MS Analysis on Protein Profile in Vascular

In clinical practice, β blocking agents compete with endogenous and ... The sample peptides bound on the SCX columns are then eluted by injected ...
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iTRAQ-Coupled 2D LC-MS/MS Analysis on Protein Profile in Vascular Smooth Muscle Cells Incubated with S- and R-Enantiomers of Propranolol: Possible Role of Metabolic Enzymes Involved in Cellular Anabolism and Antioxidant Activity Jianjun Sui, Tuan Lin Tan, Jianhua Zhang, Chi Bun Ching,* and Wei Ning Chen* School of Chemical and Biomedical Engineering, College of Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore 637459 Received November 10, 2006

Abstract: Propranolol is a nonselective β-blocker of the β-adrenergic receptors, and the S-enantiomer is more active compared with the R-enantiomer. Clinically, it has been shown to be effective in hypermetabolic burn patients by decreasing cardiac work, protein catabolism, and lipolysis. While gene expression profiles have recently been reported in children receiving propranolol treatment, variations from one individual to another may have influenced the data analysis. Using iTRAQ-coupled 2D LC-MS/MS analysis, we report here the first study of protein profile in vascular smooth muscle cells incubated separately with the two enantiomers of propranolol. Four types of cellular proteins including metabolic enzymes, signaling molecules, cytoskeletal proteins, and those involved in DNA synthesis/protein translation displayed changes. The higher protein level of a number of enzymes involved in cellular anabolism and antioxidant activity in cells incubated with the S-enantiomer, as revealed by LCMS/MS, was further supported by real-time PCR and Western blot analyses. Significantly, the increase in the anabolic activity associated with the higher level of metabolic enzymes was also supported by the higher intracellular concentration of the metabolic cofactor NAD+ which was a result of an increased oxidation of NADH. Our findings therefore provide molecular evidence on metabolic effect associated with propranolol treatment. The metabolic enzymes identified in our study may in turn be useful targets for future pharmaceutical interventions to reduce clinical side effects following propranolol treatment. Keywords: Propranolol • LC-HS/MS • Cellular Protein Profile • Anabolic Metabolic Enzymes • Antioxidant Enzymes • Intracellular NADH

Introduction In clinical practice, β blocking agents compete with endogenous and exogenous β-adrenergic agonists. Their specific * Corresponding authors. Tel: 65-63162870, E-mails: (C.B.C.) CBChing@ ntu.edu.sg and (W.N.C.) [email protected]. 10.1021/pr0605926 CCC: $37.00

 2007 American Chemical Society

effects depend on their selectivity for β1 receptors (located in the heart) or β2 receptors (located in bronchi, blood vessels, stomach, gut, and uterus). The β-adrenergic receptors belong to the family of G-protein-coupled receptors1 characterized by seven transmembrane spanning domains forming a pocket in which the agonists and competitive antagonists find their binding sites.2 The β-adrenergic receptors of the myocardium play an important role in the regulation of the heart function. Dilated cardiomyopathy, for instance, is a disease of left ventricular dysfunction accompanied by impairment of targets of the β1-adrenoceptor signal cascade.3 Propranolol, a nonselective β-blocker, is mostly used in the treatment of hypertension,4 angina,5 and for the prevention of re-infarction in patients who have suffered from myocardial infarction.6 There is an increasing amount of data showing that the interaction of propranolol with β-adrenoceptors is highly stereoselective.7,8 Generally the S-enantiomer of propranolol is more potent as antagonist of β-adrenergic receptors and accounts for most of the β-blocker effect. Despite the welldocumented beneficial effects from the propranolol treatment, concerns regarding its potential adverse metabolic effects and particularly the underlying molecular mechanism remain to be addressed. Proteomics analysis has been widely used to establish cellular signaling pathways in response to various external stimuli, including comparing normal and diseased conditions.9,10 Established methods for relative quantitation of proteins involve growth on isotope-enriched medium11 or chemical or enzymatic modifications.12 Recently, an MS/MS-based quantitation method (iTRAQ) has been developed.13 The system enables up to four samples to be analyzed within one experiment. They are differentially isotopically labeled such that all derivatized peptides will have an identical mass and LC retention time after tagging. Following collision-induced dissociation (CID) MS/MS analysis of the precursor ion, the four reporter groups appear as distinct ions (m/z 114-117). The relative concentration of the peptides is derived from the relative intensities of the reporter ions. In this study, we use a 4-plex multiplex strategy to simultaneously detect and quantify differences in expression levels of proteins in untreated vascular smooth muscle cells and those incubated with S- and R-enantiomer, respectively, which reflect pharmacologic action of eantiomers. To identify proteins from a complex mixture, the two-dimensional (2D) application is Journal of Proteome Research 2007, 6, 1643-1651

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Published on Web 04/11/2007

communications used. In this approach, a strong cation exchange (SCX) column is used for the first dimension, a reversed-phase (RP) column is used for the second, and two identical enrichment columns are used for trapping the peptides. The sample peptides bound on the SCX columns are then eluted by injected salt solution plugs of increasing concentration, trapped on a short enrichment column, and subsequently analyzed on a nanoRP column interfaced with electrospray ionization (ESI)-MS/MS. Our results indicated that protein profile in cells incubated with the S-enantiomer was different from those incubated with the R-enantiomer of propranolol. Four types of cellular proteins including metabolic enzymes, signaling molecules, cytoskeletal proteins and those involved in DNA synthesis/protein translation displayed changes. Significantly, metabolic enzymes involved in anabolism were at higher level in cells incubated with the S-enantiomer compared with those incubated with the R-enantiomer of propranolol. Our findings therefore provided molecular evidence on metabolic effect associated with propranolol treatment. Significance of our findings in understanding cellular signaling pathway in response to propranolol treatment and in design of future intervention to reduce its side effects was also discussed.

Experimental Procedures Cell Cultures. A7r5 cells, obtained from the American Type Culture Collection, were cultured in Dulbecco’s modified Eagle’s medium (DMEM, supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 U/mL). Cells were maintained at 37 °C in an atmosphere of 5% CO2. All culture media and media supplements were purchased from Life Technologies. After reaching 80% confluence the cells were incubated with the S-enantiomer, the R-enantiomer, and the racemic propranolol at a concentration of 20 µM, respectively, for 24 h in the absence of serum. MTT Assay. Cytotoxic concentrations were determined by the MTT reduction test. After chemical exposure, the medium was removed, and cells were incubated for 3 h with 5 mg mL-1 MTT dissolved in PBS. MTT was cleared out, and the formazan salts were solubilized in 100 µL DMSO. Plates were read at 570 nm against a 660 nm reference wavelength on a microplate reader (Benchmark Plus). The cell viability was expressed as a percentage of the corresponding control value. Cell Lysis, Protein Digestion, and Labeling with iTRAQ Reagents. Cells were harvested and lysed in 150 µL of 8 M urea, 4% (w/v) CHAPS, and 0.05% SDS (w/v) on ice for 20 min with regular vortexing. Protein was centrifuged at 15 000g for 60 min at 4 °C, supernatant was removed, and protein was quantified using the 2-D Quant Kit (GE Healthcare). A standard curve was made using BSA as a control. A total of 100 µg of each sample was precipitated by the addition of 4 vol of cold acetone at -20 °C for 2 h, dissolved in the solution buffer, and denatured, and cysteines were blocked as described in the iTRAQ protocol (Applied Biosystems). Each sample was then digested with 20 µL of 0.25 µg/ µL sequence grade modified trypsin (Promega) solution at 37 °C overnight and labeled with the iTRAQ tags as follows: normal A7r5 ) iTRAQ 114; A7r5 incubated with the S-enantiomer ) iTRAQ 115; A7r5 incubated with the Renantiomer ) iTRAQ 116; A7r5 incubated with the racemic propranolol ) iTRAQ 117. The labeled samples were then pooled before analysis. To verify that sample preparation techniques do not interfere with digestion and labeling procedures, the bovine serum albumin (BSA) standard solution 1644

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(Pierce) was used in the acetone precipitation, followed by enzymatic digestion with trypsin and labeling with the iTRAQ reagents as described above. These differentially labeled digests were mixed at a ratio of 1:2:1.5:2 and analyzed by LC-MS/MS. On-Line 2D NanoLC-MS/MS Analysis. In the first step of the separation, 3 µL of the combined peptide mixture was loaded onto the PolySulfoethyl A strong cation exchange column (0.32 × 50 mm, 5 µm). Buffer D consists of a series of increasing concentration of KCl salt solution as 10, 20, 30, 40, 50, 60, 80, 100, 300, and 500 mM used to elute the retained peptides in a stepwise manner from the SCX column by sequential injection. In the first run with the 10-port valve was in position 1, the flow-through with peptides that were not binding to the SCX column was trapped on the ZORBAX 300SB-C18 enrichment column I (0.3 × 5 mm, 5 µm) and washed isocratically using the loading buffer A (5% acetonitrile, 0.1% formic acid) for 100 min at 0.5 mL/min to remove any excess reagent. Meanwhile, the other ZORBAX 300SB-C18 enrichment column II was switched into the solvent path of the nanopump. Since no peptides were trapped on the column II, no peptides were expected to be detected after 100 min of running. In the second run, the 10-port valve was switched to position 2. A total of 5 µL of 10 mM KCl solution was injected into the SCX column to elute retained peptides to column II, which was washed isocratically using the loading buffer for 100 min at 0.5 mL/min to remove excess reagent. The column I which trapped the unbound peptides in the first run was switched into the solvent path of the nanopump. Peptides were eluted using the buffer B (0.1% formic acid) and the buffer C (95% acetonitrile, 0.1% formic acid) with a nanoflow gradient starting with 5% of the buffer C and increasing up to 80% of the same buffer C over 100 min at a flow rate of 500 nL/min. An increasing concentration of acetonitrile allowed the elution of the concentrated sample, and further separation was achieved onto the analytical Zorbax 300SB C-18 reversed-phase column (75 µm × 50 mm, 3.5 µm). For electrospray analysis, the HP1200 LC system (Agilent Technologies) was interfaced with a QSTAR XL (Applied Biosystems-MDS Sciex) mass spectrometry. Survey scans were acquired from m/z 300-1500 with up to two precursors selected for MS/MS from m/z 100-2000 using dynamic exclusion, and the rolling collision energy was used to promote fragmentation. The same approach applied to other 9 runs followed by valve switching, respectively. The 4-plex mixture sample was performed again to test the technical reproducibility. Data Analysis and Interpretation. Peptide identifications were performed using ProID software packages (Applied Biosystems). Each MS/MS spectrum was searched against the Rat IPI protein database, and protein identifications were accepted based on the ProtScore more than 2.0, which gives the confidence value of 99%. All protein identifications made on single unique peptides were discarded. The database allowed for iTRAQ reagent labels at N-terminal residues, internal K and Y residues, and the methylmethanethiosulfate-labeled cysteine as fixed modification, plus one missed cleavage. The analysis for the iTRAQ experiments was performed with ProQUANT 1.0.The cutoff for the confidence settings was 75, and the tolerance settings for peptide identification in ProQUANT searches were 0.15 Da for MS and 0.1 Da for MS/MS. ProQUANT pooled data from all the series of runs of increasing concentration of KCL in one experiment. All identifications

communications Table 1. List of Differentially Expressed Metabolic prot sc

Enzymesa avg. S:C (( SD)

avg. R:C (( SD)

metabolism

(IPI00778383.1) 16 kDa protein (IPI00197711.1) L-lactate dehydrogenase

0.84 ( 0.12 1.03 ( 0.07

0.92 ( 0.10 0.81 ( 0.06

7 6

(IPI00201561.2) Peroxiredoxin-2 (IPI00231368.4) Thioredoxin

1.16 ( 0.18 1.25 ( 0.12

0.87 ( 0.06 0.92 ( 0.11

4.59

(IPI00206624.1) 78 kDa glucose-regulated protein precursor (IPI00188112.1) Phosphoserine phosphatase (IPI00324633.2) Glutamate dehydrogenase 1, mitochondrial precursor (IPI00231662.6) diaphorase 1 (IPI00209115.2) Slc25a3 (IPI00212015.1) acyl-CoA dehydrogenase, mitochondrial precursor (IPI00231734.4) Fructose-bisphosphate aldolase A (PI00200661.1) Fatty acid synthase

1.11 ( 0.03

1.10 ( 0.05

0.85 ( 0.17

0.97 ( 0.02

Copper, zinc superoxide dismutase activity Catalyze conversion of L-lactate to pyruvate, and oxidation of NADH Antioxidant by reducing H2O2 Deactivate phosphofructokinase, antioxidant by reducing H2O2 Facilitate the assembly of multimeric protein complexes inside the ER Serine biosynthesis

1.29 ( 0.11

1.02 ( 0.13

1.21 ( 0.11 1.18 ( 0.11 1.37 ( 0.16

1.25 ( 0.18 1.12 ( 0.04 1.18 ( 0.14

1.23 ( 0.16

1.02 ( 0.15

1.13 ( 0.09

1.18 ( 0.12

28.57 12

4.01 4.01 4 3.53 3.44 2.29 2

protein name

Catalyze reversible oxidative deamination reaction and oxidation of NADH Involved in drug metabolism Mitochondria phosphate transporter Catalyze the first step in β-oxidation in mitochondrion Fructose-bisphosphate aldolase activity in glycolysis Catalyzes the formation of long-chain fatty acids

a S:C is the ratio of different protein expression level in the S-enantiomer-incubated cells relative to the unincubated cells; R:C is the ratio of different protein expression level in the R-enantiomer-incubated cells relative to the unincubated cells.

were manually inspected to minimize machine-related errors. Relative quantification of proteins in the case of iTRAQ was performed on the MS/MS scans and was the ratio of the areas under the peaks at 114, 115, 116, and 117 Da which were the masses of the tags that correspond to the iTRAQ reagents. The relative amount of a peptide in each sample was calculated by dividing the peak areas observed at 115.1, 116.1, and 117.1 m/z by that observed at 114.1 m/z. The calculated peak area ratios were corrected for overlapping isotopic contributions, and were used to estimate the relative abundances of a particular peptide. For proteins with two or more qualified peptide matches, three average peak area ratios (designated as 115/ 114, 116/114, and 117/114) were calculated using the peak area ratios of the peptides originating from the same protein. The standard deviation (SD), the CV for each average peak area ratio, and the average CV (CV) using the CVs for the average peak area ratios for each protein were also calculated. To account for small differences in protein loading, these ratios have been normalized using the overall ratios for all proteins in the sample, as recommended by Applied Biosystems. In the study, only protein quantification data with relative expression of >1.1 or