Endogenous Transforming Growth Factor-β Receptor-mediated Smad Signaling Complexes Analyzed by Mass Spectrometry
2006; Elsevier BV; Volume: 5; Issue: 7 Linguagem: Inglês
10.1074/mcp.m600065-mcp200
ISSN1535-9484
AutoresQilie Luo, Edward Nieves, Julia Kzhyshkowska, Ruth Hogue Angeletti,
Tópico(s)Connective Tissue Growth Factor Research
ResumoASmad proteins are the central feature of the transforming growth factor-β (TGF-β) intracellular signaling cascade. They function by carrying signals from the cell surface to the nucleus through the formation of a series of signaling complexes. Changes in Smad proteins and their complexes upon treatment with TGF-β were studied in mink lung epithelial (Mv1Lu) cell cultures. A time course of incubation with TGF-β was carried out to determine the peak of appearance of phosphorylated Smad2. Immobilized monoclonal antibody against Smad2 was then used to isolate the naturally occurring complexes. Three strategies were used to identify changes in proteins partnering with Smad2: separation by one-dimensional SDS-PAGE followed by MALDI peptide mass fingerprinting, cleavable ICAT labeling of the protein mixtures analyzed by LC-MS/MS, and nano-LC followed by MALDI MS TOF/TOF. Smad2 forms complexes with many other polypeptides both in the presence and absence of TGF-β. Some of the classes of proteins identified include: transcription regulators, proteins of the cytoskeletal scaffold and other tethering proteins, motility proteins, proteins involved in transport between the cytoplasm and nucleus, and a group of membrane adaptor proteins. Although some of these have been reported in the literature, most have not been reported previously. This work expands the repertoire of proteins known to participate in the TGF-β signal transduction processes. ASmad proteins are the central feature of the transforming growth factor-β (TGF-β) intracellular signaling cascade. They function by carrying signals from the cell surface to the nucleus through the formation of a series of signaling complexes. Changes in Smad proteins and their complexes upon treatment with TGF-β were studied in mink lung epithelial (Mv1Lu) cell cultures. A time course of incubation with TGF-β was carried out to determine the peak of appearance of phosphorylated Smad2. Immobilized monoclonal antibody against Smad2 was then used to isolate the naturally occurring complexes. Three strategies were used to identify changes in proteins partnering with Smad2: separation by one-dimensional SDS-PAGE followed by MALDI peptide mass fingerprinting, cleavable ICAT labeling of the protein mixtures analyzed by LC-MS/MS, and nano-LC followed by MALDI MS TOF/TOF. Smad2 forms complexes with many other polypeptides both in the presence and absence of TGF-β. Some of the classes of proteins identified include: transcription regulators, proteins of the cytoskeletal scaffold and other tethering proteins, motility proteins, proteins involved in transport between the cytoplasm and nucleus, and a group of membrane adaptor proteins. Although some of these have been reported in the literature, most have not been reported previously. This work expands the repertoire of proteins known to participate in the TGF-β signal transduction processes. Transforming growth factor-β (TGF-β) 1The abbreviations used are: TGF-β, transforming growth factor-β; SCX, strong cation exchange; NCBI, National Center for Biotechnology Information; PMF, peptide mass fingerprinting; cICAT, cleavable ICAT; Mv1Lu, mink lung epithelial; R-Smad, receptor-regulated Smad; Co-Smad; common partner Smad; P-Smad2, phosphorylated Smad2; IP, immunoprecipitation; mAb, monoclonal antibody; E1B-AP5, E1B-55 kDa-associated protein 5; hnRNP, heterogeneous nu-clear ribonucleoprotein; SARA, Smad anchor for receptor activation; Dab-2, disabled-2; TRAP-1, TGF-β receptor protein-1; HSP, heat shock protein; SBE, Smad binding element; E2, ubiquitin carrier protein; DEAEp, DEAE box protein; FERM, four.1, ezrin, radixin, moesin protein. 1The abbreviations used are: TGF-β, transforming growth factor-β; SCX, strong cation exchange; NCBI, National Center for Biotechnology Information; PMF, peptide mass fingerprinting; cICAT, cleavable ICAT; Mv1Lu, mink lung epithelial; R-Smad, receptor-regulated Smad; Co-Smad; common partner Smad; P-Smad2, phosphorylated Smad2; IP, immunoprecipitation; mAb, monoclonal antibody; E1B-AP5, E1B-55 kDa-associated protein 5; hnRNP, heterogeneous nu-clear ribonucleoprotein; SARA, Smad anchor for receptor activation; Dab-2, disabled-2; TRAP-1, TGF-β receptor protein-1; HSP, heat shock protein; SBE, Smad binding element; E2, ubiquitin carrier protein; DEAEp, DEAE box protein; FERM, four.1, ezrin, radixin, moesin protein. regulates a diverse set of cellular processes, including cell proliferation, recognition, differentiation, apoptosis, and determination of developmental fate. Smad proteins are the central feature of the TGF-β intracellular signaling cascade (1Shi Y. Massagué J. Mechanism of TGF-β signaling from cell membrane to the nucleus.Cell. 2003; 113: 685-700Abstract Full Text Full Text PDF PubMed Scopus (4739) Google Scholar, 2Wakefield L.M. Roberts A.B. TGF-β signaling: positive and negative effects on tumorigenesis.Curr. Opin. Genet. Dev. 2002; 12: 22-29Crossref PubMed Scopus (727) Google Scholar, 3Derynck R. Akhurst R.J. Balmain A. TGF-β signaling in tumor suppression and cancer progression.Nat. Genet. 2001; 29: 117-129Crossref PubMed Scopus (1939) Google Scholar, 4Massague J. Chen Y.G. Controlling TGF-β signaling.Genes Dev. 2000; 14: 627-644Crossref PubMed Google Scholar, 5Itoh S. Itoh F. Goumans M.J. ten Dijke P. Signaling of transforming growth factor-β family members through Smad proteins.Eur. J. Biochem. 2000; 267: 6954-6967Crossref PubMed Scopus (454) Google Scholar, 6Wrana J.L. Attisano L. 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The TGF-β superfamily: new members, new receptors, and new genetic tests of function in different organisms.Genes Dev. 1994; 8: 133-146Crossref PubMed Scopus (1726) Google Scholar). They function by carrying signals from the cell surface to the nucleus through the formation of a series of signaling complexes with one or more Smad proteins.Smads are a class of proteins that function as intracellular signaling effectors for the TGF-β superfamily of secreted polypeptides. The present picture of TGF-β signal transduction process is the following. First, a ligand, TGF-β, binds to a type II receptor, which recruits and phosphorylates a type I receptor. Second, the type I receptor then associates with a specific receptor-regulated (R-) Smad protein, Smad2, which is phosphorylated by the type I receptor on a serine residue in the carboxyl-terminal domain. Third, the phosphorylated Smad2 (P-Smad2) heterogeneously dimerizes with a common partner (Co-) Smad, Smad4, and together they translocate into the nucleus. Once in the nucleus, the Smad protein may form a complex with other transcription factors and subsequently activate target genes (13ten Dijke P. Hill C.S. New insights into TGF-β-Smad signalling.Trends Biochem. Sci. 2004; 29: 265-273Abstract Full Text Full Text PDF PubMed Scopus (1039) Google Scholar, 14Xu L. Massagué J. Nucleocytoplasmic shuttling of signal transducers.Nat. Rev. Mol. Cell. Biol. 2004; 5: 1-11Crossref Scopus (213) Google Scholar, 15Derynck R. Feng X.H. TGF-β receptor signaling.Biochim. Biophys. Acta. 1997; 1333: F105-F150Crossref PubMed Scopus (507) Google Scholar, 16Baker J.C. Harland R.M. From receptor to nucleus: the Smad pathway.Curr. Opin. Genet. Dev. 1997; 7: 467-473Crossref PubMed Scopus (96) Google Scholar).There is little knowledge obtained from actual physiological conditions to demonstrate the signal transduction processes and proteins involved in regulating gene expression in this system (17Massague J. How cells read TGF-β signals.Nat. Rev. Mol. Cell. Biol. 2000; 1: 169-178Crossref PubMed Scopus (1634) Google Scholar). The majority of knowledge on Smads was achieved using non-physiological conditions, such as cloning interesting genes to amplify the expression signals. Conceptually two types of immunoprecipitation (IP) approaches can be applied. One approach is to perform IP using lysates from cells coexpressing candidate interacting proteins that have been introduced by transient cotransfection of expression constructs (18Roberts E.C. Deed R.W. Inoue T. Norton J.D. Sharrocks A.D. Id helix-loop-helix proteins antagonize pax transcription factor activity by inhibiting DNA binding.Mol. Cell. Biol. 2001; 21: 524-533Crossref PubMed Scopus (119) Google Scholar). This method has the advantage that convenient epitope tags can be engineered into the expression constructs with the high level of protein expression obtained in transfected cells facilitating detection of protein interactions. Although this cotransfection approach is often successful, the data may not be physiologically relevant because these Smad proteins are present at far higher levels in cloned cells than would occur under normal physiological conditions. Alternatively antibodies against the native protein partners can be used to immunoprecipitate endogenous proteins from cells. This latter approach would provide information on protein interactions under physiological conditions.MS combined with several approaches for the separation of complex mixtures that precedes their mass spectrometric analysis has become the method of choice for the identification of proteins (19Aebersold R. Mann M. Mass spectrometry-based proteomics.Nature. 2003; 422: 198-207Crossref PubMed Scopus (5540) Google Scholar). Isotopic labeling combined with MS has also been used extensively to produce accurate quantitation of biological molecules (19Aebersold R. Mann M. Mass spectrometry-based proteomics.Nature. 2003; 422: 198-207Crossref PubMed Scopus (5540) Google Scholar). The development of isotope-coded affinity tag reagents allows for quantitation through isotopic labeling and simultaneously achieves a reduction in sample complexity by measuring only the Cys-containing peptides (20Gygi S.P. Rist B. Gerber S.A. Turecek F. Gelb M.H. Aebersold R. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.Nat. Biotechnol. 1999; 17: 994-999Crossref PubMed Scopus (4321) Google Scholar). The CID of peptides of interest by MS/MS would give rise to a sequence-specific fragmentation pattern from which the identity of the parent protein can be derived using either database search algorithms or de novo CID spectral interpretation (21Hansen K.C. Schmitt-Ulms G. Chalkley R.J. Hirsch J. Baldwin M.A. Burlingame A.L. Mass spectrometric analysis of protein mixtures at low levels using cleavable 13C-isotope-coded affinity tag and multidimensional chromatography.Mol. Cell. Proteomics. 2003; 2: 299-314Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar).We report here the results of direct proteomic analysis of Smad2 and its interacting partners from mink lung epithelial (Mv1Lu) cells. We identified not only Smad2 itself but also the interacting partners in both control and TGF-β-induced samples. Many of these proteins have not been identified previously as part of the Smad signal transduction pathway.EXPERIMENTAL PROCEDURESMaterials—Unless stated otherwise, all chemical reagents were of the highest purity available and purchased either from Sigma or Fisher Scientific. Protein G-Sepharose (fast flow) and the secondary antibodies used for Western blotting were from Amersham Biosciences. Monoclonal antibody (mAb) against Smad2 was ordered from BD Transduction Laboratories (Lexington, KY), mAb against Smad4 was from Santa Cruz Biotechnology (Santa Cruz, CA), polyclonal antibody anti-P-Smad2 was from Upstate Biotechnology Inc. (Waltham, MA), and TGF-β1 was from Calbiochem. Cleavable ICAT (cICAT) reagents and kits were obtained from Applied Biosystems (Framingham, MA). Siliconized 0.65-ml tubes from PGC Scientifics (Frederick, MD) were washed with methanol and water prior to use. The polysulfoethyl A strong cation exchanger was obtained from Poly LC through Western Analytical Products (Murietta, CA). BCA protein assay reagent kit and ImmunoPure® immobilized monomeric avidin gel were from Pierce.Cell Culture—The Mv1Lu cell line (CCL 64) was purchased from the American Type Culture Collection and was grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum in a water-saturated, 5% CO2 atmosphere at 37 °C in 75-cm2 polystyrene cell culture flask. Cells grown to ∼80% confluence were harvested for lysate preparation.TGF-β Stimulation—Mv1Lu cells starved for 12 h were incubated with or without platelet-derived TGF-β1 (final concentration, 100 pm) for various periods of time (0–180 min) at 37 °C. After the incubation, the cells were quickly washed twice with ice-cold PBS. The cells were lysed with 1 ml of lysing buffer containing 50 mm Tris (pH 8.0), 150 mm NaCl, 1% Nonidet P-40, 2 mm EDTA, 100 μm PMSF, 1 mm aprotinin, and 50 mm calyculin A. Calyculin A as an inhibitor of phosphatases was used only for the cells induced with TGF-β. However, both control and induced samples containing calyculin A had been screened, and there was no difference as compared with control samples without calyculin A. After centrifugation at 1.4 × 104 rpm in a 5415 C centrifuge (Brinkmann Instruments) for 15 min at 4 °C, the supernatants were collected for immunoprecipitation after clearing with protein G-Sepharose.Immunoprecipitation of Smad2 Complexes—Typically the lysate from ∼1.5 × 108 cells in 12 75-cm2 cell culture flasks was extracted using immunobeads prepared from mAb covalently cross-linked to protein G-Sepharose beads with dimethyl pimelimidate as described elsewhere (22Schneider C. Newman R.A. Sutherland D.R. Asser U. Graeves M.F. A one-step purification of membrane proteins using a high efficiency immunomatrix.J. Biol. Chem. 1982; 257: 10766-10769Abstract Full Text PDF PubMed Google Scholar). The mAb concentration in the bead is 1.2 μg (antibody)/μl (drained bead volume). For IP, a 30-μl slurry was added to 1 ml of Mv1Lu cell lysate and incubated for 2 h at 4 °C with constant gentle rocking. After sedimentation by centrifugation, the beads were washed as follows: 1 ml of ice-cold lysing buffer, 1 ml of ice-cold PBS containing 0.5 m NaCl, 1 ml of PBS containing 0.1% Tween 20, and 1 ml of PBS (three times). Bound proteins were released from the beads with 30 μl of nonreducing SDS sample buffer and heated for 5 min in an 85 °C water bath. The protein supernatant after centrifugation was separated by SDS-PAGE.In immunoprecipitation, co-isolation of nonspecifically associated proteins sometimes occurs that will possibly result in false positive identification. Therefore, it is important to remove nonspecific interacting proteins during IP. To achieve this, the following steps were carried out. First, the cell lysates were precleared using adsorbents without coupled antibodies. Second, monoclonal antibodies were used for IP. Third, efficient washing steps were designed with appropriate salt concentration and detergent solutions to remove any nonspecific interacting associations by charge or hydrophobic properties.Western Blotting—27 μl of lysate and 7 μl of a nonreducing sample buffer that was concentrated 5-fold were loaded per lane and separated by SDS-PAGE. Proteins were then transferred from the gels to PVDF membrane (Immobilon™-P, Millipore Corp., Bedford, MA) at 4 °C for 90 min using 75 V in 5% (v/v) methanol, 25 mm Tris (pH 8.4) containing 14.4% (w/v) glycine. Dilution of primary antibodies was between 1:100 and 1:1000 depending on the affinity of the antibodies. Protein signals were detected using peroxidase-linked secondary IgGs and enhanced chemiluminescence (Amersham Biosciences).SDS-PAGE and In-gel Tryptic Digestion—Proteins were released from the beads with a 5-fold concentrated nonreducing sample buffer, and the total 30-μl protein supernatant was electrophoresed in a 7.5–17.0% gradient, 1-mm-thick gel using a constant current of 400 mA for 1 h. The resolved proteins were visualized by silver staining (23Shevchencko A. Wilm M. Vorm O. Mann M. Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels.Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7771) Google Scholar). The bands corresponding to Smads and the interacting partners were excised. The gel bands were destained with a 1:1 (v/v) solution mixture of 30 mm potassium ferricyanide and 100 mm sodium thiosulfate (24Gharahdaghi F. Weinberg C.R. Meaggher D.A. Imai B.S. Mische S.M. Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: a method for the removal of silver ions to enhance sensitivity.Electrophoresis. 1999; 20: 601-605Crossref PubMed Scopus (840) Google Scholar). The proteins in the gels were digested with trypsin using a protocol modified from Hellman et al. (25Hellman U. Wernsteidt C. Gonez J. Heldin C.H. Improvement of an "in-gel" digestion procedure for the micropreparation of internal protein fragments for amino acid sequencing.Anal. Biochem. 1995; 224: 451-455Crossref PubMed Scopus (685) Google Scholar). Briefly gel pieces were completely dried down in a vacuum centrifuge, rehydrated with a trypsin solution, and allowed to incubate on ice for 45 min. After 45 min, the trypsin supernatant was removed and replaced with ∼20 μl of digestion buffer without trypsin so that the gel pieces were covered. The gel pieces were kept wet at 37 °C overnight for digestion with mixing.MALDI-TOF Mass Spectrometric Analyses—The method developed by Hellman et al. (25Hellman U. Wernsteidt C. Gonez J. Heldin C.H. Improvement of an "in-gel" digestion procedure for the micropreparation of internal protein fragments for amino acid sequencing.Anal. Biochem. 1995; 224: 451-455Crossref PubMed Scopus (685) Google Scholar) was used to extract the tryptic peptides. Aliquots of 0.5 μl were eluted from a C18 ZipTip with 10 mg/ml α-cyano-4-hydroxycinnamic acid in 50% acetonitrile/water containing 0.1% trifluoroacetic acid, applied directly onto a target plate, and allowed to air dry. The tryptic peptide masses were obtained using a Voyager-DE™ STR MALDI-TOF mass spectrometer (Applied Biosystems, Foster City, CA) at a resolution of 5000 (full width at half-maximum) and used for peptide mass fingerprinting (PMF). The database used was NCBInr (June 1, 2005). Known trypsin autocleavage peptide masses (842.51, 1045.56, and 2211.1 Da) were used for a three-point internal calibration for each spectrum. In the searches, methionine residues were assumed to be modified to methionine sulfoxide, and cysteine residues were assumed to be reduced and alkylated by iodoacetamide to carboxyamidomethyl cysteine wherever necessary. The database-fitting program Profound (26Zhang W. Chait B.T. Profound: an expert system for protein identification using mass spectrometric peptide mapping information.Anal. Chem. 2000; 72: 2482-2489Crossref PubMed Scopus (552) Google Scholar) was used to interpret MS spectra of protein digests. A protein was considered to be identified with p value of 0.05 and a 95% confidence level when the spectrum of its measured peptide mass met these previously established criteria for positive identification of proteins using MALDI-TOF mass spectrometry and automated database analysis (27Jensen O.N. Podtelejnikov A.V. Mann M. Identification of the components of simple protein mixtures by high-accuracy peptide mass mapping and database searching.Anal. Chem. 1997; 69: 4741-4750Crossref PubMed Scopus (244) Google Scholar, 28Clauser K.R. Baker P. Burlingame A.L. Role of accurate mass measurement (±10 ppm) in protein identification strategies employing MS or MS/MS and database searching.Anal. Chem. 1999; 71: 2871-2882Crossref PubMed Scopus (975) Google Scholar, 29Green M.K. Johnston M.V. Larsen B.S. Mass accuracy and sequence requirements for protein database searching.Anal. Biochem. 1999; 275: 39-46Crossref PubMed Scopus (24) Google Scholar, 30Egelhofer V. Bussow K. Luebbert C. Lehrach H. Nordhoff E. Improvements in protein identification by MALDI-TOF-MS mapping.Anal. Chem. 2000; 72: 2741-2750Crossref PubMed Scopus (47) Google Scholar). First, a minimum of five measured peptide masses must match tryptic peptide masses calculated for an individual protein in the database with a mass tolerance of 0.1 Da for monoisotopic mass and 1 Da for average mass. Second, the peptides identified by these matches must provide at least 15% sequence coverage of the identified proteins. PMF by MALDI MS is gradually reaching a 95% confidence level compared with sequencing techniques due to improved mass accuracy and sample preparation methods (31Jensen O.N. Larsen M.R. Roefstorff P. Mass spectrometric identification and microcharacterization of proteins from electrophoretic gels: strategies and applications.Proteins. 1998; : 74-89Crossref PubMed Google Scholar). As an example with Profound, proteins are ranked by the probability that "the candidate protein is the single protein" (26Zhang W. Chait B.T. Profound: an expert system for protein identification using mass spectrometric peptide mapping information.Anal. Chem. 2000; 72: 2482-2489Crossref PubMed Scopus (552) Google Scholar) (see Fig. 3). Profound uses the expectation values where the smaller the expectation value, the more likely it is a true match rather than a random match. An expectation value of 1.0 × 10−4 would indicate a similar match once in every 10,000 similarly sized databases and thus confirm the protein that matches the MS data. Profound also uses the Z score as an indicator of the quality of the search results. For example, a Z score of 1.65 indicates a 95th percentile result or a 5% random result having a higher Z score. The expectation value of Profound makes it easier to discriminate between scored sequences of top ranked proteins and other predicted results (26Zhang W. Chait B.T. Profound: an expert system for protein identification using mass spectrometric peptide mapping information.Anal. Chem. 2000; 72: 2482-2489Crossref PubMed Scopus (552) Google Scholar). For those proteins with a probability of less than 1 in PMF, repeating experiments under different experimental conditions (e.g. using a different enzyme) can significantly increase the confidence of protein identification.cICAT Labeling—Protein samples were assayed with the BCA protein assay reagent kit (Pierce). Protein samples of 15–20 μg were labeled with cICAT reagents using a modified protocol. Briefly protein samples were denatured in 8 m urea, 50 mm Tris, 5 mm EDTA, 0.05% SDS, pH 8.4. Reduction with 5 mm tributylphosphine was carried out for 30 min at 37 °C. cICAT reagents in acetonitrile were mixed together with reduced protein samples. Labeling was allowed to proceed for 2 h at 37 °C. Samples labeled with 12C and 13C cICAT reagents were combined and diluted to below 1.5 m urea. Tryptic digestion was initiated with the addition of 1% (w/v) of side chain-modified, tosylphenylalanyl chloromethyl ketone-treated porcine trypsin and allowed to proceed at 37 °C for 12 h.Cation Exchange Chromatography—Cation exchange chromatography was used to fractionate the peptide mixture. Tryptic digest samples were adjusted to below pH 3.0 with formic acid. The SCX separation was carried out using an off-line spin column (Bio-Rad). The cation exchanger (30 mg) was equilibrated with buffer A consisting of 5 mm KH2PO4, 25% ACN, pH 3.0. Samples were loaded onto columns and gently mixed for 15 min. The columns were washed with 500 μl of buffer A followed by a step gradient of different ratios of buffers A and B (500 mm KCl plus buffer A). Fractions (2 × 60 μl) were collected in 0.65-ml siliconized tubes.Avidin Affinity Chromatography—The SCX fractions were neutralized with NH4OH, and the pH was adjusted up to 8.0. The above mentioned spin column was also utilized for the avidin affinity chromatography. 120 μl of ImmunoPure immobilized monomeric avidin gel (Pierce) was suspended in a spin column. The column was blocked with 2 mm d-biotin, 50 mm NH4HCO3, pH 8.0. The column was then primed using 0.5 ml of 0.4% TFA in 30% ACN followed by 1 ml of 100 mm NH4HCO3, pH 8.4. Samples were loaded, and the flow-through was collected for LC-MS/MS analysis. The column was washed with 1 ml of 100 mm NH4HCO3, pH 8.0, followed by the same solution containing 10% methanol (v/v) and then by 1 ml of HPLC grade water. Labeled peptides were eluted with 3 × 60 μl of 0.4% TFA in 30% ACN.LC-MS Analysis—The labeled tryptic peptides were cleaved according to the standard protocol supplied by Applied Biosystems. The cleaved peptides were subjected to LC-MS/MS analysis on a QSTAR Pulsar i mass spectrometer (Applied Biosystems, Foster City, CA). Chromatographic separation of peptides was performed on a capillary and nano-HPLC system (LC Packings, San Francisco, CA). The LC eluent from a 75-μm-inner diameter× 15-cm PepMap C18 column (Dionex, Marlton, NJ) was directed to a microion spray source. Throughout the LC gradient, MS and MS/MS data were recorded continuously using a 6-s cycle time. With each cycle, MS data were accumulated for 1 s followed by two CID acquisitions of 2.5 s each on ions selected by preset selection parameters of the information-dependant acquisition method. In general, the ions selected for CID were the two most abundant obtained from the survey MS spectrum except that singly charged ions were excluded and dynamic exclusion was used to prevent repetitive selection of the same ions within a preset time. Rolling collision energies were used to adjust automatically for the charged state and the mass/charge value of the precursor ion. Searches were performed using MASCOT of the NCBInr database. In all searches, methionine sulfoxide was selected as a variable modification, and cysteine residues was selected as a fixed modification (ICAT heavy and light). The peptide precursor mass tolerance was ±100 ppm, and the MS/MS product ion mass tolerance was ±0.1 Da.Protein Quantitation—Protein expression ratios were calculated from the peak areas corresponding to ICAT-labeled peptides in the MS spectra. Ratios for each quantitation result were obtained for complete pairs based on the accuracy of the measured mass difference and the similarity of light (12C) and heavy isotopic (13C) profiles.Nano-LC-MALDI-TOF/TOF MS Analysis—Nanoflow HPLC using the Ultimate 3000 (Dionex, Sunnyvale, CA) at a flow rate of 250 nl/min was used on each SCX fraction. Separation of peptides was obtained using a gradient of 5–55% B (80% ACN + 20% H2O + 0.04% TFA) in 30 min on a 75-μm-inner diameter × 25-cm PepMap C18 column (Dionex, Sunnyvale, CA). The HPLC eluent was spotted directly onto a MALDI plate using Probot (Dionex, Sunnyvale, CA) with a sheath flow of 504 nl/min of matrix solution (10 mg/ml α-cyano-4-hydroxycinnamic acid with 50% methanol, 0.4% TFA) spotting one fraction every 68.7 s. Six external calibration spots, containing a 10-fold diluted 4700 Cal Mix (Applied Biosystems, Foster City, CA), were manually spotted.MALDI MS TOF/TOF data were acquired in an automated mode using the 4700 Proteomics Analyzer (Applied Biosystems, Foster City, CA). Initially a MALDI MS spectrum was acquired from each spot (1000 shots/spectrum), and peaks with a signal-to-noise ratio greater than 15 in each spectrum were automatically selected for MS/MS analysis (7500 shots/spectrum). A collision energy of 1 keV was used with air as the collision gas. Peak lists from all MS/MS spectra were submitted for database searching using an in-house copy of MASCOT, Version 1.8 (Matrix Science Inc., Boston, MA). The following criteria were used for all database searches: a minimum signal-to-noise ratio threshold of 5–10; masses of 0–60 Da and masses within 20 Da of the precursor ion were excluded; and a maximum of 60 peaks per spectrum were included as product ions. The mass tolerance was ±75 ppm for MS data, ±200 ppm for MS/MS precursor ions, and ±250 ppm for MS/MS product ions. All samples were searched against the NCBInr (May 1, 2005).RESULTSTime Course of Smad2 Phosphorylation—The Mv1Lu cell line has been used to study the biology of TGF-β. In these cells, TGF-β arrests cell proliferation and produces an apoptotic response (32Chalaux E. Lopez-Rovira T. Rosa J.L. Pons G. Boxer L.M. Bartrons R. Ventura F. A zinc-finger transcription factor induced by TGF-β promotes apoptotic cell death in epithelial Mv1Lu cells.FEBS Lett. 1999; 457: 478-482Crossref PubMed Scopus (90) Google Scholar). However, this response is context-dependent. To determine the best time window to analyze complexes involving phosphorylated Smad2 in the TGF-β signal transduction of the pathway, a time course of stimulation with TGF-β was carried out. As seen in Fig. 1, antibodies specific for phosphorylated Smad2 detected only one band in immunoblots. By 5 min, phosphorylated Smad2 was quickly detected and was only faintly visible after 3 h. Based on this result, 20 min of stimulation time was chosen for subsequent experiments.Immunoprecipitation with Anti-Smad Antibodies—To prevent contamination of isolated protein complexes with immunoglobulins, all antibodies were immobilized on protein G-Sepharose using dimethyl pimelimidate. Immobilization and precycling just prior to use were found to be important for minimizing nonspecific
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