be eluted from 2-ME buffer. Silver stain was then applied to the 2-DE gel. The gel

was developed for 10 s except for those from 2-ME eluant which was developed for 30 s. Validation of S-nitrosation by Image Analysis of SYPRO Ruby and Western Blots. In this modified approach, instead of using neutravidin to isolate the biotinylated proteins, the lysate either from SNAP or NAP treatment was subjected to the modified 2-DE directly. To maintain the biotin labeling on the cysteine residue, the 2-DE was performed by a reducing agent-free system. As depicted in Figure 2, analytical 2-DE was used to screen proteins with increased S-nitrosation and preparative 2-DE was used to collect each S-nitrosoprotein for subsequent MS analysis. Analytical 2-DE was performed on two sets of gels containing proteins from SNAP- and NAP-treated ECs simultaneously. One gel was stained with SYPRO Ruby (Molecular Probes, Eugene, OR), scanned using a Typhoon 9400 scanner (GE Healthcare, Piscataway, NJ) and analyzed using ImageMaster software (GE Healthcare, Piscataway, NJ) to determine the translational level of each candidate protein. The second gel was transferred onto nitrocellulose membranes, then hybridized for 2 h with a 1:4000 dilution of the streptavidin-horseradish peroxidase (HRP) conjugate (PerkinElmer Life Sciences, Waltham, MA) in TBS (25 mM Tris-HCl, pH 7.5, 500 mM NaCl). The membrane was washed three times with TTBS buffer (25 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.075% (v/v) Tween-20), then developed using SuperSignal West Femto Maximum chemiluminescence reagent (Thermo Fisher Scientific, Rockford, IL). S-nitrosoproteins were detected using a X-ray film scanner (Microtek, International, Inc., Hsinchu, Taiwan). The translational expression and the extent of Snitrosation of the S-nitrosoproteins were statistically analyzed from three separate experiments. In-Gel Protein Digestion and MS Analysis. Following image analysis of the analytical 2-DE gel, proteins which showed a significant increase in S-nitrosation after SNAP treatment were collected from the corresponding preparative 2-DE gel, which had been stained with VisPRO dye (Visual Protein Biotech., Taipei, Taiwan). Protein staining using VisPRO was achieved within 10 min and the fixation process with acetic acid was omitted, thereby facilitating subsequent in-gel digestion and peptide yield. After protein spot was excised and put into microcentrifuge tube, the gel slice was destained for 2 min using SDS-PAGE running buffer following wash with distilled water twice. Each gel slice was digested with trypsin (In-Gel Tryptic Digestion kit, Thermo Fisher Scientific, Rockford, IL) for 4 h at 37 °C. The resulting tryptic peptides were desalted using a Proteomics C18 Column (Mass Solution Biotech., Taipei, Taiwan) and then subjected to MS analysis using a CapLC-Q-TOF instrument (Micromass, Manchester, U.K.). MS data were searched against the NCBInr database using a MASCOT in-house search program (Matrixscience, London, U.K.). Peptides containing a biotinylated cysteine site were identified with the mass shift of 428.2 Da. The relative levels of biotinylation between SNAP- and NAP-treated peptides were estimated using MS/MS Automated Selected Ion Chromatogram (MASIC) software. Search parameters were specifically assigned according to chemical modification on cysteine residue: Biotin-HPDP, Methylation, acetylation, deamidation, Gln f pyro-Glu, Glu f pyro-Glu, and so forth. Other instrument parameters were set as follows: mass values, monoisotopic; protein mass, unrestricted; peptide mass tolerance, ( 0.4 Da; fragment mass tolerance, ( 0.4 Da, max missed cleavages, 1; instrument type, ESI-QUAD-TOF. Journal of Proteome Research • Vol. 8, No. 10, 2009 4837

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Figure 2. Schematic diagram of the modified approach applied to detect the S-nitrosoproteins in ECs. The biotinylated lysate that resulted from biotin switch was subjected to either analytical or preparative 2-DE. In the analytical 2-DE, the biotinylated lysates from SNAP- and NAP-treated ECs were applied to screen NO-induced S-nitrosoproteins. One pair of gels was stained with SYPRO Ruby to investigate the proteins’ translational expression; the other pair of gels was Western blotted with streptavidin-HRP to detect the proteins’ S-nitrosation. The proteins exhibited equal translational levels but increased S-nitrosation after SNAP treatment as revealed by analytical 2-DE collected from a VisPRO-stained preparative 2-DE gel. Gel spots were trypsin digested and analyzed using LC-MS/MS to identify the proteins and their S-nitrosated sites after SNAP treatment.

Results Detection of Biotinylated Proteins after Avidin Affinity Purification. One Petri dish of cultured ECs was treated with SNAP or NAP followed by biotin switch method (Figure 1A). The protein concentration after each step was determined as ∼1 mg whole-cell lysate (starting material, step 1, Figure 1A); ∼700 µg MMTS-treated lysate (after step 2, Figure 1A); ∼500 µg biotinylated lysate (after step 3, Figure 1A). After 2-ME elution from the neutravidin-agarose beads (step 4, Figure 1A), only trace amounts of S-nitrosoprotein were isolated from biotinylated lysates of both SNAP- and NAP- treated ECs (0.34 and 0.14 µg, respectively) as shown in Figure 1B. The protein content after various steps in the procedure was further analyzed using 2-DE. More than 900 protein spots were observed from whole-cell lysate, 500 from MMTS-treated lysate and 400 from the biotin-HPDP treated lysate. Less than 60 protein spots, however, were hardly observed from the 2-ME eluant of the SNAP-treated ECs after silver staining (Figure 1C). This indicates that only trace amounts of S-nitrosoproteins can be isolated using the traditional biotin switch method, making subsequent mass spectrometric analysis unfeasible. Direct Detection of Biotinylated Proteins Using Modified Biotin Switch Approach. In the present study, a modified approach to detect S-nitrosoproteins that requires minimal protein concentration was developed (Figure 2). In this modified approach, cell lysates containing biotinylated and nonbiotinylated proteins from SNAP- or NAP-treated samples were divided equally into two aliquots and subjected to analytical nonreducing (DTT-free) 2-DE. With the use of streptavidin-HRP, Western blots were performed on one pair of gels to identify the increase in S-nitrosated proteins after SNAP treatment; SYPRO Ruby staining is to ensure that any increased S4838

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nitrosation of a given protein is due to a post-translational event, rather than via an indirect effect on expression level of that protein. Protein spots that showed increased S-nitrosation were collected from the corresponding preparative 2-DE gel and stained with VisPRO. Excised protein spots from the gel were subjected to in-gel digestion and the tryptic peptides were analyzed by MS to identify the S-nitrosated sites. Cultured ECs either treated with SNAP (1 mM) or NAP (1 mM) for 30 min was subjected to biotin labeling (Figure 2). The biotinylated lysate (∼1 mg, recovered from two Petri dishes) were separated by analytical DTT-free 2-DE and stained by SYPRO Ruby to quantify the translational level of proteins. An additional biotinylated lysate was also separated by analytical DTT-free 2-DE but with streptavidin-HRP to detect Snitrosoporteins (Figure 3A). There were 15 ( 8 and 89 ( 17 S-nitrosoproteins detected in the ECs treated with NAP and SNAP, respectively. Of those SNAP-increased S-nitrosoproteins, 28 distinguishable proteins were shown on 2-DE. A significant increase (>1.5-fold increase) in S-nitrosation was notice (Figure 3B), with no change in translational level (as determined from the SYPRO Ruby stained 2-DE gel). These results confirm that the increase of spot’s density on X-ray film was indeed from the post-translational modification. Reference protein markers (indicated by triangles in Figure 3B) were used to locate and help to collect these 28 S-nitrosoproteins in the SNAP-treated ECs from the VisPRO stained preparative 2-DE gel (Figure 3C). Identification of S-nitrosoproteins and the Determination of S-NO Site. Because of the skip of protein fixation with acetic acid, a greater yield of trypsin-digested peptides was achieved (data not shown). After the analysis of nLC-MS/MS analyzer and MASCOT in-house search program, identified S-nitroso-

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Figure 3. Detection and identification of S-nitrosoproteins. With the modified biotin switch method, two 2-DE gels loaded with equal amounts of biotinylated lysate (1 mg) were subjected to SYPRO Ruby staining or Western blotted with streptavidin-HRP (A). Total S-nitrosoproteins detected in the SNAP- or NAP-treated ECs were shown as mean ( SE from three independent experiments. Twentyeight S-nitrosoproteins (arrowhead) with at least 1.5-fold increase in S-nitrosation after SNAP treatment were indicated in the Western blot and SYPRO Ruby-stained gels. S-nitrosation of those 28 S-nitrosoproteins was shown after normalizing with the translational level revealed by SYPRO Ruby-staining (B). For mass spectrometry analysis, a preparative 2-DE gel loaded with SNAP-treated endothelial proteins was stained by VisPRO and those 28 S-nitrosoproteins were individually collected for in-gel digestion and nLC-MS/MS analysis for protein identification by searching the NCBI database with MASCOT software search program (C). With the annotated function, those 28 S-nitrosoproteins were classified into five categories (D). The triangles represent the reference protein markers on the gel. Asterisk (*) (p < 0.05) indicates a significant increase of S-nitrosation on proteins as a result from three separate experiments by using Fisher’s LSD.

proteins were grouped into five categories according to their NCBInr-annotated functions (Table 1 and Figure 3D), which are cytoskeleton-related proteins (e.g., vimentin, actin and tropomyosin); heat shock proteins (e.g., HSP90, HSP70 and HSP60); gene regulation-related proteins (e.g., nucleophosmin, chromatin modifying protein, human elongation factor); protein stability-related proteins (e.g., protein disulfide isomerase) and proteins involved in energy regulation (e.g., ATP synthase). In addition, the S-nitrosated cysteine residues of eight of these

28 proteins were also identified by a MASCOT in-house algorithm, using a mass shift of 428.2 Da. (Figure 4A). These results further confirm that the S-nitrosated cysteine can be detected by this modified approach. To evaluate the relative contribution of S-nitrosation in cysteine residue, MS data was further analyzed for label-free quantitation using MASIC software. Four gel spots (no. 14-17), identified as vimentins, were selected for analysis (vimentin has only one cysteine residue, Cys328). Various modifications on Cys328 of vimentin Journal of Proteome Research • Vol. 8, No. 10, 2009 4839

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Table 1. List of S-nitrosoproteins Identified by nLC-MS/MS in EAhy 926 Cells spot no.

protein namea

accession no.b

MW (kDa)/pI Theor.c

MW (kDa)/pI exp.d

sequence coverage (%)

MOWSE score

peptides matched

1

Heat shock protein 90 (HSP90) HSPA9 protein (HSP70) HSPA9 protein (HSP70) HSPA9 protein (HSP70) Chaperonin (HSP60) Chaperonin (HSP60) Protein kinase C substrate 80K-H Prolyl 4-hydroxylase, beta subunit Sarcolectin Protein disulfide isomerase A3 precursor Protein disulfide isomeraserelated protein 5 Mitochondrial ATP synthase F1 complex Mitochondrial ATP synthase F1 complex Vimentin Vimentin Vimentin Vimentin beta actin beta actin beta actin B23 nucleophosmin Human elongation factor-1-delta Human elongation factor-1-delta Chromatin modifying protein 4B B23 nucleophosmin Tropomyosin 3 (29 kDa protein) Tropomyosin 3 isoform 2 Tropomyosin 4

15010550

90.5/4.7

100.2/4.7

9

75

3

21040386 21040386 21040386 31542947 31542947 182855 20070125 4688900 729433

73.8/6.0 73.8/6.0 73.8/6.0 61.0/5.7 61.0/5.7 59.3/4.3 57.1/4.8 51.4/5.8 56.9/6.2

70.5/5.5 70.5/5.6 70.5/5.7 62.4/5.3 62.4/5.4 74.5/4.1 57.7/4.6 52.5/5.6 54.8/5.9

23 33 10 16 26 3 10 10 9

597 883 173 296 480 62 209 208 202

12 21 5 6 11 2 5 4 4

1710248

46.2/5.0

47.4/5.1

21

463

6

32189394

56.5/5.3

49.5/5.0

25

973

10

32189394

56.5/5.3

49.5/4.9

25

432

10

340219 340219 340219 340219 4501885 4501885 4501885 825671 38522 38522 28827795 825671 55665775 24119203 4507651

53.6/5.0 53.6/5.0 53.6/5.0 53.6/5.0 41.7/5.3 41.7/5.3 41.7/5.3 30.9/4.7 31.2/5.0 31.2/5.0 24.9/4.8 30.9/4.7 29.1/4.8 29.0/4.8 28.5/4.7

51.2/5.0 48.2/4.9 46.5/4.8 45.5/4.7 42.5/5.3 42.5/5.4 42.5/4.6 37.1/4.7 33.8/4.9 33.8/4.8 33.8/4.8 33.9/4.7 32.2/4.8 30.0/4.6 28.1/4.5

8 10 13 11 36 36 17 13 12 7 11 13 32 29 17

167 178 223 206 648 631 217 156 116 98 120 129 342 325 230

4 4 4 4 11 11 4 3 3 2 2 4 8 8 4

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

a Function of the protein obtained via the MASCOT software (www.matrixscience.com) search program by querying the NCBInr database.The parameters were set at peptide mass tolerance, (0.4 Da; allowed missed cleavage,1. b Accession number from NCBInr database. c Protein molecular weight and pI annotated in database. d Protein molecular weight and pI calculated from 2-D gel.

were observed (Figure 4BC). The relative modifications in this Cys328 were shown as biotinylation (0.03-3%), methylation (50-99%) and the others (4-50%, acetylation, deamidation, Gln f pyro-Glu, Glu f pyro-Glu, etc.). These data suggest that S-nitrosation only contributes to a minor extent to the various modifications of cysteine residues. In summary, our results clearly demonstrate that the biotin switch method, coupled with Western blot and 2-DE, can effectively screen and identify S-nitrosated proteins in cells.

Discussion S-nitrosated proteins have been detected and routinely analyzed by the biotin switch method, developed by Jeffrey and Synder,13 in which the S-NO bond is selectively reduced by ascorbate and then labeled with biotin. The scarcity of Snitrosoproteins in vivo, however, has presented a challenge for the study of this post-translational modification of proteins.10-12 In the present study, we have detected only trace amounts of S-nitrosated proteins from the eluant of neutravidin agarose (140-340 ng from a 10 cm Petri dish culture; Figure 1B). Accordingly, detection of S-nitrosated proteins in cells needs a great deal of cultured dishes for each study. Approximately 300 µg of isolated S-nitrosoprotein is required for 2-DE separation and mass spectrometric analysis; this would demand an impractical number of culture dishes for each SNAP- and NAPtreated sample. In the present study, only six Petri dishes were needed for each treatment group. With the analytical 2-DE described above, biotinylated (S-nitrosated) protein mixtures 4840

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can be efficiently detected with the high affinity of streptavidin binding (1-10 pg, www.bio-rad.com/blotdetection). In addition, this modified approach benefits by using the fast staining dye, VisPRO. This allows an immediate localization of the protein positions after 2-DE gel, and easy destaining with SDSPAGE running buffer. S-nitrosoproteins, detected by the avidinbased Western blot, were individually collected from the corresponding preparative 2-DE gel. Additionally, due to the skip of fixing reaction with acetic acid in the gel staining, a greater yield of tryptic peptides from gel slice significantly enhanced the analysis of mass spectrometry. The specificity of the biotin switch method for S-nitrosated proteins has been under scrutiny due to the false positives that arose from incomplete blocking of free thiols by MMTS and by the ascorbate-dependent reduction of disulfides.19 Those concerns have been addressed in recent studies that confirmed the specificity of the biotin switch method to S-nitrosation when experiments are performed in the dark.20,21 In this study, we have performed the biotin labeling on protein lysates and the yield of the resulting labeled proteins was shown to be dependent on the amount of ascorbate-reduced free thiols in the protein pool (Supporting Information). These results confirm the specificity of biotin labeling to S-nitrosated thiols. In this study, about 40% of S-nitrosoproteins were identified as cytoskeleton-related proteins, such as vimentins, beta-actin and tropomyosin (Table 1). These results are consistent with those of earlier reports14,15 and are not surprising given the abundance of these proteins in cells.

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Figure 4. Determination of S-nitrosated sites on the S-nitrosoproteins. (A) The MS/MS data of peptide fragments were searched against the NCBInr protein database using a MASCOT in-house algorithm, applying the precursor ion m/z tolerance of 6 ppm. Eight biotinylated peptides were identified by a mass shift of 428.2 Da. Cysteine residues in each protein are indicated in parentheses and the biotinylated cysteine (i.e., originally the S-nitrosated cysteine) is shown in bold. (B) Part of the 2-DE gel showing the four vimentin spots (no. 14-17) with their corresponding molecular weight shift and isoelectric point. (C) MASIC label-free quantitation indicated diverse modifications of this cysteine (Cys328).

Interestingly, four spots were identified as vimentins (NCBI: 340219) and three spots as beta-actins (NCBI: 4501885), having shifted pI and molecular weights (spot no. 14-17 and

18-20). These differences may be due to the other posttranslational modifications, such as phosphorylation22 or glycation.23 Journal of Proteome Research • Vol. 8, No. 10, 2009 4841

research articles HSPs serve as molecular chaperones in protecting cells from damage.24 S-nitrosation of HSP90 promoted the inhibition of its ATPase and endothelial NO synthase regulatory activities.25 The S-nitrosation of HSP90 (spot no. 1) may provide a feedback mechanism for limiting endothelial NO synthase activation. Heat shock 70-kDa protein 9 (HSPA9), a mitochondrial HSP70 (mt-HSP70, spot nos. 2-4) was shown to play a role in protecting the mitochondria from ischemia/reperfusion (I/R)induced damage.26 HSP60, which is heavily induced following stress (such as heat, oxidant and shear blood flow stress23,27), was also identified in this study. Protein disulfide isomerase (PDI) is an enzyme that catalyzes the formation and dissociation of disulfide bonds within proteins. PDI catalyzes protein folding under pathophysiological conditions.28 S-nitrosation of PDI results in neurodegeneration.29 Interestingly, PDI can be reversibly S-nitrosated suggesting that this enzyme is intimately involved in the transport of NO equivalents in and out of the cells.30,31 In the current study, increased S-nitrosation of the prolyl 4-hydroxylase beta subunit of PDI (spot no. 8), PDI A3 precursor (PDA3; spot no. 10) and PDI-related protein (PDI-RP; spot no. 11) in SNAP-treated ECs was noticed. Considering the protective roles of PDI in cells under oxidative stress, S-nitrosation of these proteins may be physiologically important for maintaining endothelial integrity. Higher NO levels leads to a decreased oxygen consumption and lower ATP synthesis in cardiac muscle.32 This is mainly due to the inhibition of the mitochondrial respiratory chain enzymes. Inhibition of the mitochondrial ATPase F1 complex is beneficial by conserving ATP and by reducing Ca2+ uptake into the mitochondria.33 Pharmacological preconditioning with nitrosoglutathione results in an increased S-nitrosation of the mitochondrial ATPase F1 complex.34 Mitochondrial complex IV/cytochrome c oxidase was inhibited by S-nitrosation at two active cysteine residues (Cys196 and Cys200).35 These are consistent with our study that two mitochondria ATPase F1 complexes (spot nos. 12 and 13) were S-nitrosated following SNAP treatment. Nucleophosmin (spot nos. 21 and 25) is an acidic nucleolar phosphoprotein, and is involved in various biological processes, including mRNA processing, ribosome biogenesis and cell proliferation.36 Nucleophosmin is involved in the regulation of the cell cycle and the cellular response to DNA damage.37 In addition, we have also identified protein kinase C substrate 80K-H (spot no. 7), a protein involved in GLUT4 (glucose transporter 4) vesicle trafficking.38 Sarcolectin (spot no. 9) is a secreted endolectin identified in sarcoma as a protein that agglutinates normal and transformed cells.39 Human elongation factor 1 (EF1) delta (spot nos. 22 and 23) is a subunit of the protein complex that participates in the elongation step during the translation of mRNA for protein synthesis.40,41 A recent study has shown that S-nitrosation occurs in EF2 and EF1A-1 in human vascular smooth muscle cells.42 Whether this Snitrosation regulates this protein’s stability remains to be determined. S-nitrosation has emerged as a key post-translational modification of proteins, particularly in cells where nitric oxide levels can surge. By coupling the higher sensitivity of streptavidin-HRP on Western blot-based 2-DE, and the greater tryptic peptide recovery using VisPRO staining, this new approach overcomes problems associated with the limited amounts of S-nitrosoproteins in vivo. With minimal sample requirements, the study 4842

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Huang et al. of protein S-nitrosation in cells under various physiological conditions can be significantly advanced.

Acknowledgment. This work was supported in part by Grants NSC 96-2320-B-001-021 and NSC 98-2752-B-001-001 from the National Science Council, Taipei and a Thematic Project AS-97-FP-L07 from Academia Sinica, Taipei, Taiwan. We are grateful to the core facility laboratory of the Institute of Biomedical Sciences, Academia Sinica, and Mass Solution Biotechnology for image and mass spectrometric analysis. Supporting Information Available: Specificity of biotin labeled on the nitrosocysteine residue. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Hess, D. T.; Matsumoto, A.; Kim, S. O.; Marshall, H. E.; Stamler, J. S. Protein S-nitrosylation: purview and parameters. Nat. Rev. Mol. Cell Biol. 2005, 6 (2), 150–166. (2) Neill, S.; Bright, J.; Desikan, R.; Hancock, J.; Harrison, J.; Wilson, I. Nitric oxide evolution and perception. J. Exp. Bot. 2008, 59 (1), 25–35. (3) Liu, L.; Yan, Y.; Zeng, M.; Zhang, J.; Hanes, M.; Ahearn, G.; McMahon, T. J.; Dickfeld, T.; Marshall, H. E.; Que, L. G.; Stamler, J. S. Essential roles of S-nitrosothiols in vascular homeostasis and endotoxic shock. Cell 2004, 116 (4), 617–628. (4) Hare, J. M.; Stamler, J. S. NO/redox disequilibrium in the failing heart and cardiovascular system. J. Clin. Invest. 2005, 115 (3), 509– 517. (5) Sun, J.; Steenbergen, C.; Murphy, E. S-nitrosylation: NO-related redox signaling to protect against oxidative stress. Antioxid. Redox Signaling 2006, 8 (9-10), 1693–1705. (6) Antonio, M. R.; Santiago, L. Signaling by NO-induced protein S-nitrosylation and S-glutathionylation: convergences and divergences. Cardiovasc. Res. 2007, 75 (2), 220–228. (7) Derakhshan, B.; Hao, G.; Gross, S. S. Balancing reactivity against selectivity: The evolution of protein S-nitrosylation as an effector of cell signaling by nitric oxide. Cardiovasc. Res. 2007, 75 (2), 210– 219. (8) Hancock, J. T. The role of redox in signal transduction. Methods Mol. Biol. 2008, 476, 1–9. (9) Lima, B.; Lam, G. K.; Xie, L.; Diesen, D. L.; Villamizar, N.; Nienaber, J.; Messina, E.; Bowles, D.; Kontos, C. D.; Hare, J. M.; Stamler, J. S.; Howard, A.; Rockman, H. A. Endogenous S-nitrosothiols protect against myocardial injury. Proc. Natl. Acad. Sci. U.S.A. 2009, 106 (15), 6297–6302. (10) Stamler, J. S. S-nitrosothiols in the blood: roles, amounts, and methods of analysis. Circ. Res. 2004, 94 (4), 414–417. (11) Martinez-Ruiz, A.; Lamas, S. Detection and proteomic identification of S-nitrosylated proteins in endothelial cells. Arch. Biochem. Biophys. 2004, 423 (1), 192–199. (12) Torta, F.; Usuelli, V.; Malgaroli, A.; Bachi, A. Proteomic analysis of protein S-nitrosylation. Proteomics 2008, 8 (21), 4484–4494. (13) Jaffrey, S. R.; Erdjument-Bromage, H.; Ferris, C. D.; Tempst, P.; Snyder, S. H. Protein S-nitrosylation: a physiological signal for neuronal nitric oxide. Nat. Cell Biol. 2001, 3 (2), 193–197. (14) Hao, G.; Derakhshan, B.; Shi, L.; Campagne, F.; Gross, S. S. SNOSID, a proteomic method for identification of cysteine S-nitrosylation sites in complex protein mixtures. Proc. Natl. Acad. Sci. U.S.A. 2006, 103 (4), 1012–1017. (15) Yang, Y.; Loscalzo, J. S-nitrosoprotein formation and localization in endothelial cells. Proc. Natl. Acad. Sci. U.S.A. 2005, 102 (1), 117– 122. (16) Chen, Y. Y.; Chu, H. M.; Pan, K. T.; Teng, C. H.; Wang, D. L.; Wang, A. H. J.; Khoo, K. H.; Meng, T. C. Cysteine S-nitrosylation protects protein-tyrosine phosphatase 1B against oxidation-induced permanent inactivation. J. Biol. Chem. 2008, 283 (50), 35265–35272. (17) Jaffrey, S. R.; Snyder, S. H. The biotin switch method for the detection of S-nitrosylated proteins. Sci. STKE 2001, 86, pL1. (18) Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the folin phenol reagent. J. Biol. Chem. 1951, 193 (1), 265–275. (19) Gladwin, M. T.; Wang, X.; Hogg, N. Methodological vexation about thiol oxidation versus S-nitrosation: A commentary on “An ascorbate-dependent artifact that interferes with the interpretation of the biotin-switch assay”. Free Radic. Biol. Med. 2006, 41 (4), 557– 561.

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