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. 2016 Apr 13;94(5):114. doi: 10.1095/biolreprod.116.139337

Quantitative Proteomics Analysis of VEGF-Responsive Endothelial Protein S-Nitrosylation Using Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and LC-MS/MS1

Hong-Hai Zhang 3, Thomas J Lechuga 3, Yuezhou Chen 3, Yingying Yang 4, Lan Huang 4, Dong-Bao Chen 3,2
PMCID: PMC4939742  PMID: 27075618

Abstract

Adduction of a nitric oxide moiety (NO•) to cysteine(s), termed S-nitrosylation (SNO), is a novel mechanism for NO to regulate protein function directly. However, the endothelial SNO-protein network that is affected by endogenous and exogenous NO is obscure. This study was designed to develop a quantitative proteomics approach using stable isotope labeling by amino acids in cell culture for comparing vascular endothelial growth factor (VEGFA)- and NO donor-responsive endothelial nitroso-proteomes. Primary placental endothelial cells were labeled with “light” (L-12C614N4-Arg and L-12C614N2-Lys) or “heavy” (L-13C615N4-Arg and L-13C615N2-Lys) amino acids. The light cells were treated with an NO donor nitrosoglutathione (GSNO, 1 mM) or VEGFA (10 ng/ml) for 30 min, while the heavy cells received vehicle as control. Equal amounts of cellular proteins from the light (GSNO or VEGFA treated) and heavy cells were mixed for labeling SNO-proteins by the biotin switch technique and then trypsin digested. Biotinylated SNO-peptides were purified for identifying SNO-proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Ratios of light to heavy SNO-peptides were calculated for determining the changes of the VEGFA- and GSNO-responsive endothelial nitroso-proteomes. A total of 387 light/heavy pairs of SNO-peptides were identified, corresponding to 213 SNO-proteins that include 125 common and 27 VEGFA- and 61 GSNO-responsive SNO-proteins. The specific SNO-cysteine(s) in each SNO-protein were simultaneously identified. Pathway analysis revealed that SNO-proteins are involved in various endothelial functions, including proliferation, motility, metabolism, and protein synthesis. We collectively conclude that endogenous NO on VEGFA stimulation and exogenous NO from GSNO affect common and different SNO-protein networks, implicating SNO as a critical mechanism for VEGFA stimulation of angiogenesis.

Keywords: endothelium, nitric oxide, SILAC, S-nitrosylation, VEGFA

INTRODUCTION

Nitric oxide (NO) is an essential signaling molecule that is critical for vascular health, participating in the regulation of numerous physiological and pathological processes [1]. Cogent evidence has accumulated to demonstrate a critical role of NO derived from endothelial NO synthase (NOS3) in mediating endothelial cell proliferation and migration during angiogenesis in response to vascular endothelial growth factor (VEGFA) [2–4]. However, the pathways after NO biosynthesis by which VEGFA regulates these cellular processes are largely unknown.

Generation of the second messenger cyclic guanosine monophosphate (cGMP) is the best-defined NO signaling [5]; however, many NO bioactivities are cGMP independent. NO can directly regulate protein function post-translationally [6]. Covalent adduction of an NO moiety (NO•) to reactive cysteines is called S-nitrosylation (SNO). SNO denotes the most crucial cGMP-independent NO signaling; its significance has been compared to homologous O-phosphorylation [7]. SNO is capable of regulating the proteome because reactive cysteine(s) are often present in the catalytic active sites of numerous enzymes [8]. Thus, SNO inevitably participates in many biological pathways, such as calcium signaling, apoptosis, redox signaling, and angiogenesis [6, 7, 9, 10]. Not surprisingly, malfunctions in this critical cellular process have been implicated as a causal factor in diseases such as diabetes, hypertension, preeclampsia, sepsis, cancer, and Alzheimer's [11–14]. Large-scale identification of SNO substrates will certainly advance the understanding of diverse biological phenomena, potentially leading to intervention in any number of disease paradigms.

Although SNO has been recognized as a crucial mechanism by which NO regulates protein function directly, the fragile S-NO bond could not be measured accurately until the biotin switch technique (BST) was invented [15]. In this method, SNO groups are selectively reduced by ascorbate and then labeled with biotin, allowing nitroso-proteins to be readily displayed, affinity purified, and identified. We have previously developed a comprehensive proteomics approach involving BST for labeling SNO-proteins, two-dimensional difference in gel electrophoresis (2D-DIGE) for protein separation, and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)/mass spectrometry (MS) for analyzing the nitroso-proteomes of estrogen-treated endothelial cells and normotensive versus preeclamptic human placentas [14, 16, 17]. This method is perhaps the most powerful one to date, capable of analyzing SNO-proteins at a large scale. However, it is also tedious, semiquantitative, and not truly unbiased and powerful enough for digging out the entire nitroso-proteome. Moreover, it cannot simultaneously identify the specific SNO sites that are absolutely required for downstream functional analysis of SNO in each individual target protein.

An ideal large-scale identification approach for SNO-proteins should at least require specificity, high throughput, identification of proteins with specific SNO site(s), and unbiased quantitation. The powerful MS technology sequences thousands of peptides from complex mixtures without the need for protein separation, offering a simple and fast method for large-scale identification of proteins [18]. Among the many platforms of quantitative proteomics approaches, stable-isotope labeling by amino acids in cell culture (SILAC)/MS has emerged as a simple and powerful one [19]. SILAC, first described in 2002 [20], involves growing two populations of cells by metabolic labeling with stable isotopes: population A in a medium that contains the “light” (normal) essential amino acids (AA) and population B in a medium that contains the “heavy” ones. The heavy AA contains 2H instead of H, 13C instead of 12C, or 15N instead of 14N. Incorporation of the heavy AA into a peptide leads to a known mass shift compared with the peptide that contains the light AA (e.g., 6 Da for 13C6 vs. 12C6) but to no other chemical changes. Then the cells are mixed (A/B = 1:1), and their proteomes are extracted for peptide sequencing by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Each peptide appears as a pair in the mass spectra: one with lower mass contains the light AA from population A, and the other with higher mass contains the heavy AA from population B. Because the light and heavy AAs are chemically identical, except for their mass difference, the ratio of the peak intensities directly yields the ratio of the proteins in population A versus population B. Thus, the A/B ratio reflects the quantitative changes of the protein between the two proteomes [19].

We hypothesized herein that a quantitative proteomics approach based on BST and SILAC/MS can be developed for identifying and quantifying global changes in protein SNO with simultaneous identification of the specific SNO site(s) in each SNO-protein in paired proteomes. With this method, we show that endogenous NO on VEGFA stimulation and exogenous NO from donors differentially regulates the SNO of proteins, with the VEGFA-responsive SNO targets mostly linked to endothelial cell proliferation.

MATERIALS AND METHODS

Materials

Sodium ascorbate, neocuproine, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), ethylenediaminetetraacetic acid (EDTA), diethylene triamine pentaacetic acid (DTPA), copper chloride, bovine serum albumin (BSA), methanol, N-ethylmeleimide (NEM), trifluoroacetic acid, acetonitrile, and all other chemicals, unless specified, were from Sigma (St. Louis, MO). N-(6-[biotinamido] hexyl)-3′-(2′-pyridyldithio) propionamide (biotin-HPDP) was from Thermo Scientific (Rockford, IL). S-nitrosoglutathione (GSNO) was from Cayman (Ann Arbor, MI). Sequencing-grade trypsin was purchased from Promega Corp. (Madison, WI). SILAC RPMI-1640 (deficient in lysine and arginine) was from Invitrogen (Carlsbad, CA). L-12C614N4-Arg, L-12C614N2-Lys, L-13C615N4-Arg, and L-13C615N2-Lys were purchased from Cambridge Isotope Laboratories (Andover, MA).

Cell Isolation, Culture, and SILAC Labeling

Primary ovine fetoplacental artery endothelial cells (oFPAEC) were isolated, validated, and used in passages 6–10 as described previously [21]. The animal use protocol was approved by the Animal Subjects Committees from the University of California, San Diego, and we followed the National Research Council's Guide for the Care and Use of Laboratory Animals. For SILAC labeling, the cells were divided into two populations: one grown in SILAC RPMI-1640 medium supplemented with L-12C614N4-Arg and L-12C614N2-Lys (light medium containing natural isotopes) and the other in SILAC medium supplemented with L-13C615N4-Arg and L-13C615N2-Lys (heavy medium containing stable isotopes). Both light and heavy media were supplemented with 10% dialyzed fetal bovine serum (FBS; GIBCO, Grand Island, NY) and 1% antibiotics. Cells were cultured in SILAC medium for at least three passages to achieve maximum labeling and then used for stimulation. Prior to stimulation, subconfluent (∼80%) cells were cultured with SILAC medium containing 1% dialyzed FBS and 1% antibiotics overnight. Following 1 h of equilibration with fresh SILAC medium with 1% dialyzed FBS and 1% antibiotics, the cells were treated with VEGFA or an NO donor GSNO for up to 2 h. In this study, 10 ng/ml VEGFA was used based on our previous studies showing that VEGFA consistently stimulates in vitro angiogenesis of placental artery endothelial cells at least partially mediated by endogenously produced NO via NOS3 activation, without any notable negative effects up to 50 ng/ml [22, 23]. A relatively high level of endogenous trans-nitrosylating agent GSNO was used as an NO donor, which has been widely reported [24–26], for pharmacological comparison of exogenous NO. Cell lysates were prepared in a nondenaturing buffer [27] containing 1% protease inhibitor cocktail, and protein concentration was determined by the BCA Protein Assay Kit (Pierce, Rockford, IL).

SDS-PAGE and In-Gel Digestion

Protein samples were dissolved in SDS sample buffer and separated by 10%–12% SDS-PAGE, and bands of interest were cut out and digested in gel as previously described [28]. Briefly, minced gel pieces were washed with 25 mmol/L NH4HCO3 in 50% acetonitrile, dried in a Speedvac, and then rehydrated in 25 mmol/L NH4HCO3 solution containing trypsin. After overnight digestion at 37°C, the peptides were extracted with HPLC-grade water once, followed with 5% formic acid/50% acetonitrile three times. The combined supernatants were dried by Speedvac and then dissolved in 2% formic acid/3% acetonitrile. Peptides were stored at −20°C until LC-MS/MS was performed.

BST [16]

The oFPAEC cells (∼1 × 106) were treated with or without VEGFA/GSNO for 30 min. Equal amounts (0.5 mg/group) of proteins from VEGFA- or GSNO-treated light cells were mixed with that of the control heavy cells. Protein content of the mixtures was redetermined and then adjusted to 0.6 mg/ml protein in a blocking buffer (250 mmol/L HEPES, pH 7.7, 1 mmol/L DTPA, 0.1 mmol/L neocuproine, 50 mmol/L NEM, and 2.5% SDS). After blocking by incubation in dark at 50°C for 30 min, the proteins were precipitated by incubation with acetone (1:3, vol/vol) at −20°C for 2 h and washed with cold acetone (70%) once. The precipitated proteins were resuspended in a labeling buffer (25 mmol/L HEPES, pH 7.7, 30 mmol/L sodium ascorbate, 0.1 μmol/L CuCl2, 0.4 mmol/L biotin-HPDP, and 1% SDS); readjusted to 0.6 mg/ml; and then incubated in the dark at 37°C for 1 h with occasional agitation. The biotinylated samples were then acetone precipitated again to remove excess biotin-HPDP.

Avidin Capture of SNO-Proteins and Immunoblotting

Total biotinylated SNO-proteins were captured by incubation with 50 μl of NeutrAvidin protein-coated beads (Thermo Scientific) at 4°C overnight. The avidin-captured SNO-proteins were eluted from the beads with SDS sample buffer (100 μl) containing 100 mmol/L 2-mercaptoethenol at 37°C for 20 min. Protein samples were separated by 10%–12% SDS-PAGE and then transferred onto polyvinylidene fluoride membranes for immunoblotting with specific antibodies as previously described [21]. Anti-cofilin-1 (CFL1) antibody was from Abcam (San Francisco, CA). Anti-β-actin monoclonal antibody (1:10 000) was from Ambion (Austin, TX). Antibodies against heat shock protein-70 (HSP70, 1:500) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:500) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Band intensity was quantified by multiplying the absorbance of the surface areas using the NIH ImageJ.

Trypsin Digestion and SNO-Peptide Purification

Following BST labeling, the acetone-precipitated protein samples (1 mg/mixture) were thoroughly dissolved in 200 μl of digestion buffer (50 mmol/L NH4HCO3, 1 M urea) containing 20 μg of trypsin. After trypsin digestion at 37°C overnight, the samples were incubated with 50 μl of NeutrAvidin protein-coated beads at room temperature for 2 h for capturing the biotinylated SNO-peptides as described previously [29]. The SNO-peptides were eluted from the beads with 100 μl of 0.4% trifluoroacetic acid in 30% acetonitrile and then stored at −20°C until LC-MS/MS was performed. For LC-MS, 5 μg of SNO-peptides were loaded in each mass spec analysis.

LC-MS/MS Analysis and SNO-Peptide Identification

LC-MS/MS analysis of the purified SNO-peptides was performed by using an LTQ-Orbitrap XL MS (Thermo Scientific) coupled with an Eksigent NanoLC system (Eksigent, Dublin, CA), exactly as previously described [29]. The LC analysis was performed using a capillary column (100-μm inner diameter × 150 mm long) packed with C18 resins (GL Sciences, Torrance, CA), and the peptides were eluted using a linear gradient of 2%–40% B in 35 min; (solvent A: 100% H2O/0.1% formic acid; solvent B: 100% acetonitrile/0.1% formic acid). A cycle of one full Fourier transform (FT) MS scan mass spectrum (350–1800 m/z, resolution of 60 000 at m/z 400) was followed by 10 data-dependent MS/MS acquired in the linear ion trap with normalized collision energy (setting of 35%). Target ions selected for MS/MS were dynamically excluded for 30 sec. Monoisotopic masses of parent ions and corresponding fragment ions, parent ion charge states, and ion intensities from LC-MS/MS spectra were extracted using in-house software based on the Raw_Extract script from Xcalibur v2.4. The data were searched using the Batch-Tag within the developmental version (v5.8.0) of Protein Prospector against a decoy database consisting of a normal Swissprot database concatenated with its randomized v9 (SwissProt.2010.03.30.random.concat with total 864 896 protein entries). The mass accuracy for parent ions and fragment ions were set as ±20 ppm and 0.8 Da, respectively. Trypsin was set as the enzyme, and the maximum of two missed cleavages were allowed. Biotin-HPDP labeling of cysteine residues was selected as constant modifications with a monoisotopic mass shift of 428.192 Da. In addition, two additional variable modifications were included: 13C615N4-labeled arginine and 13C6 15N2-labeled lysine (heavy AAs). To quantify relative protein abundance changes, the Search Compare function was used to determine the L/H ratios based on the intensities of the monoisotopic masses of the parent ion peptide pairs [30]. Search Compare also corrects for the isotopic purity of the heavy AAs, which was set to 98% purity with the signal/noise threshold set at 10. The peptide peak intensities were averaged across the elution profile (30 sec). The proteins identified by one or two peptides were confirmed by manual inspection of the MS/MS spectra. The relative abundance ratios were also validated by the raw spectra. If two or more S-nitrosylation sites were identified for a specific SNO-protein, the final ratio was calculated as the average of ratios of all S-nitrosylation peptides for the protein.

Bioinformatics Analysis

Ingenuity pathway analysis (IPA; http://www.ingenuity.com) was used to obtain information regarding relationships, biological mechanisms, functions, and pathways of differentially regulated SNO-proteins. All differentially regulated SNO-proteins (focus molecules) with their corresponding Swiss-Prot accession numbers and fold change were imported into the IPA. Nodal molecules are those that were not identified but were found in the IPA to be either potential targets or related molecules to the identified molecules. The randomness of a biological function or network obtained by IPA is determined by calculating the P value using the Fischer exact test. The P value represents that the likelihood association between the set of the identified SNO-proteins, and a given process or pathway is due to random chance. The score of each network is a numerical value to approximate the degree of relevance and size of a network to the molecules in the given data set. The network is considered to be significant if the score is >2. The identified SNO-proteins were further classified in the UniProt knowledge database for searching their functions.

Experimental Replication and Statistical Analysis

All experiments were repeated at least three times using cells from different animals. Data were presented as mean ± SEM. Statistical analysis was performed by one-way ANOVA, followed by the Student-Newman-Keuls test for multiple comparisons using SigmaStat 3.5 (Systat Software Inc., San Jose, CA). Significant difference was defined as P < 0.05.

RESULTS

Effects of VEGFA and GSNO on Protein SNO in Endothelial Cells

To determine the effects of VEGFA and GSNO on protein SNO in endothelial cells, we first measured total levels of SNO-proteins in oFPAEC treated with or without VEGFA (10 ng/ml) or an NO donor GSNO (1 mM) for up to 2 h. In VEGFA-treated cells, total levels of SNO-proteins began to increase at 10 min, maximized around 30 min, and returned to baseline at 60 min (Fig. 1). In GSNO-treated cells, total levels of SNO-proteins began to increase at 10 min, reached levels comparable to maximal response to VEGFA at around 30 min, and continued to increase at least 2 h. In keeping with our recent studies showing that endogenous NO derived from NOS3 mediates VEGFA stimulation of protein SNO in endothelial cells [31], the different SNO time courses demonstrate that endogenous NO on VEGFA stimulation and exogenous NO from GSNO differentially stimulate endothelial protein SNO.

FIG. 1.

FIG. 1

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Time courses of VEGFA- and GSNO-induced endothelial protein S-nitrosylation. Subconfluent cells were treated with or without 10 ng/ml VEGFA or 1 mM GSNO for the indicated times. Total protein extracts were harvested for determining total SNO-proteins. Representative blots of total SNO-proteins and β-actin of one typical experiment are shown. Lower graphs summarize data (mean ± SEM, n = 3) from three independent experiments using cells from different animals. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. control.

Development of a Quantitative Nitroso-Proteomics Method Using BST, SILAC, and LC-MS/MS

The strategy of SILAC/BST-based quantitative nitroso-proteomics analysis method is shown schematically in Figure 2. In this method, a mass difference was introduced between the control and VEGFA/GSNO-treated cells to identify and quantify SNO via MS. Two populations of cells were labeled with light (L-12C614N4-Arg and L-12C614N2-Lys) and heavy (L-13C615N4-Arg and L-13C615N2-Lys) isotopes, respectively. The cells were cultured with the light and heavy AAs separately for more than three passages to reach saturated labeling. Similar to previous reports [32, 33] showing that isotope labeling had no influence on cell growth and properties, oFPAEC seemed to proliferate and grow morphologically normal, as the two groups of cells did not differ significantly after four passages in the SILAC labeling medium containing light or heavy AAs. As determined by LC-MS/MS (Fig. 3), the incorporation of heavy AAs continuously increased with passage and saturated at a plateau rate of ∼98% after four passages.

FIG. 2.

FIG. 2

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Schematic overview of BST SILAC and LC-MS/MS assay. Cells were labeled with light or heavy isotope-labeled amino acids. Light isotope-labeled cells were treated with VEGFA or GSNO (treatment group, L1 or L2), and heavy isotope-labeled cells were used as control group (H). Equal amounts of cell lysates from treatment and control groups were mixed for labeling SNO-proteins by BST. After tryptic digestion, the biotinylated SNO-peptides were purified by Avidin Capture and then analyzed by LC-MS/MS for protein identification. The spectral data of each paired SNO-peptides were used to calculate the ratios (L1/H and L2/H) that give the quantitative responses to treatments.

FIG. 3.

FIG. 3

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Incorporation of stable isotope into oFPAEC. Proteins from cells grown in the medium with heavy amino acids at passages 1–6 were digested and analyzed with LC-MS/MS.

The heavy cells (H) were used as the control to avoid possible excess utilization of arginine by NOS3 during NO synthesis [34]. The light cells were used for treatment with 10 ng/ml VEGFA (L1) or 1 mM GSNO (L2). Treatments with both VEGFA and GSNO induced comparable significant SNO responses at 30 min; thereafter, the VEGFA response declined, and the GSNO response continued to increase (Fig. 1). These data suggest that at this time point, VEGFA and GSNO stimulates the most common SNO-protein targets of functional significance. Thirty minutes of treatment was therefore chosen for analyzing the differential VEGFA and GSNO-responsive SNO-proteins. Following treatment, both cell populations were harvested and lysed. Equal amounts of proteins from group L1 or L2 were mixed with H; the mixtures (i.e., L1/H and L2/H) were subjected to BST and trypsin digestion. The biotinylated peptides (SNO-peptides) were affinity purified and then identified by MS. If an SNO-protein is present in the samples, the resulting Arg/Lys-containing peptides will be observed as pairs with heavy or light AAs originated from the incorporated isotopes. The abundance of SNO-proteins in the two groups could be quantified according to the ratios of the peak intensities of the paired Arg/Lys peptides determined by LC-MS/MS (i.e., R1 = L1/H and R2 = L2/H). Peptides derived from the proteins present in only one sample and peptides containing no arginine/lysine were observed as a singlet in the LC-MS spectra; however, these peptides were nitrosylated as well since only SNO-peptides were purified for LC-MS/MS.

Comparisons of the Common and VEGFA- or GSNO-Responsive Nitroso-Proteomes

Although both endogenous NO on VEGFA stimulation and exogenous NO from donors stimulate placental endothelial cell proliferation [35], endogenous NO on estradiol-17β stimulation and exogenous NO from donors differentially regulate mitochondrial SNO in human umbilical cord vein endothelial cells [29]. Thus, it is possible that endogenous NO on VEGFA stimulation and exogenous NO from donors might affect common and different proteins via SNO. We then analyzed the nitroso-proteomes of the control and VEGFA- and GSNO-treated oFPAEC by the quantitative proteomics method using BST, SILAC, and LC-MS/MS as illustrated in Figure 2.

After a database search, proteins with unique cysteine-containing SILAC pair(s) were used as a criterion for identifying SNO-proteins. A total of 387 SNO-peptide pairs were identified; there were overlaps in the mixtures of VEGFA/control (L1/H) and GSNO/control (L2/H) samples, including 72 exclusively present in former, 127 only in the later, and 188 in both groups. As a result, a total of 213 SNO-proteins were confidently identified, including 27 in L1/H, 61 in L2/H, and 125 in both groups.

The Search Compare program within the developmental version of Protein Prospector was used to calculate the relative abundance ratios of Arg/Lys-containing peptides based on ion intensities of monoisotopic peaks observed in the LC-MS spectra when the peptides were sequenced and subsequently identified during database searching. For instance, in the SILAC peptide MAASCILLHTGQK from alcohol dehydrogenase [NADP+] (AKR1A1, Q3ZCJ2) in the GSNO-treated group, the three left peaks shown in Figure 4 are the MS signals of light isotope-labeled peptides. The heavy isotope-labeled peptides on the right have a +4 m/z shift because there is a Lys in these peptides, and L-13C615N2-Lys is +8 Da heavier in mass compared to L-12C614N2-Lys. Based on the intensity of the peaks, the ratio of L/H was calculated as 2.64. Since MAASCILLHTGQK is the only cysteine-containing SILAC peptide identified, this result showed that the SNO of AKR1A1 increased by 2.64-fold by GSNO treatment. For proteins with multiple SILAC peptide pairs identified, the overall SNO ratio was calculated by averaging all single ratios. Based on this algorithm, the changes of all SNO-proteins were quantified and are summarized in Table 1. Based on a standard statistic power calculation (n = 3, 10% RSD, 95% confidence interval) [36, 37] and the likelihood of being able to validate a change by alternative methodology, we considered a fold change of 1.3 (or a ratio of 0.78) as a cutoff value for determining a significant change in S-nitrosylation level. We found a total of 182 SNO-proteins that were identified to be responsive to treatment with VEGFA and GSNO. These included 69 that were significantly enhanced by VEGFA treatment and 94 that were significantly enhanced by GSNO treatment. Moreover, since the specific SNO site(s) were labeled with biotin by BST and only biotinylated SNO-peptides were identified by LC-MS/MS, all SNO-proteins were identified with the specific SNO site(s) simultaneously. The quantitation data were from three independent experiments using cells from different animals. The identified proteins/peptides were observed in all three runs. The mean ratio was calculated and is listed in Table 2. For those whose P values were >0.05, the ratio is listed in Table 2 to indicate that the specific protein targets were detected in all three runs. For those peptides in only one or two runs, they were omitted and are listed as N/A in Table 2.

FIG. 4.

FIG. 4

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Quantification and identification of SNO peptides. Comparison of light and heavy reagent elution profiles for the alcohol dehydrogenase peptide MAASCILLHTGQK.

TABLE 1.

Ingenuity pathway analysis of nitroso-proteomes.*

graphic file with name i0006-3363-94-5-114-t01.jpg

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* 

Signaling pathways and molecular functions were analyzed by the Ingenuity Pathway Analysis software. The calculations are based on the VEGF- and GSNO-responsive protein sets, respectively. The column “Overlap” was used to describe the percentage of known components existing in the VEGF- and GSNO-responsive protein sets, respectively, against the total components of each individual classical canonical pathway. N/A, not detected.

TABLE 2.

The nitroso-proteomes in VEGF- or GSNO-treated ovine fetoplacental artery endothelial cells identified by BST-SILAC mass spectrometry.

graphic file with name i0006-3363-94-5-114-t02.jpg

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a 

N/A, not detected.

* 

Significant changes (P < 0.05 among three independent experiments).

§ 

The SNO-cysteine in the identified peptides containing two or more cysteines.

‡ 

The SNO-cysteine was not determined in the identified peptides containing two or more cysteines.

Validation of the Identified SNO-Proteins

To validate the changes of SNO-proteins identified by the quantitative proteomics method, we analyzed SNO-proteins identified to be VEGFA/GSNO-responsive SNO targets, including HSP70, GAPDH, and CFL1 in endothelial cells by immunoblotting of the purified total SNO-proteins with specific antibodies. As summarized in Figure 5, VEGFA and GSNO stimulated time-dependent changes in the SNO of each of these proteins. The VEGFA- and GSNO-induced time courses were similar with that of the total SNO responses to VEGFA and GSNO as shown in Figure 1. Both induced maximal responses at 30 min posttreatment; however, VEGFA-induced responses returns to baseline after 60 min, and the GSNO-induced response persisted at high levels up to 2 h. VEGFA stimulated SNO of HSP70, GAPDH, and CFL1 by 2.22-, 1.77-, and 1.66-fold at 30 min, respectively, whereas GSNO stimulated SNO of HSP70, GAPDH, and CFL1 by 2.47-, 2.79-, and 2.55-fold, respectively. These changes were comparable to those summarized in Table 2; VEGFA stimulated SNO of HSP70, GAPDH, and CFL1 by 2.14-, 1.31-, and 2.01-fold at 30 min, respectively, whereas GSNO stimulated SNO of HSP70, GAPDH, and CFL1 by 2.58-, 2.78-, and 2.68-fold, respectively.

FIG. 5.

FIG. 5

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Time courses of specific endothelial SNO-proteins in response to stimulation with VEGFA and GSNO. Cells were treated with VEGFA or GSNO for up to 2 h. Whole cell lysates were prepared for labeling SNO-proteins by BST. The biotinylated SNO-proteins were captured by avidin-coated beads for analyzing total and nitrosylated HSP70, GAPDH, and CFL1 by immunoblotting with specific antibodies, respectively. Lower graphs summarize data (mean ± SEM, n = 3) from three independent experiments using oFPAEC from different animals. *P < 0.05 versus control.

Bioinformatics Analysis

Functional analysis suggested that the SNO-proteins are associated with various functions, including cell cycle and proliferation, cytoskeleton and motility, metabolism, protein synthase and modification, and cellular signaling and transportation (Fig. 6). The biological function analysis indicates that EIF2 signaling is the most significant signaling pathway for both VEGFA- and GSNO-responsive SNO-proteins, with 22 and 25 focus molecules and a −lg (P value) of 20.1 and 21.5, respectively (Table 1). Gene expression/protein synthesis was identified as the TOP 1 molecular and cellular function for the VEGFA- and GSNO-responsive SNO-proteins, with the most significant network analysis score of 64, representing 31 focus molecules (Fig. 7).

FIG. 6.

FIG. 6

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Summary of the VEGFA- and GSNO-responsive endothelial SNO-proteins. Venn diagram illustrates the overlap of the identified SNO-proteins in oFPAEC treated with VEGFA or GSNO. Pie diagrams illustrate the function classification of the identified SNO-proteins from oFPAEC treated with VEGFA or GSNO, respectively.

FIG. 7.

FIG. 7

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VEGFA- and GNSO-induced endothelial SNO-protein networks. Networks of the VEGFA/GSNO-responsive endothelial SNO-proteins were generated by using IPA. Proteins were represented as nodes, and the biological relationship between two nodes is represented as a line. All lines are supported by at least one published reference. Solid lines represent a direct relationship, and dashed lines represent an indirect relationship. The solid and broken semicircles on some proteins indicate direct/indirect self-regulation, i.e., positive or negative feedback, respectively. Lines or semicircles with arrows stand for stimulation or activation on downstream targets. The green (or red) node represents VEGFA (or GSNO) stimulation, respectively, and the yellow node represents stimulation by both VEGFA and GSNO. The shape of each node represents the functional class of proteins.

DISCUSSION

In the present study, we have successfully developed a quantitative proteomics approach for analyzing global protein SNO in paired proteomes by using BST, SILAC, and LC-MS/MS. With this high-throughput approach, we show herein for the first time that endogenous NO via NOS3 activation on VEGFA stimulation and a widely used NO donor GSNO [24–26] regulate common and different target proteins through SNO, with the VEGFA-responsive SNO targets mostly linked to endothelial cell proliferation. Because endothelial NOS3-derived NO plays a key role in mediating VEGF-stimulated angiogenesis [2–4] and our recent studies showing that VEGFA stimulates SNO via NOS3-derived NO in endothelial cells [31, 38], the VEGFA-responsive endothelial SNO-proteins identified herein provide a fundamental database for future functional analysis of SNO in regulating endothelial cell biology on VEGFA stimulation, especially as it pertains to angiogenesis.

VEGFA is the primary angiogenic factor whose role in angiogenesis has been well documented to be mediated mainly by NO produced by NOS3 activation [2–4]. We have shown that endogenous NO derived from NOS3 is critical for mediating VEGFA-induced placental endothelial cell angiogenesis [21, 39, 40], and exogenous NO from donors also stimulates placental endothelial cell proliferation [35]. We also have shown that activation of mitogen-activated protein kinases and protein kinase B/Akt pathways is important for both VEGFA-stimulated placental endothelial proliferation [35, 41] and gene expression [42]. Although these signaling pathways are downstream of NOS3-NO in mediating VEGFA-induced placental angiogenesis, they seem not to be regulated by NO directly.

SNO has been increasingly recognized as a critical NO signaling mechanism for NO to directly regulate protein function to participate in nearly all major categories of biological pathways [6]. For instance, we have recently shown that VEGFA and estradiol-17β stimulates dynamic SNO of proteins in endothelial cells, which are linked to a variety of biological functions [31, 38]. We have recently shown that SNO of CFL1 on different cysteines have different functions in response to different extracellular stimuli. SNO on Cys80/139 results in increased actin-severing activity of CFL1, which mediates VEGFA-stimulated endothelial cytoskeleton remodeling and cell migration [31]. However, on estrogen stimulation, SNO of CFL1 occurs primarily on Cys80, which in turn mediates estrogen stimulation of cytoskeleton remodeling in endothelial cells [38]. Thus, our findings have further highlighted the importance of identification of the specific SNO sites for delineating the mechanism by which SNO regulates endothelial cell function in response to stimulation.

As a posttranslational modification of proteins on specific cysteine residues, SNO is capable of affecting the proteome of a cell. Similar to O-phosphorylation and other posttranslational modifications [7], SNO occurs only on specific cysteine residues with a specific surrounding sequence [6]. Thus, identification of the specific SNO sites (reactive cysteines) is a prerequisite for subsequently deciphering the functional consequences of SNO of a specific protein. However, the detection of SNO used to be troublesome because of its low level and liability. Antibodies directed against the SNO functionality have suffered from a lack of specificity and loss of sensitivity during immunodetection, as the S-NO bond is sensitive to redox changes even in in vitro assays [43, 44]. The invention of a reliable and reproducible BST for measuring SNO in 2001 [15] has greatly accelerated the understanding of protein SNO in biology and medicine, exemplified by an explosion of more than 1500 publications on this topic to date. The three-step BST method selectively converts the S-NO groups into stable biotinylated ones, allowing SNO-proteins to be readily displayed, affinity purified, and identified [15]. According to this principle, many modified BST methods have been developed. For instance, replacing the biotin tag with fluorescence tags [45] allows the SNO-proteins to be detected in cells/tissues in situ [16, 46]. We have reported a proteomics approach for analyzing SNO-proteins from paired proteomes by labeling them with two different fluorescently labeled tags and 2D-DIGE/MALDI-TOF/MS; this method allows identifying partial nitroso-proteomes in endothelial cells and human placentas and relatively quantifying changes in endothelial protein SNO in response to estrogen and placental SNO under the influence of preeclampsia [14, 16, 17]. Others also have developed different proteomics approaches based on BST for analyzing protein SNO [47–49]. Although the proteomics approaches reported to date have provided insightful databases of SNO-proteins in various tissues/cells and greatly accelerated the understanding of SNO, none can meet all the criteria of an ideal method for analyzing global protein SNO, at least including specificity, high-throughput unbiased quantitation and, most important, simultaneous identification of SNO sites.

The proteomics approach developed herein offers not only the specificity (BST labeling) and high throughput (MS/MS) required for large-scale analysis of SNO-proteins in paired proteomes but also unbiased quantitation of the changes in SNO-proteins using SILAC technology. Moreover, this method is capable of simultaneously identifying the specific SNO sites (i.e., reactive SNO-cysteine[s]) in each SNO-protein. These advantages make this method perhaps the most powerful method to date for analyzing global protein SNO in cell culture studies. However, there is a chance that some unique regulatory SNO-proteins might not be able to be identified by this method. For instance, if an SNO-protein is present in only one sample, MS/MS analysis will not give paired spectral readouts of light and heavy SNO-peptides from the paired proteomes using SILAC-based quantitative proteomics technology; then this unique SNO-protein will not be picked up. A potential solution for this limitation can be resolved by using a so-called super-SILAC mix [50, 51] that can be a mixture of many different types of cells labeled with heavy AAs as an internal reference to add a “pseudo”-readout for the sample that does not have the unique SNO-protein.

In this study, 10 ng/ml VEGF and 1 mM GSNO were chosen to treat endothelial cells for 30 min to prepare the starting materials for comparing the VEGFA- and GSNO-responsive endothelial SNO-proteomes for the following reasons. We have determined the optimal dose of VEGFA to be ∼10 ng/ml in stimulating eNOS activation and NO production, ERK1/2 and Akt1 signaling, and in vitro angiogenesis with detailed dose-response and time-course studies in many of our publications (reviewed in Chen and Zheng [52]); 1 mM GSNO has been widely used as a potent exogenous NO donor in cell culture studies [53–55]. More recently, we also have compared the effects of 10 ng/ml VEGF and 1 mM GSNO side by side of SNO of cofilin-1 (CFL1) and found that 10 ng/ml VEGF or 1 mM GSNO stimulated comparable SNO responses in CFL-1 in endothelial cells [31]. With these starting materials and the SILAC-based proteomics technology, we are able to quantitatively distinguish the VEGFA-responsive from the GSNO-responsive SNO-proteins in endothelial cells. We have first shown herein that endogenous NO by VEGFA stimulation and exogenous NO from GSNO dynamically regulate endothelial protein SNO, targeting common and different sets of SNO-proteins, including 125 common SNO-proteins in both VEGFA- and GSNO-treated cells and 27 VEGFA-responsive and 61 GSNO-responsive SNO-proteins. SNO of some unique targets are induced in even opposite changes by VEGFA and GSNO. For instance, the SNO level of actin-related protein 3 (ACTR3) is increased by GSNO but decreased by VEGFA treatment (Table 2). Thus, NO donors and endogenous NO stimulated by VEGFA and potentially other physiological stimuli induce different and even opposite biological responses. Of note, the time courses of endothelial SNO responses to stimulation with VEGFA and an NO donor GSNO are quite different (Fig. 1) [31]. Both maximize at 30 min after stimulation; however, VEGFA-induced SNO response returns to baseline, while the GSNO-induced response persists at 60 min. In this study, the responses were analyzed at only one time point (30 min) to develop/validate the SILAC-based method. However, these time courses suggest that SNO is regulated in a spatiotemporal manner in response to stimulation. Further analysis of the detailed SNO responses to VEGFA and GSNO over time is needed for revealing important spatiotemporal SNO-protein networks in endothelial cells.

Leading pathway analysis of the 182 VEGFA/GSNO-responsive SNO-proteins (ratio to control >1.3) showed that they are involved in cell cycle and proliferation, cellular cytoskeleton and motility, protein syntheses and modification, cellular signaling and transportation, and metabolism. Of note, we have confirmed herein that CFL1 is one of the VEGFA-responsive SNO target proteins as revealed in our previous studies using different approaches [31]. The primary function of CFL1 is to regulate cytoskeleton remodeling via depolymerizing/severing actin filaments [56]. Our previous work has shown that SNO on CFL1 inhibits the VEGFA-stimulated filopodium formation in endothelial cells [31]. The SNO-dependent mechanism(s) of CFL1 has been demonstrated to regulate VEGFA-induced endothelial cell migration occurring at the early stage of cell migration by affecting filopodium formation, therefore mediating the VEGFA-induced angiogenesis response [31].

In addition to CFL1, a subset of proteins was identified as VEGFA-responsive SNO-proteins whose functions are associated with actin dynamics and cytoskeleton remodeling [57], including myosin, fasin, and actin-related protein 2/3 complex (ARP2/3). Myosin is a member of the ATP-dependent motor proteins responsible for actin-based motility [58]. Myosin-based contraction of filamentous actin (F-actin) is an important determinant of endothelial cell stiffness [59], which is a mechanical property of the vessel wall that affects blood pressure, permeability, and inflammation. It has been recently reported that SNO significantly reduces the Mg2+-ATPase activity of myosin [60], suggesting myosin to be an important regulatory target for vascular wall health via SNO. Consistent with our previous reports [17, 61], fasin is another VEGFA-responsive SNO-protein that is an actin cross-linking protein present at the leading edges and borders of cells and is well known for its role in promoting cell invasion and migration in vitro [62]. ARP2/3 complex coordinates signals to the actin cytoskeleton and initiates F-actin assembly in response to stimulation [63]. Cyclase-associated protein 1 promotes rapid actin dynamics in conjunction with cofilin [64]. F-actin capping protein caps barbed ends of the actin filament to limit growth of the newly formed actin filament [65]. These findings indicate that SNO is an important mechanism for mediating VEGFA-induced actin dynamics and cytoskeleton remodeling.

There are 12 VEGFA-responsive SNO-proteins that are involved in cell cycle and proliferation, including calpain-2 catalytic subunit, calpain small subunit 1, galectin-1, poly (rC)-binding protein 2, GTP-binding nuclear protein Ran, protein S100-A11, interleukin enhancer-binding factor 3, DNA replication licensing factor MCM5, and four 14-3-3 proteins. The calpains, a ubiquitous family of calcium-dependent cytosolic cysteine proteases, are thought to initiate cytoskeletal breakdown by cleaving proteins important in linking components of the cytoskeleton together and to the cell membrane [66]. NO inhibits cytoskeletal breakdown in skeletal muscle cells by inhibiting calpain cleavage activity via SNO, thereby protecting the cells from ionophore-induced proteolysis [67]. Previous studies have shown that calpain proteolysis may proteolytically disorganize VE-cadherin and subsequently accelerate atherosclerosis [68], suggesting that SNO-mediated calpain activity may provide a therapeutic approach to protect the endothelium from injury or disease. Galectins are members of a highly conserved family of β-galactoside-binding animal lectins and can differentially affect cellular maturation and function. NO accelerates oxidization of galectin-1 [69], affecting galectin-1 function in promoting proliferation of adult neural stem cells [70]. Galectin also can enhance the survival of breast cancer cells against NO and peroxinitrite during experimental hepatic ischemia-reperfusion injury or direct treatment [71], indicating a potential protective mechanism of SNO on galectin in cell proliferation and against apoptosis. The cellular function of poly (rC)-binding protein 2 is to form ribonucleoprotein complexes with cellular mRNAs, which regulate mRNA stability and translation [72]. GTP-binding nuclear protein Ran, a Ran-GTPase, is involved in nucleocytoplasmic protein import and plays a role in the cell cycle [73]. Ran has been previously identified as an SNO-protein in mouse lung alveolar type II epithelial cells treated with exogenous NO donors [74]. DNA replication licensing factor MCM5 is a member of a family of minichromosome maintenance factors, which is responsible for restricting DNA synthesis only once per cell cycle [75]. The 14-3-3 proteins form a highly conserved family of acidic dimeric proteins with a subunit mass of approximately 30 kDa; overall, they inhibit cell cycle progression and apoptosis and may act as stimulatory or inhibitory factors in signal transduction [76]. Overall these VEGFA-responsive SNO targets are important to mediate the angiogenic effects of VEGFA.

Among the 125 proteins that are common SNO targets responsive to both VEGFA and GSNO, the SNO response to GSNO is in general greater than that of VEGFA. Interestingly, a cysteine-containing peptide, ILYSQCGDVMR (SNO site underlined), is identified in myosin light polypeptide 6 in both VEGFA- and GSNO-treated cells, whereas another cysteine-containing peptide, MCDFTEDQTAEFK (SNO site underlined), is found only in the VEGFA-treated cells. These results show that VEGFA and GSNO stimulate SNO of the same protein on different SNO sites. In keeping with our recent functional studies showing that VEGFA and estrogens regulate CFL1 function via SNO on different sites [31, 38], these results not only show the complexity of SNO in regulating protein function but also strengthen the importance of identifying specific SNO sites for delineating the function of SNO in a specific protein in response to different stimulation.

We have successfully developed a BST/SILAC-based quantitative proteomics method for unbiased analysis of global SNO with identification of the specific SNO sites simultaneously. By using this novel assay, we have identified the common and specific SNO protein targets affected by endogenous NO on VEGFA stimulation and exogenous NO from GSNO. Quantitative and leading pathway analysis of the VEGFA- and GSNO-responsive nitroso-proteomes reveals that SNO is a critical mechanism for VEGFA stimulation of endothelial cell proliferation and motility, which are critical steps for angiogenesis. With the identification of specific SNO sites in each SNO-protein, the VEGFA- and GSNO-responsive endothelial nitroso-proteomes identified herein provide fundamental databases for delineating the functional significance of SNO in endothelial cell biology.

Footnotes

1

Supported in part by National Institutes of Health (NIH) grants R03 HD84972, R21 HL98746, and RO1 HL70562 (to D.B.C.), RO1 GM074830 (to L.H.), and American Heart Association (AHA) grant 13SDG13910006 (to H.-H.Z.). The content is solely the responsibility of the authors and does not necessarily the official views of the NIH and AHA.

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