Proteomic Identification of Endothelial Proteins Isolated in Situ from Atherosclerotic Aorta via Systemic Perfusion Jiang Wu,*,† Wei Liu,† Eric Sousa,† Yongchang Qiu,† Debra D. Pittman,‡ Vasu Maganti,‡ Jeffrey Feldman,‡ Davinder Gill,† Zhijian Lu,† Andrew J. Dorner,† Robert Schaub,‡ and Xiang-Yang Tan*,† Biological Technologies, Cardiovascular and Metabolic Diseases, Wyeth Research, Cambridge, Massachusetts 02140 Received August 16, 2007
The functional and structural alterations of vascular endothelium contribute to the initiation, progression, and complications of atherosclerotic plaque formation, but limited information is known about the molecular composition and pathways underlying pathological changes during atherosclerosis. We have developed an affinity proteomic strategy for in situ isolation and differential mapping of vascular endothelial proteins in normal and atherosclerotic aorta tissues. The selective labeling was carried out by perfusion of the blood vessels with an active biotin reagent for covalent modification of accessible vascular endothelial proteins. The biotinylated proteins were then enriched by streptavidin affinity chromatography, separated by SDS-PAGE, and subsequently characterized by LC-MS/MS. The described procedure led to the identification of 454 distinct proteins in normal and atherosclerotic aorta tissues. A majority of the proteins are plasma membrane associated and extracellular matrix proteins, and 81 showed altered expressions in atherosclerotic aorta tissue. The differentially expressed proteins are involved in immune and inflammatory responses, cell adhesion, and lipid metabolism. The method provides a new avenue for investigating the endothelial dysfunction and development of atherosclerosis. Keywords: atherosclerosis • apolipoprotein E deficient • biotinylation • endothelium • proteomics
Atherosclerosis is a chronic multifactorial inflammatory disease that remains a major cause of morbidity in the Western world.1 It is characterized by chronic endothelium damage, accumulation of low-density lipoprotein (LDL), recruitment of inflammatory cells, migration and proliferation of smooth muscle cells, as well as thrombus formation and calcification.2,3 Even though many important mechanisms associated with atherosclerosis have been characterized, more information is needed to reveal the molecular composition and pathways underlying pathological changes of atherosclerosis. Vascular endothelium is the blood-tissue interface that plays an important role in controlling the passage of blood proteins and cells into the tissue. It is also involved in many physiological processes, including vascular homeostasis, coagulation, inflammation, as well as tissue growth and repair. Impairment of endothelial function, an early marker for atherosclerosis, leads to enhanced expression of adhesion molecules (e.g., VCAM-1, ICAM-1, PECAM-1, P-selectin) as well as release of cytokines and chemokines.4 These changes further mediate platelet aggregation, leukocyte adhesion, smooth muscle cell proliferation and migration, and thrombogenesis in the vessel wall which promote atherosclerotic plaques.5,6
Endothelial cells overlying atherosclerotic plaques display altered protein expression that consequently results in increased cell adhesion, altered permeability, and release of inflammatory mediators. A variety of approaches have been reported to the identification of proteins expressed in cultured endothelial cells.7–9 However, tissue disassembly and cell culture may result in phenotypic changes and loss of native cellular function and protein expression.10,11 In vivo global mapping of the vascular endothelium in native tissues of different disease states has been more challenging because endothelial cells represent only a small percentage of the cells in the tissue. Schnitzer and colleagues described a novel approach to direct proteomic mapping of microvascular endothelium in situ using colloidal silica coating of endothelium lining vascular structures in tumors and in normal organs.10,12 Biotinylation of endothelial surface proteins in situ was explored to probe organ-specific targets accessible from blood circulation.13,14 This approach was also applied to the identification of endothelial antigens in surgically removed human kidney cancer specimens.15 To date, proteomic mapping of atherosclerotic plaque has been limited due to the heterogeneous cellular composition of the plaque and the lack of human tissues.16,17
* To whom correspondence should be addressed. Wyeth Research, 87 Cambridge Park Drive, Cambridge, MA 02140. Phone: 617-665-8052or 617665-8157. Fax: 617-665-8435. E-mail:
[email protected] or
[email protected]. † Biological Technologies. ‡ Cardiovascular and Metabolic Diseases.
We report preliminary results on an affinity-based proteomic approach to identify differentially expressed vascular endothelial surface proteins in aorta tissues of apolopoprotein E deficient (ApoE-/-) mice and age-matched control wild type (WT) C57BL/6J mice. The affinity enrichment of the endothelial
Introduction
4728 Journal of Proteome Research 2007, 6, 4728–4736 Published on Web 11/13/2007
10.1021/pr070537c CCC: $37.00
2007 American Chemical Society
Proteomics of Atherosclerotic Endothelial Proteins surface proteins was carried out by in situ perfusion of the blood vessels with an active biotin reagent followed by purification via streptavidin chromatography. In-gel digestion and subsequent LC-MS/MS analysis resulted in the identification of 454 proteins in normal and atherosclerotic aorta tissues. The functional implications of some differentially expressed proteins in the progression of atherosclerosis are discussed.
Materials and Methods Animals. All the animal experiments were performed according to protocols approved by Wyeth Institutional Animal Care and Use Committee (IACUC). ApoE-/- mice on a C57BL/ 6J background and WT C57BL/6J mice were purchased from Jackson Laboratories (Bar Harbor, ME) and raised to 39–40 weeks old in the Wyeth animal facility. Mice were fed with normal chow diet containing 4.5% fat (0.02% cholesterol) by weight. While the aorta tissues of WT mice were free of visible plaque, significant atherosclerotic lesions had progressively developed in the aorta tissues of the ApoE-/- mice at 39–40 weeks. In Situ Biotinylation of Vascular Endothelium. The procedure for in situ biotin labeling of vascular endothelium was adopted from a previous study with minor modifications.14 Briefly, the mice aged 39–40 weeks were euthanized with inhalation of CO2, and the chest cavity was open. For the systemic perfusion of vascular beds, a 25-gauge butterfly needle was inserted into the left heart ventricle, and the right atrium was cut off to allow outflow of the perfusion solutions. After injecting 200 µL of phosphate-buffered saline (PBS), pH 7.4, containing 3 mg/mL of sodium nitroprusside dihydrate and 50 units of heparin to keep vasodilation and prevent platelet aggregation, the vascular beds were perfused with 20 mL of PBS, pH 7.4, to flush away the blood components. Successful perfusion was evidenced by the outflow of clear perfusion solution from the right atrium and the whitish color of the tissues. The perfusion solution was then changed to 20 mL of PBS, pH 7.4, containing 1 mg/mL of Sulfo-NHS-LC-biotin (Pierce, Rockford, IL) at a flow rate of 1 mL/min to label the accessible molecules containing primary amine groups. The vascular beds were subsequently perfused with 20 mL of PBS, pH 7.4, to wash out excess biotinylation reagent and then with 20 mL of PBS, pH 7.4, containing 5 mg/mL of glycine to quench unreacted biotinylation reagent. The aorta tissue was removed from the aortic arch to the thoracic aorta for either preparation of tissue homogenates or histochemical analysis. Extraction and Purification of Biotinylated Endothelial Proteins. The biotin-labeled aorta tissues from ten ApoE-/mice or ten control WT mice were pooled and grounded on dry ice, respectively. The specimens were suspended in 5 volumes (mL/g) of lysis buffer (PBS, pH 7.4, containing protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN), 1% Triton-X100, and 0.1% SDS) and then placed on a rotator for 1 h at 4 °C. The tissue homogenates were centrifuged at 4500 rpm for 15 min at 4 °C. The supernatant containing extracted soluble proteins was collected and dialyzed with a 3.5 kDa MWCO dialysis cassette against PBS, pH 7.4, at 4 °C to further remove unbound biotin. The dialyzed samples were filtered through 40 µm nylon filter to remove tissue debris. The concentration of the extracted total proteins was measured with the BCA protein assay kit (Pierce, Rockford, IL). Equivalent amounts of the extracted aorta homogenate proteins from ApoE-/- mice and WT mice were subjected to affinity enrichment, respectively, with streptavidin-agarose beads (Pierce,
research articles Rockford, IL). The specimens and streptavidin-agarose beads were mixed for 1 h at room temperature to capture biotinylated proteins. The supernatants were removed, and the beads were washed 3 times with PBS, pH 7.4, containing 0.1% Triton-X100 and 3 times with PBS, pH 7.4, respectively. Immunohistochemical Staining of Tissues. Frozen tissues were cut into 5 µm sections on a Leica CM3050-S cryomicrotome. Tissue sections were fixed with cold acetone for 5 min. After the acetone was completely volatilized, the sections were blocked with 2% BSA in PBS, pH 7.4. For assessment of biotinylated vascular vessels, the tissue sections were incubated with 10 µg/mL of horseradish peroxidase (HRP) conjugated streptavidin (Pierce, Rockford, IL) at room temperature for 1 h. The slides were washed with PBS followed by staining with 3-amino-9-ethyl carbazole (AEC) substrate solution (Zymed Laboratories, South Sanfrancisco, CA) for color reaction. The sections were counterstained with hematoxylin and mounted for microscopy observation. Immunoblotting. Equivalent amounts of the protein extracts from ApoE-/- mice and WT mice were separated by SDSPAGE and electrotransferred to a nitrocellulose membrane for immunodetection with HRP-conjugated streptavidin to assess biotinylated proteins. The nitrocellulose membrane was blocked with 5% skim milk in Tris-buffered saline, pH 7.2, containing 0.1% Tween 20 (TBS-T), followed by incubation with HRPconjugated streptavidin diluted in the blocking buffer at room temperature for 1 h. After being completely rinsed in TBS-T, blots were developed using ECL Western blotting detection reagents (Pierce, Rockford, IL), and the chemiluminescent signal was captured on Hyperfilm. The signals on Hyperfilm were quantified using Quantity One software, version 4.4.1 (BioRad, Hercules, CA). SDS-PAGE Separation and In-Gel Digestion of Biotinylated Proteins. For proteomic analysis, the proteins bound to the streptavidin-agarose beads were reconstituted by boiling in 50 µL of the SDS-PAGE loading buffer (4% SDS, Tris-HCl, pH 8.5) at 90 °C for 10 min. The proteins were reduced with 20 mM dithiotreitol at 60 °C for 1 h and alkylated with 50 mM iodoacetamide at ambient temperature in the dark for 30 min. Protein separation was carried out on a 10% tricine minigel (Invitrogen, Carlsbad, CA), and the gel was stained using Colloidal Coomassie Blue. The whole gel lane (including the streptavidin band) was cut evenly into 20 slices. Each slice was minced into 1 × 1 mm size pieces and subsequently subjected to in-gel digestion with modified trypsin (0.53 µg/gel slice) in a digestion robot (DigestPro, ABIMED, Analysen-Technik GmbH, Langenfeld, Germany). Finally, the tryptic digests were concentrated in a speedvac to a final volume of ∼40 µL prior to mass spectrometric analysis. NanoLC-MS/MS. Tryptic digests were analyzed by an automated nanoLC-/MS/MS system using a Famos autosampler (LC Packings, San Francisco, CA) and an Agilent 1100 HPLC binary pump coupled to an LTQ ion trap mass spectrometer equipped with a nanospray ionization source (ThermoFinnigan, San Jose, CA). The digest solution (10 µL) was injected onto a reversedphase PicoFrit column (New Objective, Woburn, MA) packed with Magic C18 media (5 µm particle, 200 Å pore size, 75 µm × 10 cm). Peptides were eluted at a flow rate of 0.2 µL/min using a 90 min linear gradient from 2% to 55% B (mobile phase A, 0.1% formic acid aqueous solution; mobile phase B, 90% acetonitrile and 0.1% formic acid). The spray voltage was 1.8 kV. The heated capillary temperature was maintained at 180 °C, and the collision energy for MS/MS was set at 35 units. Journal of Proteome Research • Vol. 6, No. 12, 2007 4729
research articles Automated data-dependent MS analysis was carried out using the dynamic exclusion feature built into the MS acquisition software (Xcalibur 1.3, ThermoFinnigan, San Jose, CA). Each MS full scan (m/z 350–1600) was followed by MS/MS acquisition of the four most intense precursor ions detected in the prior MS scan to obtain as many CID spectra as possible. Each sample was analyzed twice. Protein Identification and Annotation. SpectrumMill v3.1 (Agilent) was used for database searches and for data processing. The mass spectrometric raw data were searched against the human subset of the NCBI nonredundant protein database (135 279 protein entries, updated as of July, 2005). Search parameters included a static modification on cysteine residues (carbamidomethylation), S/N > 25, sequence tag length >1, precursor ion mass range of 600–3000 Da, retention time ranging from 0 to 135 min, a 70% minimum matched peak intensity, ( 2.0 Da tolerance on precursor ions, ( 0.7 Da tolerance on product ions, one missed tryptic cleavage, and ESI-ion trap scoring parameters as defined by the searching algorithm. Scans for the precursor ions m/z ( 0.7 Da within a ( 45 s time window were merged. Other default parameters from the software were used. All of the database search results were further validated by applying the designated protein and peptide scores as well as the following user-defined criteria: (1) protein validation mode, protein score >18, peptide SPI (scored percent intensity) >70% for all charge states, peptide score >7 for peptide charge +1, >8 for peptide charge +2, and >9 for peptide charges g+3; (2) peptide validation mode, peptide SPI >70% and peptide score >13 for all charge states. In short, a protein can be considered positively identified if either multiple spectra of moderate quality or better or at least one spectrum of high quality was obtained. Search criteria used here would result in a false positive rate of
