Article pubs.acs.org/jpr
Quantitative Proteome of Medulla Oblongata in Spontaneously Hypertensive Rats Dongmei Sun,† Yu Cheng,‡ Danfeng Zhou,† Tanshu Liu,† Shaoqin Chen,† Jing Liang,† Chunzhi Tang,† and Xinsheng Lai*,† †
College of Acupuncture and Moxibustion, Guangzhou University of Chinese Medicine, Guangzhou 510405, China School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China
‡
S Supporting Information *
ABSTRACT: We performed an extensive quantitative proteomic analysis on the pooled medulla sample of the 11-week-old spontaneously hypertensive rats (SHR) compared to age-matched normotensive Wistar rats, using iTRAQ technology coupled with nano two-dimentional liquid chromatography followed by high resolution mass spectrometric abundance indexes techniques. Many differentially expressed proteins identified were involved in energy metabolism, such as mitochondrial part, pyruvate dehydrogenase complex, and respiratory chain. These proteins were included in citrate cycle (TCA cycle), pyruvate metabolism and oxidative phosphorylation. The proteomic analysis and subsequent Western blotting on two independent cohorts of animials indicated that the dysregulation of energy metabolism existed in the medulla of the SHR rats. The differentially expressed proteins in the dysregulation of energy metabolism in the medulla of SHR rats included down-regulated ATP6V1D, ATP6VOA1, ATP5L, DLD proteins and up-regulated AK1 protein. MAO-A protein also exhibited decreased regulation, as well as the other 3 abovementioned energy-relative proteins (ATP6V1D, ATP5L and DLD proteins) belonging to the heterocycle metabolic process. A receiver operating characteristic curve (ROC) analysis on 4 of the differentially expressed proteins respectively resulted in an area under the receiver operating characteristic curve (AUC) of 0.95, 0.90, 0.92, and 0.81 for differentiating the SHR rats from the normotensive rats. This dysfunction in energy metabolism localizes to the medulla, the lower part of brain stem, and is, therefore, likely to contribute to the development, as well as to pathophysiological complications of hypertension. KEYWORDS: quantitative proteome, medulla, spontaneously hypertensive rats/SHR rats, iTRAQ, energy metabolism
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pressure control.3,6 Up to now, the flux of the medulla in hypertension has not been well unveiled. Lopez-Campistrous reported that the brain respiratory complexes exhibit assembly defects that impair the function of the mitochondrial respiratory chain in spontaneously hypertensive rats (SHR),1 using proteomic profiling method and ATP production analysis. Tian’s study on renal regional proteomes in a salt-induced hypertension rat model also uncovered the differential proteins involved in the energy metabolism.7 Their studies confirmed that dysfunction of mitochondria emerged as a common hallmark of hypertension.1,8−10 Decreased expression of mitochondrial components and transcription factors involved in mitochondrial biogenesis were repeatedly reported, as well as defects in the assembly of respiratory complexes.1,9,11,12 Moreover, abnormal mitochondrial respiration can result in disturbance of the oxidative pathways from mitochondrial ATP synthesis1,13 and subsequent failure of cellular energetic processes.
INTRODUCTION
Hypertension (the state characterized by sustained high blood pressure) remains a poorly understood, multifactorial condition affecting >25% of the adult population in developed countries.1 Of the forms of hypertension, essential hypertension (also called primary hypertension or idiopathic hypertension) is the most common type, affecting 95% of hypertensive patients.2 That the sympathetic nervous system participates in control of arterial pressure is indisputable; the controversy regards its role in the pathogenesis of human hypertension.3 Of the sympathetic nervous system, the medulla oblongata, also referred as the medulla, which is the lower half of the brain stem, plays an important role on blood pressure control. The medulla contains the cardiac, respiratory, vomiting and vasomotor centers and deals with autonomic, involuntary functions, such as breathing, heart rate and blood pressure.4 Reis et al. focused their studies on one region of the medulla, the intermediate third of the nucleus of the solitary tract (NTS).3,5 Doba and Reis first demonstrated the consequences of impaired function of neurotransmission in the NTS on blood © 2012 American Chemical Society
Received: July 19, 2012 Published: November 21, 2012 390
dx.doi.org/10.1021/pr3009385 | J. Proteome Res. 2013, 12, 390−395
Journal of Proteome Research
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Tissue Collection
Although Lopez-Campistrous’ study localized the mitochondrial dysfunction to the brain stem, Lopez-Campistrous mainly focused his proteomic profiling study on whole brain samples and did not pay much attention to the proteome of the brain stem.1 Considering the important role of the medulla in the previous studies of hypertension, we decided to explore the molecular basis of the flux of the medulla in this disease. The current work investigates the quantitative proteomic dysfunction in the medulla in SHR rats compared to the normal controls (n = 5 in each group). We have applied various complementary techniques, including quantitative proteomic profiling analysis based on iTRAQ technology coupled with nano two-dimentional liquid chromatography followed by high resolution mass spectrometric abundance indexes and Western blotting assay. To validate the research results, we performed the Western blotting test on the second independent cohort of SHRs and normal controls (n = 5 in each group). Our findings consistently provide novel evidence on reduced proteins functioning in the heterocycle metabolic process in the medulla. These proteins influence mitochondrial respiratory complexes, oxidative pathways from mitochondrial ATP synthesis, and ATPase as well in hypertension. We applied ROC analysis on these proteins. These reduced proteins are present in the medulla and can, therefore, impair the systemic control of blood pressure.
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The animals were anesthetized with an overdose of sodium phenobarbital (50 mg/kg body weight) by intraperitoneal injection on the day to collect the tissue (at 11 weeks of age) and perfused intracardially with 50 mL physiological saline. The medulla was quickly and carefully dissected by an experienced surgeon with reference to the rat brain atlas, and the capsule was removed. The medulla was contemporarily preserved using liquid nitrogen immediately after the dissection. When all the medullas were collected, the tissues were stored at −80 °C until analysis. Tissue Preparation
Total proteins were extracted according to our lab protocols. In brief, the tissue was homogenized with solubilization buffer (9 M urea, 2.5 M thiourea,12.5 mM DTT, 0.5% Pharmalyte 3−10, 0.1% Pefabloc SC and 150 U/mL Benzonase) and centrifuged for 60 min at 20 000g to pellet any debris. The protein concentration of the supernatant was determined according to Bradford. Peptide Labeling by the iTRAQ Chemical Reagent
In order to identify proteins that are differentially expressed in medulla oblongata of rat brain stem, extracted proteins from medulla oblongata were labeled with isobaric tags for relative and absolute quantification (iTRAQ, Applied Biosystems, California, USA) for the relative quantitation of the proteins. The extracted proteins from the medulla samples of 5 rats in each group (i.e., the SHR group and the normal Wistar group respectively) were pooled separately. For each sample, proteins were precipitated with isopropanol, and pellets were redissolved in the dissolution buffer (0.5 M triethylammonium bicarbonate, 0.1% SDS). Then proteins were quantified by BCA protein assay, and 100 μg of protein was denatured, alkylated, and digested as described in the iTRAQ protocol (Applied Biosystems, California, USA). The trypsin digested samples were briefly spun to collect the sample at the bottom of the tube. Meanwhile the iTRAQ labels were thawed at room temperature and spun to collect the reagent at the bottom of the tube. 70 μL ethanol (HPLC-grade, Sigma Aldrich, Gillingham, UK) was added to each label and thoroughly mixed by vortexing. Then samples were labeled with the iTRAQ tags as follows: Sample A (pooled sample from the control group), 113.1 tag; and Sample B (pooled sample from the SHR group), 116.1 tag. Labeled peptides from two samples were combined into one tube and dried in a vacuum concentrator. A SepPac C18 cartridge (Waters Corporation, Milford, MA) was used to exchange the buffer, and to remove trypsin and the hydrolyzed unbound iTRAQ reagents from the labeled peptides.
MATERIALS AND METHODS
Materials
Material related to proteomics, including urea, dithiothreitol (DTT), 2-mercaptoethanol, bio-lyte3/10, bromophenol blue, mineral oil, 12.5% Tris-HCL, 1.5 M Tris-HCL(pH 8.8), potassium chloride, isopropanol, hydrochloric acid (supplied at 37% (v/v) (∼4.1 M), thiouea, the protease inhibitor cocktail and a matrix solution of α-cyano-4-hydroxycinnamic acid in acetonitrile/methanol were purchased from Sigma-Aldrich Inc. (St Louis, MO). Sequencing-grade modified trypsin was from Promega (cat. no. V5280). All iTRAQ reagents and buffers were of Applied Biosystems (Foster City, CA). All reagents were analytical grade or equivalent. Antibodies for proteins MAO-A, AK1 and DLD were purchased from Santa Cruz Biotechnology, Inc., USA. Antibodies for proteins ATP6V1D ATPase and ATP5L ATP synthase were from Proteintech Group, Inc., USA. Antibody for protein ATP6V0A1 was from Abcam, UK. Ethanol and acetonitrile were HPLC grade from Thermo Fisher, USA. Male SHR and normal Wistar rats were purchased from Beijing Vital River Laboratory Animals Co., Ltd. (Beijing, China) at 9 weeks of age and housed 3 weeks at the Laboratory Animal Center in Guangzhou University of Chinese Medicine, Guangzhou, China. The first cohort of the rats included 5 rats in each group. The second independent cohort of rats to validate also included 5 rats in each group. This study was approved by the Ethics Committee of Guangzhou University of Chinese Medicine, Guangzhou, China.
Peptide Fractionation by the Strong Cation Exchange (SCX) Column
The concentrated iTRAQ labeled sample was added to 2 mL of diluent buffer (10 mM potassium phosphate, 25% acetonitrile (ACN)), and the pH was adjusted below 3 with phosphoric acid. The samples were subjected to cation exchange chromatography using a PolySULFOETHYL A HPLC column (2.1 × 100 mm i.d, PolyLC Inc., Columbia, MD, USA). A gradient flow of 0.2 mL/min was used, and 24 0.4 mL fractions were collected in 1.5 mL microfuge tubes. Buffer A contains 10 mM potassium phosphate with 25% ACN, pH < 3, adjusted with phosphoric acid, and buffer B is a high salt buffer containing 10 mM potassium phosphate, 500 mM potassium
Systolic Blood Pressure Measurement
Systolic blood pressure in conscious male 11-week-old spontaneously hypertensive rats (SHRs) or age-matched Wistar rats (normotension control) was measured using a computerized rat tail-cuff technique (Chengdu TME Technology Co., Ltd.; n = 5 in each group). 391
dx.doi.org/10.1021/pr3009385 | J. Proteome Res. 2013, 12, 390−395
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because of unequal mixing during combining different labeled samples. For iTRAQ quantitation, the peptide for quantification was automatically selected by Pro Group algorithm (at least one peptide with 99% confidence) to calculate the reporter peak area, error factor (EF), and p-value.
chloride, 25% ACN, pH < 3, adjusted with phosphoric acid. The following gradient was applied: 5 min 100% buffer A; 5− 45 min increasing to 30% buffer B; 45−50 min increasing to 80% buffer B, maintained for 5 min; 56 min 100% buffer A, maintained for 15 min. Fractions were collected started at 6 min and at 2-min intervals. To remove excess KCL that was added during the peptide fractionation process, after the ACN that is present in the buffers was removed by volatilization, a SepPac C18 cartridge (Waters Corporation, Milford, MA) was used for desalting and dried in a vacuum concentrator for the next step.
Western Blotting
Differentially expressed protein markers determined by iTRAQ were validated using Western blot in medulla oblongata samples. For Western blots, total proteins were extracted from each sample, and 50 μg of the total protein were separated with 12% gradient SDS-PAGE and transferred to PVDF membrane (Amersham Bioscience). The membranes were blocked with 5% BSA in TBST buffer (137 mM NaCl, 20 mM Tris-HCl, pH 7.6, 0.1% Tween-20) and then incubated for 1 h at room temperature with primary antibody, followed by washing and incubation with corresponding HRP-conjugated secondary antibody. The washed membranes were developed with enhanced chemiluminescence reagent (Amersham Bioscience), and the images were captured with LAS-3000 instrument (Fuji, Japan) and were calculated with Gray analysis.
Reversed Phase LC−MS/MS Analysis
The peptides were resuspended with 20 μL solvent A (A: water with 0.1% formic acid; B: ACN with 0.1% formic acid), separated by nanoLC and analyzed by online electrospray tandem mass spectrometry. The experiments were performed on a Nano Aquity UPLC system (Waters Corporation, Milford, USA) connected to an LTQ Orbitrap XL mass spectrometer (Thermo Electron Corp., Bremen, Germany) equipped with an online nanoelectrospray ion source (Michrom Bioresources, Auburn, USA). A 18 μL peptide sample was loaded onto the Captrap Peptide column (Michrom Bioresources, Auburn, USA), with a flow of 20 μL/min for 5 min and subsequently eluted with a three-step linear gradient. Starting from 5% A to 45% B in 70 min, increased to 80% B in 1 min, and then hold on 80% B for 4 min. The column was re-equilibrated at initial conditions for 15 min. The column flow rate was maintained at 500 nL/min, and column temperature was maintained at 35 °C. The electrospray voltage of 1.9 kV versus the inlet of the mass spectrometer was used. LTQ Orbitrap XL mass spectrometer was operated in the data-dependent mode to switch automatically between MS and MS/MS acquisition. Survey full-scan MS spectra (m/z 300− 1600) were acquired in the Obitrap with a mass resolution of 60 000 at m/z 400, followed by four sequential HCD-MS/MS scans. The automatic gain control (AGC) was set to 500 000 ions to prevent overfilling of the ion trap. The minimum MS signal for triggering MS/MS was set to 10 000. In all cases, one microscan was recorded. The lock mass option was enabled, and the polydimethylcyclosiloxane ion signal (protonated (Si(CH3)2O)6; m/z 445.120025) was used for internal calibration of the mass spectra. MS/MS scans were acquired in the Obitrap with a mass resolution of 7500. The dissociation mode was HCD (higher energy C-trap dissociation). Dynamic exclusion was used with two repeat counts, 10-s repeat duration, and the m/z values triggering MS/MS were put on an exclusion list for 120 s. For MS/MS, precursor ions were activated using 45% normalized collision energy and an activation time of 30 ms.
ROC Analysis
On the basis of the data of the Gray analysis from the Western blotting results, ROC analysis was performed by SPSS software (IBM, SPSS 19.0).
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RESULTS
Arterial Blood Pressure
The 11-week-old spontaneously hypertensive rats were with elevated blood pressure (162 ± 7.36 mmHg) compared against those from age-matched normotensive (112 ± 2.90 mmHg) Wistar rats (n = 5 in each group). Differential Proteome Profiles
Quantitative proteomic profiles in the medulla were compared between 11-week-old SHR rats and age-matched normotensive rats. The quantitative proteomic discovery experiments with iTRAQ were performed in triplicates on pooled sample from each group (n = 5). A summary of the throughput of the triplicated proteomic analysis was shown in Table 1. In total 474 identified proteins Table 1. Summary of the Throughput of the Triplicated Proteomic Analysisa proteins detected first run second run third run in total a
Peptide Sequencing and Data Interpretation
Protein identification and quantification for the iTRAQ experiment was performed with the ProteinPilot software version 3.0 (AppliedBiosystems,USA). The Paragon Algorithm in ProteinPilot software was used for peptide identification and isoform specific quantification. The data search parameters are shown in Supporting Information Table S1. To minimize false positive results, a strict cutoff for protein identification was applied with the unused ProtScore 1.3, which corresponds to a confidence limit of 95%, and at least two peptides with the 95% confidence were considered for protein quantification. The FDR level was
