Global Profiling of Surface Plasma Membrane Proteome of Oviductal

Jul 25, 2006 - Tree Root Walk, Sheffield S10 2SF, United Kingdom, Royal Veterinary College, London, United Kingdom, and. Institute of Zoology, London,...
0 downloads 0 Views 269KB Size
Download PDF
Global Profiling of Surface Plasma Membrane Proteome of Oviductal Epithelial Cells Edita Sostaric,† Aristophanes S. Georgiou,† Chi H. Wong,† Paul F. Watson,‡ William V. Holt,§ and Alireza Fazeli*,† Academic Unit of Reproductive and Developmental Medicine, University of Sheffield, The Jessop Wing, Level 4, Tree Root Walk, Sheffield S10 2SF, United Kingdom, Royal Veterinary College, London, United Kingdom, and Institute of Zoology, London, United Kingdom Received July 25, 2006

In mammalian reproduction, many important events occur within the female reproductive tract, especially within the oviduct. These include transport and final maturation of the female and male gametes, fertilization, embryonic development, and transport of the embryo to the uterus. The plasma membrane molecules of oviductal epithelia that are in direct contact with gametes and embryo(s) and potentially mediate these processes are poorly characterized, and their function is poorly understood. Defining the oviductal cell surface proteome could provide a better understanding of the basis of reproductive processes taking place within the oviduct. We aimed to provide a detailed profile of the surface plasma membrane proteome of the oviductal epithelium by biotinylation of proteins at the cell surface, followed by highly specific purification of these proteins using avidin. This approach for enrichment of oviductal cell surface proteome was validated by immunohistochemistry, gel electrophoresis, and western blot analysis experiments. The enriched molecules were identified using two different technologies: (i) the combination of 2D gel electrophoresis with mass spectrometry and (ii) 1D gel electrophoresis with mass spectrometry (a modified multidimensional protein identification technology (MudPIT) technique). The number of proteins identified using the MudPIT approach was approximately 7 times the number of proteins identified by 2D gel electophoresis using the same samples (40 versus 276, respectively). Some of the proteins found at the surface of oviductal cells had previously been reported as present in the oviduct and to have known functions in relation to reproductive processes. The other category of proteins that were highly represented in the oviductal surface proteome were various members of the family of heat-shock proteins. To the best of our knowledge, this is the first comprehensive study to identify and characterize proteins at the surface of the epithelium of the mammalian oviduct. Keywords: reproduction • oviductal epithelium • surface proteome • biotinylation

Introduction The plasma membrane is a cellular compartment that provides a physical boundary between the cell and its environment. Membrane proteins are integral components of the plasma membrane and are poised at the interface between the cell and the surrounding environment. These proteins perform key biological functions, such as cell-to-cell recognition and the transport of ions and solutes. In addition, they act as receptors for relaying the diverse signals with which cells come into contact. During differentiation, the cell surface proteome is altered to accommodate cellular needs. In nearly all biological systems, eukaryotic cells regulate the abundance and distribution of cell surface membrane proteins in response to contact * To whom correspondence should be addressed. Tel.: 0114-2268195. Fax: 0114-2268538. E-mail: [email protected]. † University of Sheffield. ‡ Royal Veterinary College. § Institute of Zoology. 10.1021/pr060366w CCC: $33.50

 2006 American Chemical Society

with an adjacent cell. Events such as the export and internalization of whole receptor molecules after ligand binding are well-characterized phenomena1-3 that influence many important functions of the cells. Defining the surface membrane proteome is the key to understanding the role of membrane proteins in these fundamental biological processes. Plasma membrane protein expression has profound biological effects and may, for example, underlie phenotypic and functional differences between normal and tumor cells.4 For these reasons, profiling of the plasma membrane protein expression is an area of intense interest.5 In mammalian reproduction, many important phenomena occur within the female reproductive tract, especially within the oviduct. These include transport and final maturation of the female and male gametes, fertilization, embryonic development, and transport of the embryo to the uterus. The physiological interaction between gametes and embryos within the oviduct involves intimate and specific contact with oviductal Journal of Proteome Research 2006, 5, 3029-3037

3029

Published on Web 10/19/2006

research articles epithelia that affects both oviduct epithelium and gametes. The presence of gametes in the oviduct alters oviductal gene transcription6 and induces specific alterations of the oviductal secretory proteomic profile.7 Furthermore, attachment of spermatozoa to oviductal epithelia promotes sperm viability, motility, and modulates sperm capacitation.8-11 Moreover, the oviduct synchronizes the release of a sufficient number of live and competent spermatozoa needed for fertilization.12 The oviduct epithelium enhances the quality of the oocyte cytoplasmic maturation in vitro and subsequent blastocyst cell proliferation13 and embryo development.14 The plasma membrane molecules of oviductal epithelia that mediate these processes are poorly characterized, and their function is poorly understood. Defining the oviductal cell surface proteome might provide a better understanding of the manner in which the cell surface proteome in this tissue is regulated and how it responds to a variety of intracellular and extracellular signals. This knowledge may help us to understand the basis of reproductive processes taking place within the oviduct. Biotinylation of proteins at the cell surface, followed by highly specific purification of these proteins using avidin, has previously been used successfully for the enrichment and identification of surface membrane proteins.15-17 This is an effective technology for achieving genuine membrane protein purification, something that has proved very difficult to achieve using other less specific technologies. Using this technology (biotin tagging of the cell surface proteome), we aimed to profile the entire surface plasma membrane proteome of the oviductal epithelium and to characterize the molecules involved in gamete/embryo-oviduct interaction. We used two different technologies for the identification of biotinylated surface proteins: (i) the combination of 2D gel electrophoresis with mass spectrometry and (ii) 1D gel electrophoresis with mass spectrometry (a modified multidimensional protein identification technology (MudPIT) technique). Finally, this approach for global profiling of oviductal cells surface proteome was validated by immunohistochemistry, gel electrophoresis, and western blot analysis experiments. To the best of our knowledge, this is the first study to identify and characterize proteins at the surface of the epithelium of the mammalian oviduct in such a comprehensive manner.

Materials and Methods Tissue Collection and Oviductal Epithelial Cell Isolation and Culture. Sow reproductive tracts were obtained from a local abattoir (G. Wood and sons, Mansfield, UK) on the day of slaughter and transported at ambient temperature to the laboratory. Sows used in the study were healthy, and their ovaries showed no signs of abnormal follicular development. On arrival at the laboratory, the oviducts attached to ovaries containing large follicles (8-12 mm in diameter) with signs of recent ovulation, and no corpora lutea were selected and used in the further experiments. The source animals were considered as being in the follicular and ovulatory stages of the reproductive cycle. The oviducts were trimmed from the surrounding tissue and rinsed with PBS. Each oviduct was flushed with 10 mL of PBS (Gibco, Invitrogen, Paisley, UK) and then sealed at the isthmic end using cotton thread (approximately 5 mm above cutting line). Oviducts were filled with 0.25% (w/v) collagenase (Sigma-Aldrich, Dorset, UK) in Hank’s balanced salt solution (HBSS) (Gibco) using a syringe, and then immediately sealed at the ampullar end using cotton thread (approximately 5 mm above cutting line). Collagenase-filled oviducts were 3030

Journal of Proteome Research • Vol. 5, No. 11, 2006

Sostaric et al.

incubated in HBSS at 39 °C, 5% CO2 for 2 h. Oviducts were removed from HBSS, cut open at one end, and the oviductal fluid was drained and collected in a centrifuge tube. Isolated oviductal epithelial (OE) cells were washed three times with HBSS (37 °C) by centrifugation at 300g for 5 min. The pellet was gently mixed with 2 mL of red blood cell lysis buffer (Sigma-Aldrich) for 1 min, further diluted with 20 mL of HBSS, and centrifuged at 300g for 5 min. Pelleted OE cells were washed twice with HBSS and finally resuspended in medium 199 (M199) (Gibco) supplemented with 10% FCS (Gibco), 100 units/mL penicillin (Gibco), 100 units/mL streptomycin (Gibco), and 100 units/mL amphotericin (Gibco) and were grown in 50 cm2 tissue flasks or six well culture dishes (Nunc, VWR International Ltd., Leics, UK) at 39 °C, 5% CO2. Verification of OE Cells Epithelial Nature. To verify that OE cells used in current experiments were of epithelial nature, we performed immunostaining using an epithelial cell specific antibody, monoclonal anti-pan cytokeratin clone C-II (mouse; Sigma). The OE cells grown for 18 h in six-well culture dish were fixed overnight at 4 °C in 2% formaldehyde. The cells were stained using a Vectastain Elite ABC peroxidase kit (Vector Laboratories Ltd., UK), and to avoid nonspecific binding, an avidin/biotin blocking kit (Vector) was used. Briefly, the fixed cells were three times washed with PBS and blocked for 1 h at room temperature in PBS containing 0.2% v/v horse serum and 25% v/v avidin supplied in the blocking kit. The blocking was removed, and cells were incubated for 1 h with monoclonal anti-pan cytokeratin antibody diluted 1:1000 in diluent media (Dakocytomation Ltd., UK) and 250 mL of biotin per mL of diluted antibody. The cells were three times washed with PBS, and antibody was visualized by incubation with 3,3′diamino benzidine (DAB) (Vector) for 10 min. The cells were washed in distilled water for 3 min, counterstained in 10% hemeatoxylin for 10 min, and washed three times with tap water. Staining was analyzed under a bright-field microscope BH-2 (Olympus). Percentage of positive stained OE cells was estimated in 200 counted cells per well. Biotinylation of the OE Cell Surface Proteins. After 18 h of culture, the OE cells that had adhered to the culture flask were washed five times with prewarmed PBS (37 °C) and then incubated with 0.5 mg/mL EZ-Link sulfo-NHS-LC-biotin (Pierce, Perbio Science, Cramlington, UK) in PBS for 20 min at 37 °C in 5% CO2. Following biotinylation, cells were washed with PBS. Excess biotin was quenched with 50 mM Tris-HCl (pH 7.5) to terminate the biotinylation reaction, and cells were washed three times with PBS. Determination of OE Cell Viability after Biotinylation Process. The OE cells viability after biotinylation was assessed using the LIVE/DEAD Viability/Cytotoxicity Kit (Invitrogen Ltd., Paisley, UK) according to the manufacturer’s instructions. Briefly, the cells were washed three times with prewarmed (37 °C) phosphate buffered saline (PBS) (Gibco), incubated for 20 min with 2 µM calcein AM and 4 µM Ethidium homodimer-1 (EthD-1) at room temperature, and washed three times with PBS. Staining was analyzed under a fluorescence microscope (Olympus). Percentage viability was estimated in 200 cells per well. Solubilization and Enrichment of OE Cells Biotinylated Proteins. Biotinylated OE cells were collected by scraping them from the culture flasks into PBS containing protease inhibitors. Igepal CA-630 (NP-40) (Sigma-Aldrich) was added to a final concentration of 2% (v/v), and OE cells were homogenized by sonication in ice for 15 min, and centrifuged at 22 000g for 30

Oviductal Epithelial Cells Surface Proteome Profile

min at 4 °C. The supernatant, containing solubilized proteins from the whole cells lysate, was collected and stored at -80 °C until used for purification of biotinylated surface proteins. Biotinylated OE cell surface proteins were purified using an ImmunoPure Immobilized monomeric avidin (Pierce). The immobilized monomeric avidin column was prepared by washing the gel-bed (1 mL volume) with ten volumes of PBS. Non-reversible biotin binding sites of the column were blocked by washing with 2 mM D-biotin (Pierce) in PBS. The column was then washed with 10 mL of 0.1 M glycine (pH 2.8) and finally equilibrated with 10 mL of PBS. Solubilized whole cell lysate proteins were passed through the column five times. Unbound solubilized proteins were removed from the column by washing with 20 mL of 1% NP-40 in PBS followed by 10 mL of PBS. The bound biotinylated proteins were eluted from the column with 20 mL of 0.1 M glycine (pH 2.8) and concentrated with Vivaspin concentrators (5000 kDa molecular weight cutoff; Sigma-Aldrich). The amount of total protein present in the solubilized whole cell lysate, and the amount recovered after elution of biotinylated proteins from the avidin column, was determined using a bicinchoninic acid assay (BCA) as previously described in ref 18. Detection of Proportion of Biotinylated Proteins before and after Biotinylated Protein Enrichment. To visualize the degree of enrichment of biotinylated proteins, samples containing 50 or 300 µg proteins of protein fractions before and after enrichment of biotinylated proteins were separated by 2D SDSPAGE. Plus-one 2D clean up kit (Amersham Biosciences, Buckinghamshire, UK) was used according to the manufacturer’s instructions to purify, desalt, and remove all impurities from protein samples. The resulting protein pellets after clean up were resuspended in 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 18 mM dithiothreitol (DTT), 2% (v/v) IPG buffer, and 0.005% (w/v) bromophenol blue (Pierce). Fifty or 300 µg of total protein, in a 350 µL volume, was used to rehydrate 18 cm, pH 3-10 immobilized pH gradient (IPG) strips (Amersham). Proteins were resolved in the first dimension by isoelectric focusing (IEF) using the IPGphor Isolectric Focusing system (Amersham; 500 V (0.5 kV h), 1000 V (0.8 kV h), 8000 V (13.5 kV h), and 8000 V (6.2 kV h)). The focused strip was incubated in 10 mL of equilibration buffer (50 mM Tris/HCl, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, 0.005% (w/v) bromophenol blue) containing 65 mM DTT for 15 min and subsequently in 10 mL of equilibration buffer containing 135 mM iodoacetamide for 15 min. The second dimension was performed using 12.5% SDS-PAGE performed in the EttanDalt vertical system (Amersham) at 25 °C. Gels were fixed overnight in 7% (v/v) acetic acid in 40% (v/v) methanol at room temperature. The resultant 2D gels containing 50 µg of proteins were rinsed with water and stained with Silver staining kit (Pharmacia Biotech AB, Uppsala, Sweden) according to the manufacturer’s protocol. The gels containing 300 µg of proteins were blotted onto polyvinylidene difluoride membranes (PVDF) (Millipore Corp., Bedford, MO). The membranes were blocked for 5 min at room temperature with 0.3% (v/v) Tween-20 in PBS (Gibco) and then overnight in TPBS (containing 0.05% (v/v) Tween-20 and 1% (w/v) BSA in PBS). To visualize the biotinylated proteins, the membranes were incubated for 1 h at room temperature with neutravidin-horse radish peroxidase (neutravidin-HRP; Pierce) diluted (1:250 000) in TPBS and washed five times for 10 min each in TPBS. Biotinylated proteins were visualized by use of

research articles SuperSignal West Dura Extended Duration Substrate (Pierce) followed by exposure of blots to X-ray film (Hyperfilm ECL, Amersham). Verification of Enrichment of Cell Surface Proteome by Western Blot Analysis. To verify the enrichment of cell surface proteome after enrichment of the biotinylated proteins, both samples before and after enrichment of biotinylated proteins containing 50 µg of total protein were separated by SDS-PAGE using 12% polyacrylamide gels (Laemmli et al., 1970) and transferred to a PVDF membrane for Western blot analysis. The membranes were blocked (as described previously) and incubated for 2 h at room temperature with primary antibodies diluted in TPBS, anti-cytochrome C, 1:2000 dilution (Mouse IgG) (BD Biosciences, Oxford, UK), P23, 1:2000 dilution (Rabbit IgG) (anti-Golgi antibody: a generous gift from Prof. B. Helms from Utrecht University, The Netherlands), anti-calnexin, 1:2000 dilution (Rabbit IgG) (Stressgen, Victoria, Canada), and N-cadherin, 1:100 dilution (sc-1502; Goat IgG) (Santa Cruz Biotechnology, Inc., Heidelberg, Germany), and subsequently with horseradish peroxidase-conjugated secondary antibodies (anti-mouse IgG, anti-rabbit IgG, or anti-goat IgG (Sigma)) for 1 h (final concentrations 1:5000 µg/mL) in TPBS. After every incubation step, the membranes were washed four times for 10 min each in TPBS. Antibody binding on the Western blots was visualized by use of SuperSignal West Dura Extended Duration Substrate (Pierce) followed by exposure to X-ray film (Amersham). Identification of Biotinylated Proteins. Two different methodologies were used for the identification of biotinylated proteins. In one, proteins were subjected to 2D gel electrophoresis and mass spectrometry analysis. In the other, a modified multidimensional protein identification technology (MudPIT) in combination with 1D gel electrophoresis and mass spectrometry was used for identification of proteins. (i) 2D Protein Gel Electrophoresis and Mass Spectrometry. Enriched, biotinylated protein samples, containing 300 µg of total protein, were separated by 2D SDS-PAGE and fixed as described previously. 2D gels were stained with colloidal brilliant blue Coomassie G50 (Coomassie) (Sigma-Aldrich) for 2 h at room temperature. Gels stained with Coomassie were destained in 10% acetic acid in 25% (v/v) methanol for 1 min at room temperature, washed, and stored in 25% (v/v) methanol. Gel images were captured using an Image Scanner II flat bed scanner with LabScan software (Amersham). All visible protein spots were manually excised from the Coomassie stained 2D gels. All gel spots were washed in 25% (v/v) methanol and incubated for 1 h at 37 °C in Coomassie destain solution containing 40% (v/v) acetonitrile in 200 mM ammonium bicarbonate. The solution was removed, and gel pieces were incubated with acetonitrile (ACN) for 15 min at room temperature. After ACN was removed, gel pieces were dried in a vacuum centrifuge. Proteins, in dried gel pieces, were digested with 20 ng/µL of sequencing grade modified trypsin (Promega, Southampton, UK) in 50 mM ammonium bicarbonate at 37 °C for 12 h. The supernatant from the trypsin digest was transferred to a siliconized microcentrifuge tube. The proteins from the gel pieces were sequentially extracted three times by incubation with peptide extraction solutions: 25 mM ammonium bicarbonate (10 min at room temperature), 5% formic acid (15 min at 37 °C), and ACN (15 min at 37 °C). The original supernatant and the supernatants from the three sequential extractions were combined and dried in a vacuum centrifuge. The peptides were dissolved in 12 µL of 0.1% (v/v) formic acid Journal of Proteome Research • Vol. 5, No. 11, 2006 3031

research articles in 3% (v/v) ACN in water. Samples were centrifuged for 5 min at 12 000g, and the supernatants were subjected to liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Digested peptides were separated using a reverse phase CapLC system (Waters, Manchester, UK). Peptides were delivered to a PepMap C18 microguard column (300 µm internal diameter × 1 mm) (LC-packings Dionex, Leeds, UK) at 5 µL/ min. A splitter then reduced the flow rate to approximately 200 nL/min, and peptides were transferred to the analytical column (PepMap C18; 75 µm internal diameter × 15c m column (LCpackings Dionex)). The peptides were eluted with a linear gradient from 0-80% buffer B (0.1% formic acid in 95% (v/v) ACN in water) over 60 min. The column eluent was sprayed directly into the ESI source of a Q-TOF micro (Waters). Spray voltage was set to 3000 V, and the source temperature was 80 °C. The data acquisition on the MS was performed using information dependent acquisition (IDA). After each TOF-MS scan, three peaks with charge states two or three within the range of 400-1500 m/z were selected for tandem mass spectrometry. (ii) 1D Protein Electrophoresis and Multidimensional Protein Identification Technology (MudPIT). Protein samples containing 20 or 200 µg protein of biotinylated enriched fraction were mixed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (final concentration: 62.5 mM Tris, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, and 0.002% bromphenol blue, pH 6.8). After being boiled for 15 min, samples were separated by SDS-PAGE using a 4-20% gradient mini gel (Pierce) and stained with colloidal brilliant blue Coomassie for 2 h at room temperature. The gel was destained in 10% acetic acid in 25% (v/v) methanol for 1 min at room temperature, washed, and stored in 25% (v/v) methanol. The 4-20% gradient minigel (20 or 200 µg of protein) was cut in its length to 20 bands each approximately 2 mm and diced into smaller pieces. Protein bands were digested, prepared, and analyzed by mass spectrometry (as described previously). Three MS runs were performed for each sample (band). In all cases, MS spectra were searched against the Mass Spectrometry Data Base (MSDB) in a sequence query search using MASCOT 2.0 software (www.matrixscience.com). The taxonomy was limited to filter for only mammalian match, and trypsin was used as the cleavage enzyme, with one missed cleavage site allowed. The peptide tolerance was set to 1.0 Da, and the MS/MS tolerance was set to 0.3 Da. Carbamidomethyl modification of cysteine, oxidized methionine, and biotin K and N-term were set as variable modifications. The MASCOT MudPIT scoring algorithm was used with an ion score cutoff of 20, with required bold red only. Peptides with an ion score below a statistical significance of p < 0.05 were manually removed from further analysis. Further peptide analysis was performed as follows: (i) The results of three separate MS runs were combined in one protein list with all of the respective peptides identified during the MS run (for 20 or 200 µg of protein concentrations, this was performed separately); (ii) redundant peptide sequences for identical proteins of different runs were removed by leaving one sequence with the highest peptide score; thereafter (iii) identified proteins from 20 and 200 µg of protein sample concentrations were combined into one list; (iv) any redundant proteins identified between these two groups were omitted from the list based on the same criteria as mentioned above, that is, omitting peptide sequences 3032

Journal of Proteome Research • Vol. 5, No. 11, 2006

Sostaric et al.

Figure 1. Light microscopic micrograph of primary isolated oviductal epithelial cells. After 18 h in culture, cells that remained attached to the bottom of the tissue culture flask were stained with anti-pan cytokeratin. The majority of the cells were cytokeratin positive (brown) and therefore epithelial cells.

with the low peptide scores; and (v) finally the inter-species redundancy of proteins was removed. In addition, each protein was examined for the presence of hypothetical trans-membrane domains and signal sequences using the TMbase database (www.ch.embnet.org)19 and SOUSI prediction system (http://sosui.proteome.bio.tuat.ac.jp),20 respectively.

Results Oviductal Epithelial Cell Isolation. Cells isolated from oviductal tissue included a population of oviductal epithelial cells, blood cells, and cell debris. To purify viable oviductal epithelial cells, primary isolated cells were treated with red blood cell lysis buffer, washed, and kept for 18 h in cell culture. During this time, oviductal epithelial cells started to attach to the bottom of the tissue flask and partially started to proliferate. By washing the tissue flask with PBS, all unattached oviductal epithelial cells including all blood cells and cell debris were removed. Using this approach, we were able to purify a population of viable oviductal epithelial cells suitable for biotinylation. Verification of OE Cells Epithelial Nature and Determination of Their Viability after Biotinylation. After 18 h of culture, isolated cells were washed with PBS to remove unattached cells and cell debris. The remaining attached cells were stained with anti-pan cytokeratin or were biotinylated and stained with a combination of Calcein AM and EthD-1 to assess their viability. The cells that remained attached in the culture dish contained >99% (99.98 ( 0.01) of cytokeratin positive (epithelial) cells (Figure 1) and were highly viable (>99%; 99.94 ( 0.01) after biotinylation. Detection of Proportion of Biotinylated Proteins before and after Biotinylated Protein Enrichment. The protein fractions containing 50 or 300 µg of proteins from before and after enrichment of biotinylated proteins were separated by 2D gel electrophoresis. Gels containing 50 µg of proteins were silver stained. Proteins from gels containing 300 µg of proteins were transferred to PVDF membranes, and the biotinylated proteins were visualized by hybridization with neutravidin-HRP. There were distinctive differences between protein patterns of gels obtained from samples before and after enrichment of biotinylated proteins as visualized in silver stained gels (Figure 2). A large number of proteins from sample fraction before enrichment could not be matched to the proteins of samples after enrichment. In addition, the staining intensity of proteins that had matched between the fractions was markedly different. Marked differences between the two fractions were also visible when biotinylated proteins were visualized on the PVDF

Oviductal Epithelial Cells Surface Proteome Profile

Figure 2. Detection of proteins separated with 2D gel electrophoresis. Equal amounts of proteins from before (A) and after (B) avidin purification were separated with 2D gel electrophoresis, and proteins were visualized by silver staining. Protein patterns of gels stained after avidin purification distinctively changed, resulting in enrichment or diminishing of proteins.

membranes. The enriched biotinylated protein sample contained a larger number and larger quantities of biotinylated proteins (Figure 3). The 2D pattern of the enriched biotinylated protein fraction visualized by silver staining was very similar to the 2D pattern of enriched biotinylated proteins transferred to PVDF membranes and visualized by neutravidin-HRP, implying that nearly all of the enriched proteins represent biotinylated proteins (see Figures 2 and 3). This was not the case for protein samples before enrichment. Verification of Enrichment of Cell Surface Proteome by Western Blot Analysis. Using antibodies and western blot analysis, molecules specific for mitochondria (anti-cytochrome C), endoplasmic reticulum (calnexin), and Golgi (P23) were not detected in the enriched biotinylated protein samples, but the abundance of a plasma membrane protein (N-Cadherin) was increased after enrichment (Figure 4). Identification of Enriched Biotinylated Proteins Using 2D Gel Electrophoresis and Mass Spectrometry. All 90 visible, individual protein spots were excised from Coomassie Blue stained 2D gels. Protein spots were digested and subjected to identification by nano-LC/MS/MS. Forty proteins were identified. Identification of Enriched Biotinylated Proteins by the Combination of 1D Gel Electrophoresis and MudPIT Approach. Two different amounts of enriched biotinylated proteins (20 and 200 µg) were used for MudPIT analysis in combination with SDS-PAGE. Twenty gel slices of equal size

research articles

Figure 3. Detection of biotinylated cell surface proteins. Equal amounts of protein samples from before (A) and after (B) avidin purification were separated by 2D gel electrophoresis, and proteins were transferred to PVDF membrane. Biotinylated proteins were visualized by hybridization with neutravidin-HRP. The majority of proteins purified with avidin (B) are biotinylated proteins.

Figure 4. Western blot analysis of avidin purification. The abundance of cytoplasmic proteins (cytochrome C, calnexin, and P23) diminished in proteins isolated by avidin chromatography, and at the same time the abundance of a plasma membrane protein (N-cadherin) increased. Biotinylated protein sample before enrichment (A) and after enrichment (B).

(∼1.5 mm) in the mass range between 0 and 250 kDa were cut from each of these protein concentrations and subjected to ingel digestion. The resulting peptides extracted from each gel slice were analyzed in triplicate by nano-LC/MS/MS for protein identification. This approach allowed us to identify a total number of 839 unique peptides and 276 unique proteins, of which some were only identified in gel slices obtained from 20 µg and some were only identified in gel slices obtained from 200 µg (Figure 5a and b). Increasing protein concentration from 20 to 200 µg resulted in an over 4-fold increase in peptide identification and 2-fold increase in the number of proteins Journal of Proteome Research • Vol. 5, No. 11, 2006 3033

research articles

Sostaric et al.

Figure 5. Venn diagrams presenting number of peptides (A) and proteins (B) identified by the MudPIT approach.

Figure 8. Graph presenting a virtual two-dimensional map of all unique proteins identified by the MudPIT (O) and 2D (0) approaches. The calculated PI of the proteins was plotted against their calculated molecular weight on a logarithmic scale. Box shows the typical limitations of proteins resolution by 2D gel electrophoresis.

Figure 6. Analysis of identified proteins by the MudPIT approach. Percentages of peptides and proteins identified in each nanoLC/MS/MS analysis from the total identified peptides and proteins. 9 depicts percentage of identified peptides in 20 µg protein samples, ^ depicts percentage of identified peptides in 200 µg protein samples, $ depicts percentage of identified proteins in 20 µg protein samples, and * depicts percentage of identified proteins in 200 µg protein samples.

Figure 7. Venn diagram presents number of proteins identified by the 2D and MudPIT approaches.

identified. Figure 6 shows the average percentage of peptides or proteins found after each round of nano-LC/MS/MS analysis. The majority of proteins identified in this study were uniquely recognized by the MudPIT approach. However, despite the low number of proteins identified by the 2D approach, nearly one-half of them were not detected by MudPIT (Figure 7). The molecular weight and PI range of proteins identified by MudPIT were more diverse than those identified by the 2D approach (Figure 8). In total, the number of unique proteins identified by 2D and MudPIT approach was 287 (Table 1, Supporting Information).

Discussion Current proteomic technologies are well suited for the preparation and investigation of cytoplasmic samples. However, the preparation of samples for membrane protein identification is very difficult. The main problems in isolation of pure plasma membrane fractions are the loss of membranespecific structure upon cell lysis, and heavy contamination of plasma membrane-rich fraction prepared by ultracentrifugation 3034

Journal of Proteome Research • Vol. 5, No. 11, 2006

Figure 9. All uniquely identified proteins categorized according to their transmembrane and signal sequence properties. Proteins presented as others had no transmembrane and signal sequence properties. White depicts proteins with transmembrane domain and signal sequence (9%), light gray depicts proteins with transmembrane domain (56%), dark gray depicts proteins with signal sequence (3%), and black depicts others (33%).

with other membrane components.21 Therefore, several methods have been developed to obtain relatively homogeneous preparations of plasma membranes, including coating of intact cells with silica derivatives.22 Focusing on the cell surface proteome and employing protein (peptide) tagging technologies is an effective strategy for enriching cell surface proteome. This will overcome the technical problems involved in the preparation of clean plasma membrane fractions. The high affinity and specificity of avidin-biotin interactions have been exploited for diverse applications such as immunology, histochemistry, in situ hybridization, and affinity chromatography.23-27 In the current investigation using this technology, we successfully enriched the porcine oviductal cell surface proteome. Several lines of evidence support the feasibility of our approach. There were distinct differences in the pattern of proteins in gels before and after avidin purification of biotinylated samples (Figure 2). This, in combination with abundant representation of biotinylated proteins in the purified samples (Figure 3), indicates enhancement of biotinylated proteins. As biotin protein tagging is targeted toward the cell surface proteome, one expects higher representation of membrane proteome markers in the enriched samples and diminishing of the cytoplasmic markers in the biotinylated enriched samples. This was exactly the case when western blot analysis was used to quantify the abundances of plasma membrane and cytoplasmic biomarkers before and after enrichment of biotin labeled samples (Figure 4). Furthermore, the bioinformatic analysis of the identified proteins in enriched samples demonstrated that the majority possessed a predicted transmembrane domain or signal peptide sequence (Figure 9). This

Oviductal Epithelial Cells Surface Proteome Profile

further verifies the feasibility of our strategy, leading to successful purification of the oviductal cell surface proteome. The number of proteins identified using the MudPIT approach was approximately 7 times the number of proteins identified by 2D gel electophoresis using the same samples (Figure 7). Considering the total amount of protein that was required to conduct MudPIT experiments and the range of proteins identified (Figure 8) as compared to 2D analysis, we conclude that the ability of MudPIT to identify proteins in enriched cell surface proteome is far superior to that of the 2D. This finding is not surprising considering the welldocumented shortcomings of 2D PAGE for membrane protein analysis.28 Despite the superior performance of MudPIT as compared to 2D, this technology still needs further optimization. It is clearly apparent that increasing the protein concentration in samples used increased the number of peptides identified and subsequently the number of proteins identified (Figure 5). However, the gain in the increase of identified peptides/ proteins is not necessarily of the same magnitude as the increase in protein concentration. In the present investigation, a 10-fold increase in the sample protein concentration (from 20 to 200 µg) led to an approximately 4-fold increase in the identified peptides and 2-fold increase in the number of proteins recognized. Interestingly, even after two successive sample injections into the mass spectrometer, an additional 15-20% of new peptides were found in a third injection irrespective of sample protein concentration (Figure 6). One expects further identification of peptides by increasing the number of sample injections into the mass spectrometer. It seems that the finite duty cycle and limited overall dynamic range of current mass spectrometry instruments do not allow every precursor peptide ion species to be sampled as peptides elute from the chromatography column. Therefore, MudPIT is biased toward preferential detection of higher abundance peptides. In contrast, lower abundance peptides can go undetected. To overcome this “under-sampling” problem, repeated injection of the same protein sample is suggested as a means for obtaining complete proteomic coverage.22,29,30 Our results are completely in agreement with previous studies, indicating the need for multiple sampling of proteins during MudPIT. However, on the basis of our findings, one can speculate that in the case of limited availability of samples a strategy to identify more peptides could be the dilution of the protein sample to increase its volume (therefore reducing the sample concentration) and increasing the number of injections into the mass spectrometer. This may lead to a higher number of proteins/peptides identified as compared to that using a relatively high concentration of proteins and a limited number of mass spectrometer rounds of analysis. Some of the proteins we found at the surface of oviductal cells in the current investigation have previously been reported to be present in the oviduct and to have known functions in relation to reproductive processes. For example, oviduct specific glycoprotein (OGP) has been demonstrated to be the major secretory glycoprotein in the oviduct and is expressed exclusively by the oviduct. This protein, under oestrogen control during oestrus and the follicular phase of the reproductive cycle, is conserved across all species examined. OGP binding to the extracellular matrix, the zona-pellucida, and membranes of gametes and embryos indicates a potential biological role during fertilization and development. Functional studies indicate that OGP affects sperm capacitation, motility, and viability.

research articles Moreover, evidence indicates that OGP has direct effects on egg penetration, fertilization, and development that may be mediated through interactions with the oocyte (for review, see ref 31). Recently, we have looked at alteration of oviductal secretory proteins in response to gametes7 and found specific changes in oviductal secretory proteins. Some of the proteins altered in response to gametes that were reported in our previous study were also detected in the present investigation, for example, triosephosphate isomerase 1, heat-shock 70 kDa protein 1, elongation factor 1-beta, non-selenium glutathione phospholipid hydroperoxide peroxidase, dimethylarginine dimethylaminohydrolase 2, and peroxiredoxin 2. It is possible that these proteins at the time of biotinylation were at the surface of the oviductal cells or they form a part of oviduct peripherally associated membrane proteins and therefore were detected in our present investigation. This can also be the case for those proteins that were identified in the current investigation at the surface of the oviductal epithelial cells to have a signal peptide (Table 1 and Figure 9). Another category of proteins that were highly represented in the oviductal surface proteome was various members of the family of heat-shock proteins. Several recent cell surface proteomic studies in other cell types have also seen a similar enrichment of chaperones and heat-shock proteins on the cell surface.15,16,32,33 The majority of heat-shock proteins do not encode transmembrane domains or, for that matter, signal sequences (which target the nascent polypeptide into the secretory pathway) within their genomic structure. It is not known how these proteins are targeted to the cell surface. However, it may be possible that they do not use the classical secretory pathway to target the cell surface proteome (endoplasmic reticulum to Golgi to plasma membrane). Other examples of unconventional transport of proteins to the cell surface are basic fibroblast growth factor, interleukin-1beta (IL-1beta), galectin-3, thioredoxin, and HIV-Tat.34-38 Shin et al.16 hypothesized that heat-shock proteins reach the cell surface by being a companion of misfolded proteins or peptide fragments out of the cytosol via a nonclassic pathway. Alternatively, they may be actively transported from their site of synthesis in the cytosol to help maintain structural integrity among the individual components of various receptor complexes. Not all proteins identified are classically linked with cell surface localization. Some are more easily identified as cytoplasmic or intracellular organelle proteins (Figure 9). The isolation procedure was intended to eliminate all but the cell surface proteins. One can conclude either the enrichment was only partially successful or, more likely, these proteins are linked to the cell surface proteins and are surface-associated proteins. Therefore, they are being enriched together with cell surface proteome. A complementary strategy for reduction of co-purification of cytosolic proteins with cell surface proteome can be the enrichment of cell surface biotinylated peptides rather than biotinylated proteins. One can digest the enriched cell surface proteome preparations and purify the biotinylated peptides using avidin columns.39 This approach should greatly reduce the enrichment of associated cytosolic proteins. The major potential drawback is that the protein representation of the peptides is vastly reduced and is only limited to biotinylated tagged peptides. This may reduce the peptide coverage in identified proteins by mass spectrometry. Many of the proteins identified in the present investigation have cell surface associated functions to do with cell adhesion, Journal of Proteome Research • Vol. 5, No. 11, 2006 3035

research articles receptor action, or cell chaperone effects. These functions may all be involved in the special activities of the oviduct epithelium. The mammalian oviduct is the venue of important events leading to the establishment of pregnancy. These events include final maturation and transport of the female and male gametes, fertilization, cleavage-stage embryonic development, and transport of the embryo to the uterus. In mammals, the physiological interaction between gametes, embryos, and oviductal epithelia involves intimate and specific contact between different cell types with oviductal epithelial surface.9,40-43 Understanding the proteomic profile at the surface of oviduct is essential for understanding the function of this tube during the reproductive process. For example, recently Boilard et al.44 demonstrated the binding of two molecules (HSP 60 and GRP78) from bovine oviductal epithelium to bull sperm. They have speculated that these proteins may be involved in the maintenance of sperm viability in the female reproductive tract in cattle or in the capacitation process where spermatozoa become competent at fertilizing the egg. Characterizing the oviduct surface proteome can lead us to understand the molecules that are involved in gamete/embryo interaction. It seems a number of molecules produced by the oviduct are involved in gamete/ embryo interactions with the oviduct. Interestingly, Boilard et al.44 detected more than six oviductal proteins interacting with bull spermatozoa, but were only able to recognize the identity of two of them as discussed above. During the oestrous cycle, the mammalian oviduct undergoes significant endocrine-induced morphological, biochemical, and physiological changes. These changes establish an essential microenvironment within the oviduct for interaction with gametes and embryos. In the current investigation, all oviducts used to obtain cell surface proteome samples were derived from sows at the oestrus stage of the reproductive cycle. We chose this particular phase of reproductive cycle because gametes are mainly present in the female reproductive tract at this phase of the cycle and fertilization occurs in oestrus. Future studies should be directed toward understanding the potential changes in the oviduct surface epithelium in different stages of the cycle. In conclusion, here we report the first global profile of oviductal surface proteome at oestrus in sows. This knowledge can help us to understand key events taking place in oviduct between gametes/embryo and reproductive tract, leading to conception and establishment of pregnancy.

Acknowledgment. We thank Mrs. C. Bruce and S. E. Elliott for technical support, and BBSRC, Sygen PLC, and Genus PLC for financial support. Supporting Information Available: Proteins identified by the 2D and MudPIT approaches (Table 1). This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Conner, S. D.; Schmid, S. L. Regulated portals of entry into the cell. Nature 2003, 422, 37-44. (2) Murphy, A. S.; Bandyopadhyay, A.; Holstein, S. E.; Peer, W. A. Endocytotic cycling of PM proteins. Annu. Rev. Plant Biol. 2005, 56, 221-51. (3) Duvernay, M. T.; Filipeanu, C. M.; Wu, G. The regulatory mechanisms of export trafficking of G protein-coupled receptors. Cell Signalling 2005, 17, 1457-65. (4) Eccles, S. A. Monoclonal antibodies targeting cancer: ‘magic bullets’ or just the trigger? Breast Cancer Res. 2001, 3, 86-90. (5) Santoni, V.; Molloy, M.; Rabilloud, T. Membrane proteins and proteomics: un amour impossible? Electrophoresis 2000, 21, 1054-70.

3036

Journal of Proteome Research • Vol. 5, No. 11, 2006

Sostaric et al. (6) Fazeli, A.; Affara, N. A.; Hubank, M.; Holt, W. V. Sperm-induced modification of the oviductal gene expression profile after natural insemination in mice. Biol. Reprod. 2004, 71, 60-5. (7) Georgiou, A. S.; Sostaric, E.; Wong, C. H.; Snijders, A. P.; Wright, P. C.; Moore, H. D.; Fazeli, A. Gametes alter the oviductal secretory proteome. Mol. Cell. Proteomics 2005, 4, 1785-96. (8) Dobrinski, I.; Smith, T. T.; Suarez, S. S.; Ball, B. A. Membrane contact with oviductal epithelium modulates the intracellular calcium concentration of equine spermatozoa in vitro. Biol. Reprod. 1997, 56, 861-9. (9) Fazeli, A.; Elliott, R. M.; Duncan, A. E.; Moore, A.; Watson, P. F.; Holt, W. V. In vitro maintenance of boar sperm viability by a soluble fraction obtained from oviductal apical plasma membrane preparations. Reproduction 2003, 125, 509-17. (10) Smith, T. T.; Nothnick, W. B. Role of direct contact between spermatozoa and oviductal epithelial cells in maintaining rabbit sperm viability. Biol. Reprod. 1997, 56, 83-9. (11) Suarez, S. S. Formation of a reservoir of sperm in the oviduct. Reprod. Domest. Anim. 2002, 37, 140-3. (12) Hunter, R. H.; Rodriguez-Martinez, H. Capacitation of mammalian spermatozoa in vivo, with a specific focus on events in the Fallopian tubes. Mol. Reprod. Dev. 2004, 67, 243-50. (13) Kidson, A.; Schoevers, E.; Langendijk, P.; Verheijden, J.; Colenbrander, B.; Bevers, M. The effect of oviductal epithelial cell coculture during in vitro maturation on sow oocyte morphology, fertilization and embryo development. Theriogenology 2003, 59, 1889-903. (14) Hill, J. A. Maternal-embryonic cross-talk. Ann. N.Y. Acad. Sci. 2001, 943, 17-25. (15) Jang, J. H.; Hanash, S. Profiling of the cell surface proteome. Proteomics 2003, 3, 1947-54. (16) Shin, B. K.; Wang, H.; Yim, A. M.; Le Naour, F.; Brichory, F.; Jang, J. H.; Zhao, R.; Puravs, E.; Tra, J.; Michael, C. W.; Misek, D. E.; Hanash, S. M. Global profiling of the cell surface proteome of cancer cells uncovers an abundance of proteins with chaperone function. J. Biol. Chem. 2003, 278, 7607-16. (17) Zhao, Y.; Zhang, W.; Kho, Y.; Zhao, Y. Proteomic analysis of integral plasma membrane proteins. Anal. Chem. 2004, 76, 181723. (18) Smith, P. K.; Krohn, R. I.; Hermanson, G. T.; Mallia, A. K.; Gartner, F. H.; Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Klenk, D. C. Measurement of protein using bicinchoninic acid. Anal. Biochem. 1985, 150, 76-85. (19) Hofmann, K.; Stoffel, W. TMbase - A database of membrane spanning proteins segments. Biol. Chem. Hoppe-Seyler 1993, 374, 166. (20) Gomi, M.; Sonoyama, M.; Mitaku, S. High performance system for signal peptide prediction: SOSUIsignal. Chem-Bio Inf. J. 2004, 4, 142-147. (21) Pasquali, C.; Fialka, I.; Huber, L. A. Subcellular fractionation, electromigration analysis and mapping of organelles. J. Chromatogr., B: Biomed. Sci. Appl. 1999, 722, 89-102. (22) 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, 985-92. (23) Bayer, E. A.; Wilchek, M. Application of avidin-biotin technology to affinity-based separations. J. Chromatogr. 1990, 510, 3-11. (24) Chapman-Smith, A.; Cronan, J. E., Jr. The enzymatic biotinylation of proteins: a post-translational modification of exceptional specificity. Trends Biochem. Sci. 1999, 24, 359-63. (25) Diamandis, E. P.; Christopoulos, T. K. The biotin-(strept)avidin system: principles and applications in biotechnology. Clin. Chem. 1991, 37, 625-36. (26) Wilbur, D. S.; Pathare, P. M.; Hamlin, D. K.; Stayton, P. S.; To, R.; Klumb, L. A.; Buhler, K. R.; Vessella, R. L. Development of new biotin/streptavidin reagents for pretargeting. Biomol. Eng. 1999, 16, 113-8. (27) Wilchek, M.; Bayer, E. A. Foreword and introduction to the book (strept)avidin-biotin system. Biomol. Eng. 1999, 16, 1-4. (28) Rabilloud, T. Membrane proteins ride shotgun. Nat. Biotechnol. 2003, 21, 508-10. (29) Kislinger, T.; Gramolini, A. O.; MacLennan, D. H.; Emili, A. Multidimensional protein identification technology (MudPIT): technical overview of a profiling method optimized for the comprehensive proteomic investigation of normal and diseased heart tissue. J. Am. Soc. Mass Spectrom. 2005, 16, 1207-20. (30) Liu, H.; Sadygov, R. G.; Yates, J. R., III. A model for random sampling and estimation of relative protein abundance in shotgun proteomics. Anal. Chem. 2004, 76, 4193-201.

research articles

Oviductal Epithelial Cells Surface Proteome Profile (31) Buhi, W. C. Characterization and biological roles of oviductspecific, oestrogen-dependent glycoprotein. Reproduction 2002, 123, 355-62. (32) Coonrod, S. A.; Calvert, M. E.; Reddi, P. P.; Kasper, E. N.; Digilio, L. C.; Herr, J. C. Oocyte proteomics: localisation of mouse zona pellucida protein 3 to the plasma membrane of ovulated mouse eggs. Reprod. Fertil. Dev. 2004, 16, 69-78. (33) Stein, K. K.; Go, J. C.; Lane, W. S.; Primakoff, P.; Myles, D. G. Proteomic analysis of sperm regions that mediate sperm-egg interactions. Proteomics 2006, 6, 3533-43. (34) Chang, H. C.; Samaniego, F.; Nair, B. C.; Buonaguro, L.; Ensoli, B. HIV-1 Tat protein exits from cells via a leaderless secretory pathway and binds to extracellular matrix-associated heparan sulfate proteoglycans through its basic region. AIDS 1997, 11, 1421-31. (35) Mehul, B.; Hughes, R. C. Plasma membrane targetting, vesicular budding and release of galectin 3 from the cytoplasm of mammalian cells during secretion. J. Cell Sci. 1997, 110, 1169-78. (36) Mignatti, P.; Morimoto, T.; Rifkin, D. B. Basic fibroblast growth factor, a protein devoid of secretory signal sequence, is released by cells via a pathway independent of the endoplasmic reticulumGolgi complex. J. Cell. Physiol. 1992, 151, 81-93. (37) Rubartelli, A.; Bajetto, A.; Allavena, G.; Wollman, E.; Sitia, R. Secretion of thioredoxin by normal and neoplastic cells through a leaderless secretory pathway. J. Biol. Chem. 1992, 267, 241614. (38) Rubartelli, A.; Sitia, R. Interleukin 1 beta and thioredoxin are secreted through a novel pathway of secretion. Biochem. Soc. Trans. 1991, 19, 255-9.

(39) Nunomura, K.; Nagano, K.; Itagaki, C.; Taoka, M.; Okamura, N.; Yamauchi, Y.; Sugano, S.; Takahashi, N.; Izumi, T.; Isobe, T. Cell surface labeling and mass spectrometry reveal diversity of cell surface markers and signaling molecules expressed in undifferentiated mouse embryonic stem cells. Mol. Cell. Proteomics 2005, 4, 1968-76. (40) Fazeli, A.; Duncan, A. E.; Watson, P. F.; Holt, W. V. Sperm-oviduct interaction: induction of capacitation and preferential binding of uncapacitated spermatozoa to oviductal epithelial cells in porcine species. Biol. Reprod. 1999, 60, 879-86. (41) Green, C. E.; Bredl, J.; Holt, W. V.; Watson, P. F.; Fazeli, A. Carbohydrate mediation of boar sperm binding to oviductal epithelial cells in vitro. Reproduction 2001, 122, 305-15. (42) Hunter, R. H.; Nichol, R. Transport of spermatozoa in the sheep oviduct: preovulatory sequestering of cells in the caudal isthmus. J. Exp. Zool. 1983, 228, 121-8. (43) Talbot, P.; Shur, B. D.; Myles, D. G. Cell adhesion and fertilization: steps in oocyte transport, sperm-zona pellucida interactions, and sperm-egg fusion. Biol. Reprod. 2003, 68, 1-9. (44) Boilard, M.; Reyes-Moreno, C.; Lachance, C.; Massicotte, L.; Bailey, J. L.; Sirard, M. A.; Leclerc, P. Localization of the chaperone proteins GRP78 and HSP60 on the luminal surface of bovine oviduct epithelial cells and their association with spermatozoa. Biol. Reprod. 2004, 71, 1879-89.

PR060366W

Journal of Proteome Research • Vol. 5, No. 11, 2006 3037