Identification of Vascular Surface Proteins by in Vivo Biotinylation: A Method Sufficiently Sensitive To Detect Changes in Rat Liver 2 Weeks after Partial Hepatectomy Lauren A. McCann, Miriam C. Haywood, Bin Hai Ren,† Ann M. Simpson,† Michael Guilhaus,‡ Valerie C. Wasinger,‡ Mark J. Raftery,‡ and Ross A. Davey*,§ Bill Walsh Cancer Research Laboratories, Royal North Shore Hospital and University of Sydney, St. Leonards NSW 2065, Australia, Department of Medical & Molecular Biosciences, University of Technology Sydney, Broadway NSW 2007, Australia, and Bioanalytical Mass Spectrometry Facility, University of NSW, Sydney, NSW 2052, Australia Received January 22, 2007
We have developed a methodology to selectively isolate and identify proteins associated with the luminal surface of blood vessels using in vivo biotinylation, streptavidin-affinity chromatography, and SDS-PAGE/LC-MS/MS. This had sufficient sensitivity to identify 32 proteins with changed expression in rat livers at 2 weeks or 5 weeks after partial hepatectomy, well after the 7 day tissue remodeling period. This method could be adapted to study other angiogenic tissues including tumors. Keywords: Angiogenesis • vascular surface proteins • endothelial cells • in vivo biotinylation • liquid chromatographytandem mass spectrometry • liver regeneration
Introduction
blood vessels, and these would mainly be proteins on the surface of endothelial cells.
Endothelial cells lining all blood vessels not only play an important role in specifically delivering nutrients from the blood to the tissue, but they also respond to signals from hypoxic or malnourished tissues by proliferating and forming new blood vessels, a process known as angiogenesis. Angiogenesis is an essential requirement for tumor progression, and it is also involved in many other diseases such as rheumatoid arthritis, arthrosclerosis, hemangioma, and endometriosis.1 Since the process of angiogenesis involves endothelial cells leaving their normal dormant state to rapidly divide, it was hypothesized that the pattern of proteins expressed on the endothelial cell surface would differ between dormant and angiogenic states. These differences could potentially be used as targets for novel treatments of diseases in which angiogenesis is involved, including cancer.
We adapted a previous method for the in vivo biotinylation of proteins4 and optimized the protein labeling and isolation using the rat partial hepatectomy model. This model is relevant to tumor angiogenesis because it contains endothelial cells that were recently proliferating, with sufficient material for our proposed proteomics approach. In this model, two-thirds of a rat liver is surgically removed and the liver grows back to the preoperative size within 7 days. In order for the liver to regenerate, the normally quiescent hepatocytes and endothelial cells are activated to proliferate and restore the tissue mass. This process requires angiogenesis to occur.5,6
There are many antiangiogenic cancer treatments under development. Those involving antibodies such as bevacizumab2 (antivascular endothelial growth factor, VEGF) or DC1013 (antiVEGF2 receptor) will only be effective if the target protein is accessible. In our search for potential cancer treatment targets, we propose to solve this issue of access by labeling and identifying only those proteins that are accessible by intravenous injection by intravenously injecting a biotinylation reagent with poor penetration across cell membranes. This strategy would label proteins associated with the luminal surface of * To whom correspondence should be addressed. Tel: +61-2-9926-7456. Fax: +61-2-9926-5253. E-mail:
[email protected]. § Royal North Shore Hospital and University of Sydney. † University of Technology Sydney. ‡ University of NSW.
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Published on Web 06/30/2007
We describe here a method to isolate and identify proteins associated with the endothelial cell surface in this liver regeneration model. We also report the protein changes that persist 2 or 5 weeks after partial hepatectomy as an indication of the method’s sensitivity.
Materials and Methods Liver Regeneration Model. Fisher 344 rats (ARC, Murdoch, WA, Australia) were housed and treated according to our institutional Animal Care and Ethics Committee. Rats weighing from 200-400 g were anesthetized using isofluorane/N2O/O2, and two-thirds of the liver was surgically removed as previously described by opening the abdomen through a midline incision and a 70% hepatectomy performed by resecting the left lateral and median liver lobes.7 Sham operations involved opening the abdomen with same midline incision as above. All rats were given 0.1-0.5 mg/kg buprenorphine analgesia and were housed in separate cages for 48h. 10.1021/pr070032m CCC: $37.00
2007 American Chemical Society
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In Vivo Labeling of Vascular Surface Proteins. Several groups of two untreated rats were used to optimize the perfusion and biotinylation. To determine the protein changes that persisted following liver regeneration, groups of two rats at 2 and 5 weeks after partial hepatectomy or 2 weeks after sham operation were anesthetized and their livers perfused. An incision was made in the midline of abdomen, and a 22 gauge cannula was retrogradely inserted into the abdominal aorta below the renal arteries. Cardiac and circulatory arrest was induced by phrenotomy and immediate clamping of the thoracic aorta. The suprahepatic vena cava was opened to allow venting of flushing solution, and the liver was perfused via the aortic catheter with 60-100 mL of heparinized 0.9% saline at 4 °C over 5 min. A 15 mL amount of 0.5 mg/mL sulfosuccinimidyl-6-(biotinamindo)-hexanoate (Sulfo-NHS-LC-Biotin) (Pierce, IL) in Dulbecco’s PBS was then perfused into the liver over approximately 8 min, and the unreacted reagent was flushed out with 40 mL of saline. The liver was carefully removed, and for some samples a 2 mm slice was cut through the center and placed in fixative (Histochoice, Amresco, OH). The remaining liver was snapfrozen in liquid nitrogen. Protein Isolation and Identification. Frozen tissue was homogenized in 9 volumes of 0.9% Triton X-100, 0.09 M NaCl, 0.018 M Tris, pH 7.4, 1 mg/mL DNase-bovine pancreatic (Roche, Sydney, Australia), 0.25 mg/mL RNase A (Roche), 0.05 M MgCl2, and protease inhibitors (EDTA-free, Roche). The homogenate was centrifuged at 10 000 rpm for 10 min, and 0.8-1.2 mL of the clear supernatant was applied to a monomeric avidin affinity column (ImmunoPure Immobilized Monomeric Avidin, Pierce). The column was washed extensively with 1% Triton X-100/TBS to remove any unbound proteins. The biotinylated proteins were then eluted with 2 mM biotin (Sigma, Sydney, Australia) in 1% Triton X-100/TBS and precipitated with methanol/chloroform.8 The precipitated proteins were collected by centrifugation at 10 000 g for 5 min, dissolved in 100 µL sample buffer (0.25 M Tris, 10% glycerol, 2% SDS, 5% 2-mercaptoethanol, bromophenol blue to color), and incubated at 97 °C for 2 min, and 20 µL was separated on a 12% polyacrylamide minigel.9 Gels were either stained with silver to detect protein10 or the proteins were electroblotted to nitrocellulose9 (0.45 µm, BioRad, Sydney, Australia), and the biotinylated proteins were detected using horse radish peroxidase (HRP)-conjugated streptavidin (Neutravidin HRP, Pierce) and ECL (Amersham, Sydney, Australia). Images of stained gels and blots were captured with a Scanmaker 8700 Microtek scanner (Microtek, Sydney, Australia). Silver-stained protein profiles were compared between the normal (2 week sham-operated) and 2 and 5 week regenerated liver samples (n ) 2), and protein bands that had a corresponding band on the Western blot and were consistently changed in intensity in both samples at a given time point were excised from the gels for protein identification. Gel pieces were reduced and alkylated with iodoacetamide, washed with water, dehydrated with acetonitrile, and dried. Gel pieces were rehydrated in 50 mM ammonium bicarbonate containing ∼100 ng trypsin per band (Promega, Sydney, Australia) for 15 h at 37 °C, after which the supernatant was collected. Peptides were further extracted from each band with three washes each of ammonium bicarbonate and 5% formic acid/50% acetonitrile.10 Supernatants were centrifuged and collected after each wash, and the pooled supernatants were dried using a vacuum centrifuge and then resuspended 10 µL of 0.1% formic acid.
Digested proteins were separated by nanoLC using an Ultimate HPLC and Famos autosampler system (LC-Packings, Amsterdam, Netherlands). Samples (5 µL) were concentrated and desalted using a micro C18 precolumn (500 µm × 2 mm, Michrom Bioresources, Auburn, CA) with H2O:CH3CN (98:2, 0.1% formic acid) at 20 µL/min. After a 4 min wash, the precolumn was switched (Switchos, LC Packings) into line with a fritless nanocolumn manufactured according to Gatlin et al.11 Peptides were eluted using a linear gradient of H2O:CH3CN (95: 5, 0.1% formic acid) to H2O:CH3CN (50:50, 0.1% formic acid) at a flow rate of ∼200 nL/min over 30 min. High voltage (2300 V) was applied to low volume tee (Upchurch Scientific) and a column tip positioned ∼1 cm from the orifice of an API QStar Pulsar i hybrid tandem mass spectrometer (Applied Biosystems, Foster City, CA). Positive ions were generated by electrospray, and the QStar was operated in information dependent acquisition mode (IDA). A TOF-MS survey scan was acquired (m/z 350-1700, 1 s). The two largest multiply charged ions (counts > 15) were sequentially selected by Q1 for MS/MS analysis. Nitrogen was used as collision gas and an optimum collision energy chosen (based on charge state and mass). Tandem mass spectra were accumulated for 2.5 s (m/z 65-2000). Peak lists were generated using Mascot Distiller (Matrix Science, London, England), using the default parameters, and submitted to the Mascot database search program (version 2.1, Matrix Science). Search parameters: precursor and product ion tolerances ( 0.25 and 0.2 Da, respectively; Met(O)- and Cys-carboxyamidomethylation specified as variable modification, enzyme specificity was trypsin, one missed cleavage was possible, and the NCBInr ((mammalian taxonomy) database (September 2006) was searched. Confidence in identified proteins was based on scores >51, meaning these proteins were present with 95% confidence (P < 0.05; see Supporting Information). Localization of Biotinylated Proteins in Tissue Slices. Sections of fixed tissue were incubated in 3% H2O2 to block endogenous peroxidase, rinsed with water, incubated with proteinase K for 6 min, and washed with water and TBS/0.1% Tween. Sections were then incubated in protein block serum free (Dako, Sydney, Australia) for 10 min, rinsed in TBS/0.1% Tween, and incubated overnight in a 1:5000 dilution of streptavidin horseradish peroxidase (Neutravidin HRP, Pierce). Sections were rinsed with TBS/0.1% Tween and developed with liquid DAB for 2 min as per kit instructions (Dako). Sections were then washed with water and counterstained with Han’s hematoxylin followed by Scott’s blue solution, for 2 min each. After being washed with water, sections were mounted with xylene mounting medium.
Results Biotinylation Labeling of Proteins. To determine the effectiveness of biotinylation to label vascular surfaces, streptavidin peroxidase histochemistry was performed on sections of perfused and biotinylated liver. Figure 1 shows a section of biotinylated liver stained with streptavidin, with the brown staining indicating that biotin was localized to the luminal surface of major blood vessels, the microvasculature, and the sinusoids (Figure 1B). Under these conditions there was no detectable endogenous biotin staining in liver sections which were perfused with saline only (Figure 1A), nor was there any staining of biotinylated sections where the streptavidinperoxidase incubation was omitted, which appeared identical to Figure 1A and was therefore not shown. This indicates that the perfusion and biotinylation were effective in removing Journal of Proteome Research • Vol. 6, No. 8, 2007 3109
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Figure 1. Localization of biotinylated protein in perfused and labeled liver. The livers of anesthetized rats were perfused with saline alone (A) or saline followed by the biotinylation reagent (B) as described in Materials and Methods. Thin sections of fixed treated livers were incubated with streptavidin-HRP to detect biotinylated proteins as described in Materials and Methods, and the images were captured at a magnification of ×40-×10.
Figure 2. Affinity purification of biotinylated proteins. Extracts of biotinylated livers were run on monomeric avidin affinity columns to isolate the biotinylated protein fraction. The unbound and bound fractions and the total homogenate were separated by PAGE, and the protein was detected by silver stain (A). Separated proteins on a duplicate gel were electrotransferred onto a nitrocellulose filter, and the biotinylated protein was detected by streptavidin-HRP/ECL (B) as described in Materials and Methods.
blood proteins and selectively labeling the endothelial cell surfaces of the liver with biotin. Isolation and Identification of Proteins. Proteins that had been labeled with biotin were isolated from the liver homogenate using monomeric streptavidin affinity chromatography. The effectiveness of the isolation of the biotinylated proteins 3110
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Figure 3. Comparison of biotinylated proteins isolated from normal and regenerating liver. Livers were perfused and biotinylated at 2 weeks (2W) or 5 weeks (5W) after partial hepatectomy, along with the 2 week sham-operated (normal) livers (N), the biotinylated proteins isolated by avidin-affinity chromatography, and these were separated by PAGE and detected by silver staining (A). Separated proteins on a duplicate gel were electrotransferred onto a nitrocellulose filter, and the biotinylated protein was detected by streptavidin-HRP/ECL (B) as described in Materials and Methods.
was determined by comparing the protein profiles of the total homogenate, the unbound and bound (biotinylated) fractions as detected by silver staining (Figure 2A), and the profile of biotinylated proteins determined by Western blot using streptavidin-HRP/ECL detection (Figure 2B). Figure 2A shows that similar protein profiles were seen in the total liver homogenate and the unbound fractions, while the profile of the biotin eluted bound fraction was quite different. Figure 2B shows that the biotinylated proteins can be seen mostly in the bound fraction, with some biotinylated proteins detected in the total liver homogenate and only very faint banding detected in the unbound fraction. In addition, most of the silver-stained protein bands detected in the bound fraction had an equivalent biotinylated band on the streptavidin-HRP/ECL Western blot, suggesting that there was little contamination of the biotinylated proteins with unbiotinylated proteins. This indicated that the streptavidin affinity chromatography was effective in purifying the biotinylated proteins away from the unbiotinylated proteins. The biotinylated proteins were then isolated from duplicate samples of 2 week sham-operated control liver and 2 and 5 week regenerated liver and separated by SDS-PAGE. Figure 3A shows many differences were detected by silver staining between the sham-operated controls (N) and regenerated liver samples (2W, 5W). These differences were also seen on the Western blot detected with streptavidin-HRP/ECL (Figure 3B). The silver-stained bands showing consistent differences between treatments were cut from separate gels for protein identification using in-gel trypsin digestion and LC-MS/MS. Table 1 lists the 32 proteins that were identified in one or two but not all three of the normal, 2 week, or 5 week regenerated
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Table 1. Proteins Identified in 2 Week Sham-Operated (normal) Liver (N), 2 Week (2W) or 5 Week (5W) Regenerated Liver, but Not in All Three Samples (n ) 2) accession number
protein
1MABB NP_001013128 NP_058690 NP_113692 P20760 P20761 P01836 S26927 AAA91895 NP_110477 AAD09199 NP_579833 AAH82009 NP_478120 NP_445837 NP_445770 AAA40723 NP_001033064 NP_599153 NP_062016 NP_446205 NP_062160 NP_113886 NP_033773 NP_002323 NP_640347 NP_058768 NP_036716 NP_056644 NP_033395 ABC87985 NP_446429
rat liver F1-ATPase chain B transferrin complement C3 complement C4a immunoglobulin G2a immunoglobulin G2b immunoglobulin kappa immunoglobulin heavy chain V region elongation factor-1 alpha betaine-homocysteine methyltransferase semicarbazide-sensitive amine oxidase peroxisomal trans 2-enoyl CoA reductase heat shock protein 90 beta calcium transporting ATPase prolargin (proline arginine rich and leucine rich repeat protein, PRELP) hemopexin alpha-1-macroglobulin alpha-1-inhibitor III albumin fibronectin 1 C-type lectin superfamily member 13 apolipoprotein B ribophorin 2 AHNAK nucleoprotein isoform 1 low-density lipoprotein-related protein 1 beta 3 tubulin carbamoyl-phosphate synthetase 1 hydroxy-delta-5-steroid dehydrogenase adenine nucleotide translocator transforming growth factor-beta macrophage mannose receptor 1 cadherin 17
liver samples. The probability scores for each protein and the peptides identified are available as Supporting Information. Proteins which were identified in all three of these samples and were excluded included the biotin binding proteins pyruvate carboxylase, propionyl CoA carboxylase, methylcrotonyl CoA carboxylase, and acetyl CoA carboxylase.
Discussion Endothelial cells of blood vessels that have recently divided during angiogenesis behave differently from endothelial cells which have been dormant for some time. We hypothesized that there may be differences in the proteins expressed on the surface of these endothelial cells relative to dormant cells and that these differences could be potential targets in the treatment of angiogenesis dependent diseases such as cancer. Liver regeneration following partial hepatectomy provided a good model to study this process because of its abundance of tissuecontaining endothelial cells that had recently divided. Using this rat liver regeneration model, we developed a methodology for the in vivo biotinylation, isolation, and identification of endothelial cell surface proteins and showed that this method has sufficient sensitivity to detect changes in these proteins 2 weeks after partial hepatectomy relative to the sham-operated controls. Other groups have used the approach of biotinylation to identify endothelial cell surface proteins in vivo4,12,13 and in vitro,14,15 as well as labeling and studying other cells16 or organisms17 in culture. There have also been other approaches to identify vascular surface proteins, including using silica beads to coat endothelial cell surfaces in vitro and in vivo,18 which were used to isolate the surface proteins. Another approach did not identify vascular proteins directly, but rather
found in
N N N
N N N N N
2W 2W 2W 2W 2W 2W 2W 2W 2W 2W 2W 2W 2W 2W 2W 2W 2W 2W 2W 2W 2W 2W 2W
5W 5W 5W 5W 5W 5W 5W 5W 5W 5W 5W 5W 5W 5W 5W
identified peptides that bound on the vasculature of specific organs using phage display.19 We believe our method may be as sensitive, if not more sensitive, than these other in vivo studies, as we were able to identify a set of proteins associated with endothelial cells which had initiated angiogenesis soon after hepatectomy 2 to 5 weeks before. These protein changes would probably be more subtle than those detectable in the other in vivo studies, and this indicates the method’s sensitivity. Our approach of biotinylation, like the others above, involves perfusion to remove blood proteins before biotin labeling. This perfusion may alter the distribution of proteins on the vascular surface. The extent of this effect would be difficult to determine. However it is likely that similar perfusion-induced changes would occur in both the normal and regenerated liver, making this less of an issue in our comparative study. In addition, our strategy of in vivo biotinylation and affinity chromatography isolation was relatively specific since the vast majority, if not all proteins which were isolated (silver-stained bands in Figure 2A), were also biotinylated as detected in Figure 2B. The histochemical staining of biotinylated proteins in the liver sections also showed that biotin labeling was detected preferentially on the surface of endothelial cells either as part of blood vessels or as part of the sinusoids (Figure 1B). There was slight cytoplasmic staining, but it was not convincing, and this suggests that the proteins that were selectively labeled and isolated were associated with the cell membrane. Since we identified 32 proteins differentially expressed between regenerated and normal liver (Table 1), this suggests this method is sufficiently sensitive for reliable mass spectrometry identification. Many of the proteins identified were proteins considered to be associated with the cell surface, such as low-density lipoprotein related protein 1, rat liver F1 ATPase, Journal of Proteome Research • Vol. 6, No. 8, 2007 3111
research articles and calcium transporting ATPase, while others were not cell surface proteins but could have conceivably been labeled because they bind to the cell surface, such as transferrin and hemopexin. Of the proteins that were identified by this method, most could be associated with endothelial cells through their role in angiogenesis or with liver regeneration. We did identify proteins which are generally localized to the blood, including albumin and alpha 1 macroglobulin, and this may be because all the blood may not have perfused out of all areas of the liver. However, since there were only a few blood proteins identified, and they were not identified in all the samples, a more feasible explanation is that these few blood proteins were preferentially bound to the endothelial cell surface in these samples. In addition, there were proteins identified that are thought to be intracellular such as the mitochondrial protein carbamoyl-phosphate synthetase and the cytosolic protein HSP-90beta. We can only speculate on how these were isolated by our methodology, and there are four likely possibilities. The first is that a small fraction of these intracellular proteins may also be located externally. A possible example of this could be HSP90-beta, a protein that is mainly cytoplasmic and nuclear but could also be membrane associated because recently its association partner, HSP-90-alpha and the client protein matrix metalloprotease 2 (MMP2), were reported on the cell surface.20 The second possibility is that contrary to popular belief, the biotinylation reagent may have access to the cytosol. It is probably true that little if any of this hydrophilic reagent crosses the lipid bilayer of the plasma membrane. However, there are well-defined mechanisms such as pinocytosis that internalize external substances and do not involve the transmembrane path. Others have also reported labeling intracellular proteins with biotinylation reagents designed to only label the cell surface.4 The third possibility is these proteins come from leaky or dying cells and become associated with the vascular cell surface; however, this would be difficult to determine in vivo. Finally, these proteins could be localized inside cells under normal conditions, but could change localization to be present on the surface of cells during angiogenesis, which may not have been previously described. Two proteins identified as increased in regenerated liver were fibronectin and prolargin. These proteins are normally considered to be localized in the extracellular matrix. However, they may also be expressed on the luminal side of the endothelial cell surface since they bind to perlecan,21,22 which is found on the endothelial cell surface,23 and has been shown to induce bFGF-mediated neovascularization in vivo.24 In addition, a form of the type 3 domain of fibronectin (ED-B) is a marker of angiogenesis in normal and tumor tissues,25 and an antibody to this domain localizes to tumor blood vessels in both animal models and cancer patients.26 Other proteins that were identified in regenerated liver were low-density lipoprotein-related protein (LRP1, CD91), otherwise known as alpha-2-macroglobulin receptor, and two proteins which bind to this receptor, hemopexin, a matrix protein related to the matrix metalloproteases (MMPs) and alpha I inhibitor III. LRP1 has been reported to be the receptor for endocytosis of thrombospondin-1 into endothelial cells, a protein which inhibits endothelial cell proliferation.27 Therefore, the increased expression of LRP in 5 week regenerated liver may be explained by upregulation of this receptor, allowing an increased amount of thrombospondin 1 to be taken up into endothelial cells, thereby decreasing endothelial proliferation and angiogenesis when the liver has stopped regenerating. 3112
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In addition, the inhibition of endothelial cell proliferation by thrombospondin 1 is thought to be partially due to transforming growth factor-beta (TGFβ),28 another protein which was identified as increased in 5 week regenerated liver. TGFβ is thought to suppress growth during liver regeneration, as it has been shown to inhibit hepatocyte proliferation and is secreted by endothelial cells.29 This effect is reflected by our results, since TGFβ was detected in normal and 5 week regenerated liver, but not in 2 week regenerated liver. We have developed a method to label vascular proteins in vivo and selectively isolate them from the rest of the proteins in a tissue. This method has sufficient selectivity and sensitivity to reliably identify these proteins. With minor adaptations, it will be possible to study the vasculature of many other normal organs, tissues, and tumors. Any proteins identified using this method have the advantage of being accessible to treatments delivered intravenously.
Supporting Information Available: Table S1 contains a list of the number and sequence of peptides identified for each of the proteins listed in Table 1. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Folkman, J. In Harrison’s Principles of Internal Medicine; Braunwald, E., Fauci, A. S., Kasper, D. L., Hauser, S. L. et al., Eds.; McGraw-Hill: New York, 2001; pp 517-530. (2) Collins, I.; Workman, P. New approaches to molecular cancer therapeutics. Nat. Chem. Biol. 2006, 2, 689-700. (3) Lamszus, K.; Brockmann, M. A.; Eckerich, C.; Bohlen, P.; May, C.; Mangold, U.; Fillbrandt, R.; Westphal, M. Inhibition of glioblastoma angiogenesis and invasion by combined treatments directed against vascular endothelial growth factor receptor-2, epidermal growth factor receptor, and vascular endothelialcadherin. Clin. Cancer Res. 2005, 11, 4934-4940. (4) Rybak, J. N.; Ettorre, A.; Kaissling, B.; Giavazzi, R.; Neri, D.; Elia, G. In vivo protein biotinylation for identification of organ-specific antigens accessible from the vasculature. Nat. Methods 2005, 2, 291-298. (5) Taniguchi, E.; Sakisaka, S.; Matsuo, K.; Tanikawa, K.; Sata, M. Expression and role of vascular endothelial growth factor in liver regeneration after partial hepatectomy in rats. J. Histochem. Cytochem. 2001, 49, 121-130. (6) Greene, A. K.; Wiener, S.; Puder, M.; Yoshida, A.; Shi, B.; PerezAtayde, A. R.; Efstathiou, J. A.; Holmgren, L.; Adamis, A. P.; Rupnick, M.; Folkman, J.; O’Reilly, M. S. Endothelial-directed hepatic regeneration after partial hepatectomy. Ann. Surg. 2003, 237, 530-535. (7) Higgins, G. F.; Anderson, R. M. Experimental pathology of the liver. I. Restoration of the liver of the white rat following partial surgical removal. Arch. Pathol. 1931, 12, 186-202. (8) Wessel, D.; Flugge, U. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal. Biochem. 1984, 138, 141-143. (9) Henness, S.; Davey, M. W.; Harvie, R. M.; Banyer, J.; Wasinger, V.; Corthals, G.; Davey, R. A. Changes in gene expression associated with stable drug and radiation resistance in small cell lung cancer cells are similar to those caused by a single x-ray dose. Radiat. Res. 2004, 161, 495-503. (10) Schevchenko, A.; Wilm, M.; Vorm, O.; Mann, M. Mass Spectrometric Sequencing of Proteins from Silver-Stained Polyacrylamide Gels. Anal. Chem. 1996, 68, 850-858. (11) Gatlin, C. L.; Kleemann, G. R.; Hays, L. G.; Link, A. J.; Yates, J. R. Protein identification at the low femtomole level from silverstained gels using a new fritless electrospray interface for liquid chromatography-microspray and nanospray mass spectrometry. Anal. Biochem. 1998, 263, 93-101. (12) Castronovo, V.; Waltregny, D.; Kischel, P.; Roesli, C.; Elia, G.; Rybak, J. N.; Neri, D. A chemical proteomics approach for the identification of accessible antigens expressed in human kidney cancer. Mol. Cell. Proteomics 2006, 5, 2083-2091. (13) Rybak, J. N.; Scheurer, S. B.; Neri, D.; Elia, G. Purification of biotinylated proteins on streptavidin resin: a protocol for quantitative elution. Proteomics 2004, 4, 2296-2299.
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Identification of Vascular Surface Proteins (14) Scheurer, S. B.; Rybak, J. N.; Roesli, C.; Brunisholz, R. A.; Potthast, F.; Schlapbach, R.; Neri, D.; Elia, G. Identification and relative quantification of membrane proteins by surface biotinylation and two-dimensional peptide mapping. Proteomics 2005, 5, 27182728. (15) Chen, W. N.; Yu, L. R.; Strittmatter, E. F.; Thrall, B. D.; Camp, D. G.; Smith, R. D. Detection of in situ labeled cell surface proteins by mass spectrometry: application to the membrane subproteome of human mammary epithelial cells. Proteomics 2003, 3, 1647-1651. (16) Shin, E. Y.; Lee, J. Y.; Park, M. K.; Chin, Y. H.; Jeong, G. B.; Kim, S. Y.; Kim, S. R.; Kim, E. G. Overexpressed alpha3beta1 and constitutively activated extracellular signal-regulated kinase modulate the angiogenic properties of ECV304 cells. Mol. Cell 1999, 9, 138-145. (17) Sabarth, N.; Lamer, S.; Zimny-Arndt, U.; Jungblut, P. R.; Meyer, T. F.; Bumann, D. Identification of surface proteins of Helicobacter pylori by selective biotinylation, affinity purification, and two-dimensional gel electrophoresis. J. Biol. Chem. 2002, 277, 27896-27902. (18) Durr, E.; Yu, J.; Krasinska, K. M.; Carver, L. A.; Yates, J. R.; Testa, J. E.; Oh, P.; Schnitzer, J. E. Direct proteomic mapping of the lung microvascular endothelial cell surface in vivo and in cell culture. Nat. Biotechnol. 2004, 22, 985-992. (19) Arap, W.; Kolonin, M. G.; Trepel, M.; Lahdenranta, J.; Cardo-Vila, M.; Giordano, R. J.; Mintz, P. J.; Ardelt, P. U.; Yao, V. J.; Vidal, C. I.; Chen, L.; Flamm, A.; Valtanen, H.; Weavind, L. M.; Hicks, M. E.; Pollock, R. E.; Botz, G. H.; Bucana, C. D.; Koivunen, E.; Cahill, D.; Troncoso, P.; Baggerly, K. A.; Pentz, R. D.; Do, K. A.; Logothetis, C. J.; Pasqualini, R. Steps toward mapping the human vasculature by phage display. Nat. Med. 2002, 8, 121-127. (20) Eustace, B. K.; Sakurai, T.; Stewart, J. K.; Yimlamai, D.; Unger, C.; Zehetmeier, C.; Lain, B.; Torella, C.; Henning, S. W.; Beste, G.; Scroggins, B. T.; Neckers, L.; Ilag, L. L.; Jay, D. G. Functional proteomic screens reveal an essential extracellular role for hsp90 alpha in cancer cell invasiveness. Nat. Cell Biol. 2004, 6, 507514.
(21) Hopf, M.; Gohring, W.; Kohfeldt, E.; Yamada, Y.; Timpl, R. Recombinant domain IV of perlecan binds to nidogens, lamininnidogen complex, fibronectin, fibulin-2 and heparin. Eur. J. Biochem. 1999, 259, 917-925. (22) Bengtsson, E.; Morgelin, M.; Sasaki, T.; Timpl, R.; Heinegard, D.; Aspberg, A. The leucine rich repeat protein PRELP binds perlecan and collagens and may function as a basement membrane anchor. J. Biol. Chem. 2002, 277, 15061-15068. (23) Mertens, G.; Cassiman, J. J.; Van den, B. H.; Vermylen, J.; David, G. Cell surface heparan sulfate proteoglycans from human vascular endothelial cells. Core protein characterization and antithrombin III binding properties. J. Biol. Chem. 1992, 267, 20435-20443. (24) Aviezer, D.; Hecht, D.; Safran, M.; Eisinger, M.; David, G.; Yayon, A. Perlecan, basal lamina proteoglycan, promotes basic fibroblast growth factor-receptor binding, mitogenesis, and angiogenesis. Cell 1994, 79, 1005-1013. (25) Castellani, P.; Viale, G.; Dorcaratto, A.; Nicolo, G.; Kaczmarek, J.; Querze, G.; Zardi, L. The fibronectin isoform containing the ED-B oncofetal domain: a marker of angiogenesis. Int. J. Cancer 1994, 59, 612-618. (26) Santimaria, M.; Moscatelli, G.; Viale, G. L.; Giovannoni, L.; Neri, G.; Viti, F.; Leprini, A.; Borsi, L.; Castellani, P.; Zardi, L.; Neri, D.; Riva, P. Immunoscintigraphic detection of the ED-B domain of fibronectin, a marker of angiogenesis, in patients with cancer. Clin. Cancer Res. 2003, 9, 571-579. (27) Godyna, S.; Liau, G.; Popa, I.; Stefansson, S.; Argraves, W. S. Identification of the low density lipoprotein receptor-related protein (LRP) as an endocytic receptor for thrombospondin-1. J. Cell Biol. 1995, 129, 1403-1410. (28) Murphy-Ullrich, J. E.; Schultz-Cherry, S.; Hook, M. Transforming growth factor-beta complexes with thrombospondin. Mol. Biol. Cell. 1992, 3, 181-188. (29) Bucher, N. L. Liver regeneration: an overview. J. Gastroenterol. Hepatol. 1991, 6, 615-624.
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