Identification of Novel Natural Substrates of Fibroblast Activation Protein-alpha by Differential Degradomics and Proteomics
2018; Elsevier BV; Volume: 18; Issue: 1 Linguagem: Inglês
10.1074/mcp.ra118.001046
ISSN1535-9484
AutoresHui Zhang, Elizabeth Hamson, Maria Magdalena Koczorowska, Stefan Tholen, Sumaiya Chowdhury, Charles G. Bailey, Angelina J. Lay, Stephen M. Twigg, Quintin Lee, Ben Roediger, Martin L. Biniossek, Matthew B. O’Rourke, Geoffrey W. McCaughan, Fiona M. Keane, Oliver Schilling, Mark D. Gorrell,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoFibroblast activation protein-alpha (FAP) is a cell-surface transmembrane-anchored dimeric protease. This unique, constitutively active serine protease has both dipeptidyl aminopeptidase and endopeptidase activities and can hydrolyze the post-proline bond. FAP expression is very low in adult organs but is upregulated by activated fibroblasts in sites of tissue remodeling, including fibrosis, atherosclerosis, arthritis and tumors. To identify the endogenous substrates of FAP, we immortalized primary mouse embryonic fibroblasts (MEFs) from FAP gene knockout embryos and then stably transduced them to express either enzymatically active or inactive FAP. The MEF secretomes were then analyzed using degradomic and proteomic techniques. Terminal amine isotopic labeling of substrates (TAILS)-based degradomics identified cleavage sites in collagens, many other extracellular matrix (ECM) and associated proteins, and lysyl oxidase-like-1, CXCL-5, CSF-1, and C1qT6, that were confirmed in vitro. In addition, differential metabolic labeling coupled with quantitative proteomic analysis also implicated FAP in ECM-cell interactions, as well as with coagulation, metabolism and wound healing associated proteins. Plasma from FAP-deficient mice exhibited slower than wild-type clotting times. This study provides a significant expansion of the substrate repertoire of FAP and provides insight into the physiological and potential pathological roles of this enigmatic protease. Fibroblast activation protein-alpha (FAP) is a cell-surface transmembrane-anchored dimeric protease. This unique, constitutively active serine protease has both dipeptidyl aminopeptidase and endopeptidase activities and can hydrolyze the post-proline bond. FAP expression is very low in adult organs but is upregulated by activated fibroblasts in sites of tissue remodeling, including fibrosis, atherosclerosis, arthritis and tumors. To identify the endogenous substrates of FAP, we immortalized primary mouse embryonic fibroblasts (MEFs) from FAP gene knockout embryos and then stably transduced them to express either enzymatically active or inactive FAP. The MEF secretomes were then analyzed using degradomic and proteomic techniques. Terminal amine isotopic labeling of substrates (TAILS)-based degradomics identified cleavage sites in collagens, many other extracellular matrix (ECM) and associated proteins, and lysyl oxidase-like-1, CXCL-5, CSF-1, and C1qT6, that were confirmed in vitro. In addition, differential metabolic labeling coupled with quantitative proteomic analysis also implicated FAP in ECM-cell interactions, as well as with coagulation, metabolism and wound healing associated proteins. Plasma from FAP-deficient mice exhibited slower than wild-type clotting times. This study provides a significant expansion of the substrate repertoire of FAP and provides insight into the physiological and potential pathological roles of this enigmatic protease. Fibroblast activation protein-alpha (FAP) 1The abbreviations used are:FAPfibroblast activation proteinAPTTactivated partial thromboplastin timesBNPB-type natriuretic peptideCAFcancer associated fibroblastC1qT6complement C1q tumor necrosis factor-related protein 6CCL-2/MCP-1monocyte chemoattractant protein-1CCMcell conditioned mediumCCRC-C motif chemokine receptorCN-Itype 1 collagenCSFcolony-stimulating factorCXCLC-X-C motif chemokine ligandDPPdipeptidyl peptidaseDTT1,4-dithiothreitolECMextracellular matrixFGFfibroblast growth factorGFPgreen fluorescent proteinGKOgene knockoutIAAiodoacetamideILinterleukinlimmalinear models for microarray dataLOX-Llysyl oxidase homologMALDImatrix assisted laser desorption ionisationMEFmouse embryonic fibroblastMMPmatrix metalloproteinaseNPYneuropeptide YPEPprolyl endopeptidasePMSFphenylmethylsulfonylfluoridePTprothrombin timesPYYpeptide YYSILACstable isotope labeling with amino acids in cell cultureTGFtransforming growth factorTAILSterminal amine isotopic labeling of substratesTIMPinhibitor of matrix metalloproteinase. is a unique post-proline peptidase, which possesses both endopeptidase activity and dipeptidyl aminopeptidase (DPP) activity, allowing it to cleave a peptide bond two or more residues from the N terminus of a peptide or protein (1Park J.E. Lenter M.C. Zimmermann R.N. Garin-Chesa P. Old L.J. Rettig W.J. Fibroblast activation protein: A dual-specificity serine protease expressed in reactive human tumor stromal fibroblasts.J. Biol. Chem. 1999; 274: 36505-36512Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar, 2Aertgeerts K. Levin I. Shi L. Snell G.P. Jennings A. Prasad G.S. Zhang Y. Kraus M.L. Salakian S. Sridhar V. Wijnands R. Tennant M.G. Structural and kinetic analysis of the substrate specificity of human fibroblast activation protein {alpha}.J. Biol. Chem. 2005; 280: 19441-19444Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). FAP is expressed at the cell surface and also shed. Both forms are enzymatically active and have similar substrate preferences (3Lee K.N. Jackson K.W. Christiansen V.J. Lee C.S. Chun J.G. McKee P.A. Antiplasmin-cleaving enzyme is a soluble form of fibroblast activation protein.Blood. 2006; 107: 1397-1404Crossref PubMed Scopus (126) Google Scholar). Although FAP has attracted much interest because of potential roles in fibrosis, cell migration and apoptosis and as a potential marker for many cancers (4Hamson E.J. Keane F.M. Tholen S. Schilling O. Gorrell M.D. Understanding Fibroblast Activation Protein (FAP): substrates, activities, expression and targeting for cancer therapy.Proteomics Clin. Appl. 2014; 8: 454-463Crossref PubMed Scopus (210) Google Scholar, 5Polak N. Gorrell M.D. Fibroblast activation protein.in: Choi S. FAP. Springer International Publishing, Cham2018: 1676-1681in: Choi S. Encyclopedia of Signaling Molecules. Springer International Publishing, Cham2018: 1676-1681Google Scholar, 6Juillerat-Jeanneret L. Tafelmeyer P. Golshayan D. Fibroblast activation protein-alpha in fibrogenic disorders and cancer: more than a prolyl-specific peptidase?.Expert Opin. Ther. Targets. 2017; 21: 977-991Crossref PubMed Scopus (35) Google Scholar), few of its natural substrates have been identified (1Park J.E. Lenter M.C. Zimmermann R.N. Garin-Chesa P. Old L.J. Rettig W.J. Fibroblast activation protein: A dual-specificity serine protease expressed in reactive human tumor stromal fibroblasts.J. Biol. Chem. 1999; 274: 36505-36512Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar, 4Hamson E.J. Keane F.M. Tholen S. Schilling O. Gorrell M.D. Understanding Fibroblast Activation Protein (FAP): substrates, activities, expression and targeting for cancer therapy.Proteomics Clin. Appl. 2014; 8: 454-463Crossref PubMed Scopus (210) Google Scholar, 7Keane F.M. Nadvi N.A. Yao T.-W. Gorrell M.D. Neuropeptide Y, B-type natriuretic peptide, substance P and peptide YY are novel substrates of fibroblast activation protein-α.FEBS J. 2011; 278: 1316-1332Crossref PubMed Scopus (90) Google Scholar, 8Dunshee D.R. Bainbridge T.W. Kljavin N.M. Zavala-Solorio J. Schroeder A.C. Chan R. Corpuz R. Wong M. Zhou W. Deshmukh G. Ly J. Sutherlin D.P. Ernst J.A. Sonoda J. Fibroblast activation protein cleaves and inactivates fibroblast growth factor 21.J. Biol. Chem. 2016; 291: 5986-5996Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 9Zhen E.Y. Jin Z. Ackermann B.L. Thomas M.K. Gutierrez J.A. Circulating FGF21 proteolytic processing mediated by fibroblast activation protein.Biochem. J. 2016; 473: 605-614Crossref PubMed Scopus (79) Google Scholar, 10Coppage A.L. Heard K.R. DiMare M.T. Liu Y. Wu W. Lai J.H. Bachovchin W.W. Human FGF-21 is a substrate of fibroblast activation protein.PLoS ONE. 2016; 11: e0151269Crossref PubMed Scopus (44) Google Scholar, 11Lee K.N. Jackson K.W. Christiansen V.J. Chung K.H. McKee P.A. A novel plasma proteinase potentiates alpha2-antiplasmin inhibition of fibrin digestion.Blood. 2004; 103: 3783-3788Crossref PubMed Scopus (109) Google Scholar). fibroblast activation protein activated partial thromboplastin times B-type natriuretic peptide cancer associated fibroblast complement C1q tumor necrosis factor-related protein 6 monocyte chemoattractant protein-1 cell conditioned medium C-C motif chemokine receptor type 1 collagen colony-stimulating factor C-X-C motif chemokine ligand dipeptidyl peptidase 1,4-dithiothreitol extracellular matrix fibroblast growth factor green fluorescent protein gene knockout iodoacetamide interleukin linear models for microarray data lysyl oxidase homolog matrix assisted laser desorption ionisation mouse embryonic fibroblast matrix metalloproteinase neuropeptide Y prolyl endopeptidase phenylmethylsulfonylfluoride prothrombin times peptide YY stable isotope labeling with amino acids in cell culture transforming growth factor terminal amine isotopic labeling of substrates inhibitor of matrix metalloproteinase. Only three of the identified endopeptidase substrates of FAP are physiological substrates, namely denatured type I collagen (1Park J.E. Lenter M.C. Zimmermann R.N. Garin-Chesa P. Old L.J. Rettig W.J. Fibroblast activation protein: A dual-specificity serine protease expressed in reactive human tumor stromal fibroblasts.J. Biol. Chem. 1999; 274: 36505-36512Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar, 12Christiansen V.J. Jackson K.W. Lee K.N. McKee P.A. Effect of fibroblast activation protein and alpha2-antiplasmin cleaving enzyme on collagen types I, III, and IV. Arch. Biochem.Biophys. 2007; 457: 177-186Google Scholar), α2-antiplasmin (3Lee K.N. Jackson K.W. Christiansen V.J. Lee C.S. Chun J.G. McKee P.A. Antiplasmin-cleaving enzyme is a soluble form of fibroblast activation protein.Blood. 2006; 107: 1397-1404Crossref PubMed Scopus (126) Google Scholar) and fibroblast growth factor-21 (FGF-21) (8Dunshee D.R. Bainbridge T.W. Kljavin N.M. Zavala-Solorio J. Schroeder A.C. Chan R. Corpuz R. Wong M. Zhou W. Deshmukh G. Ly J. Sutherlin D.P. Ernst J.A. Sonoda J. Fibroblast activation protein cleaves and inactivates fibroblast growth factor 21.J. Biol. Chem. 2016; 291: 5986-5996Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 9Zhen E.Y. Jin Z. Ackermann B.L. Thomas M.K. Gutierrez J.A. Circulating FGF21 proteolytic processing mediated by fibroblast activation protein.Biochem. J. 2016; 473: 605-614Crossref PubMed Scopus (79) Google Scholar, 10Coppage A.L. Heard K.R. DiMare M.T. Liu Y. Wu W. Lai J.H. Bachovchin W.W. Human FGF-21 is a substrate of fibroblast activation protein.PLoS ONE. 2016; 11: e0151269Crossref PubMed Scopus (44) Google Scholar). By screening known DPP4 substrates (7Keane F.M. Nadvi N.A. Yao T.-W. Gorrell M.D. Neuropeptide Y, B-type natriuretic peptide, substance P and peptide YY are novel substrates of fibroblast activation protein-α.FEBS J. 2011; 278: 1316-1332Crossref PubMed Scopus (90) Google Scholar), and then measuring cleavage in plasma (13Wong P.F. Gall M.G. Bachovchin W.W. McCaughan G.W. Keane F.M. Gorrell M.D. Neuropeptide Y is a physiological substrate of fibroblast activation protein: Enzyme kinetics in blood plasma and expression of Y2R and Y5R in human liver cirrhosis and hepatocellular carcinoma.Peptides. 2016; 75: 80-95Crossref PubMed Scopus (26) Google Scholar), we have previously identified four physiological DPP-type substrates of FAP, namely neuropeptide Y (NPY), substance P, peptide YY (PYY) and B-type natriuretic peptide (BNP). NPY was found to be the most efficiently cleaved substrate of both human and mouse FAP, whereas all four peptides were found to be efficiently cleaved by endogenous DPP4, indicating that the in vivo degradomes of FAP and DPP4 differ (13Wong P.F. Gall M.G. Bachovchin W.W. McCaughan G.W. Keane F.M. Gorrell M.D. Neuropeptide Y is a physiological substrate of fibroblast activation protein: Enzyme kinetics in blood plasma and expression of Y2R and Y5R in human liver cirrhosis and hepatocellular carcinoma.Peptides. 2016; 75: 80-95Crossref PubMed Scopus (26) Google Scholar). Identification of additional FAP substrates is essential to fully elucidate its role as a protease. FAP is implicated in fibrinolysis because human α2-antiplasmin has 4-fold increased activity following FAP mediated cleavage (14Lee K.N. Jackson K.W. Christiansen V.J. Dolence E.K. McKee P.A. Enhancement of fibrinolysis by inhibiting enzymatic cleavage of precursor alpha2-antiplasmin.J. Thromb Haemost. 2011; 9: 987-996Crossref PubMed Scopus (32) Google Scholar). Inhibition of FAP increases the levels of active FGF-21 in obese mice more than in lean mice, demonstrating a metabolic benefit of FAP inhibition (15Sánchez-Garrido M.A. Habegger K.M. Clemmensen C. Holleman C. Müller T.D. Perez-Tilve D. Li P. Agrawal A.S. Finan B. Drucker D.J. Tschöp M.H. DiMarchi R.D. Kharitonenkov A. Fibroblast activation protein (FAP) as a novel metabolic target.Mol. Metabolism. 2016; 5: 1015-1024Crossref PubMed Scopus (48) Google Scholar), which might be exploited for treating diabetes. Up-regulated expression of FAP is predominantly associated with disease states, including but not limited to tumorigenesis, fibrotic conditions and atherosclerosis (5Polak N. Gorrell M.D. Fibroblast activation protein.in: Choi S. FAP. Springer International Publishing, Cham2018: 1676-1681in: Choi S. Encyclopedia of Signaling Molecules. 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The overall aim of this study was to identify novel substrates of FAP using an unbiased, global, degradomic technique (terminal amine isotopic labeling of substrates, or TAILS), a more targeted data mining approach, and a proteome comparison (stable isotope labeling by amino acids in cell culture, or SILAC). Through our identification of FAP substrates and subsequent investigation of the biological consequence of cleavage of these proteins, knowledge and understanding of the physiological role of FAP was enhanced. A total of five replicate analyses were conducted for the differential proteomic investigation of the FAP enzyme-active (e+) and FAP enzyme-inactive (e−) mouse embryonic fibroblasts (MEFs) (see below for details of cell generation). A total of three replicate analyses were conducted for the differential N-terminomic investigation of the FAP e+ and FAP e− MEFs (see below for details of cell generation). Statistical analysis using linear models for microarray data (limma) (23Ritchie M.E. Phipson B. Wu D. Hu Y. Law C.W. Shi W. Smyth G.K. limma powers differential expression analyses for RNA-sequencing and microarray studies.Nucleic Acids Res. 2015; 43: e47Crossref PubMed Scopus (15626) Google Scholar) allows for the use of linear models to assess differential expression in the context of multifactor designed experiments. In addition, limma enables analyses of complex experiments involving comparisons between many peptides simultaneously in a small sample size, including multiple testing correction. Following the calculation of moderated-t-tests, proteins or cleavage sites with a p value < 0.05 and a quantitative alteration > 50% (either increase or decrease), were considered as being significantly affected. MEFs were isolated from FAP gene knockout (GKO) mouse embryos aged 13.5 days. In this FAP GKO mouse strain, FAP has been deleted in both alleles and exons 3 to 26 are absent (24Niedermeyer J. Kriz M. Hilberg F. Garin-Chesa P. Bamberger U. Lenter M.C. Park J. Viertel B. Puschner H. Mauz M. Rettig W.J. Schnapp A. Targeted disruption of mouse fibroblast activation protein.Mol. Cell. Biol. 2000; 20: 1089-1094Crossref PubMed Scopus (91) Google Scholar, 25Niedermeyer J. Enenkel B. Park J.E. Lenter M. Rettig W.J. Damm K. Schnapp A. Mouse fibroblast-activation protein: Conserved Fap gene organization and biochemical function as a serine protease.Eur. J. Biochem. 1998; 254: 650-654Crossref PubMed Scopus (58) Google Scholar). Primary MEFs were immortalized by transduction with the pFU-SV40-LTpuro lentiviral vector encoding the SV40 large T antigen then selected in puromycin (26Gall M.G. Chen Y. Ribeiro A.J.V.d. Zhang H. Bailey C.G. Spielman D. Yu D.M. Gorrell M.D. Targeted inactivation of Dipeptidyl Peptidase 9 enzyme activity causes mouse neonate lethality.PLoS ONE. 2013; 8: e0078378Crossref PubMed Scopus (36) Google Scholar). These MEFs were then divided into three cultures and transduced with a lentiviral vector pCCLteteGFP to express green fluorescent protein (GFP) as an empty vector control, or linked to one of two human FAP (hFAP) constructs using a picornaviral 2A peptide (27Guan F.H.X. Bailey C.G. Metierre C. O'Young P. Gao D. Khoo T.L. Holst J. Rasko J.E.J. The antiproliferative ELF2 isoform, ELF2B, induces apoptosis in vitro and perturbs early lymphocytic development in vivo.J. Hematol. Oncol. 2017; 10: 75Crossref PubMed Scopus (9) Google Scholar). PCR was used to amplify enzyme active (hFAP e+) and inactive (hFAP e−; serine724alanine) constructs from GFP-FAP (28Wang X.M. Yu D.M.T. McCaughan G.W. Gorrell M.D. Fibroblast activation protein increases apoptosis, cell adhesion and migration by the LX-2 human stellate cell line.Hepatology. 2005; 42: 935-945Crossref PubMed Scopus (136) Google Scholar) using the primers hFAP-5′BmgBI 5′-GTGGAAGAAAACCCCGGGCCCATGAAGACTTGGGTAAAAATCG-3′ and hFAP-3′BstBI 5′-AAAAGATTCGAAGTTTAAACTTAGTCTGACAAAGAGAAACAC-3′. PCR fragments (2324bp) were generated using Phusion polymerase (Finnzymes; Thermo Scientific, Waltham, MA) phosphorylated with T4 polynucleotide kinase (NEB, Ipswich, MA) and digested with BstBI. The pCCLteteGFP2Aluc2 lentiviral vector was cut with BmgBI/ClaI to clone in hFAP e+ and hFAP e− genes. Transduced MEFs were selected by GFP-based cell sorting using a BD Biosciences FACS Aria flow cytometer and expanded as pools. An endopeptidase activity assay was performed on the cells and secretomes of FAP e+ and FAP e− MEFs using the fluorogenic substrate Z-Gly-Pro-AMC (1 mm; Bachem, Bubendorf, Switzerland), in the presence or absence of a non-selective DPP inhibitor (Val-boro-Pro at 50 μm (kind gift from WW Bachovchin, Boston, MA) and a PEP-selective inhibitor (S17092 at 1 μm; Sigma-Aldrich, St Louis, MO). Cell conditioned medium (CCM) was generated from three biological replicates of FAP e+ and e− MEF cell lines. Cells were grown in 15 cm dishes to a confluency of ∼70% in 20 ml culture medium. After 16 h incubation in serum-free supplemented DMEM (Thermo Scientific) without phenol red, CCM was harvested in the presence of the following protease inhibitors: 0.01 mm trans-epoxysuccinyl-l-leucylamido (4-guanidino) butane (E64; Sigma-Aldrich), 5 mm ethylene-diaminetetraacetic acid (EDTA; Sigma-Aldrich) and 1 mm phenylmethylsulfonylfluoride (PMSF; Sigma-Aldrich). CCM was centrifuged at 1500 rpm and filtered using a 0.2 μm filter (Amicon; EMD Millipore, Billerica, MA) to remove dead cells and debris. HEPES buffer (Sigma-Aldrich) (pH 7.5) was added to a final concentration of 100 mm and the CCM mildly denatured and reduced with 5 mm dithiothreitol (DTT) (Sigma-Aldrich) at room temperature for 1–2 h. Free sulfide groups were alkylated by incubation with 20 mm iodoacetamide (IAA) (Sigma-Aldrich) for 2 h at room temperature in the dark, then 5 mm DTT was added for 15 min at room temperature to quench any remaining IAA. TAILS was performed as described previously (29Tholen S. Biniossek M.L. Gessler A.L. Müller S. Weisser J. Kizhakkedathu J.N. Reinheckel T. Schilling O. Contribution of cathepsin L to secretome composition and cleavage pattern of mouse embryonic fibroblasts.Biol. Chem. 2011; 392: 961-971Crossref PubMed Scopus (28) Google Scholar, 30Tholen S. Biniossek M.L. Gansz M. Gomez-Auli A. Bengsch F. Noel A. Kizhakkedathu J.N. Boerries M. Busch H. Reinheckel T. Schilling O. Deletion of cysteine cathepsins B or L yields differential impacts on murine skin proteome and degradome.Mol. Cell. Proteomics. 2013; 12: 611-625Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 31Knopf J.D. Tholen S. Koczorowska M.M. De Wever O. Biniossek M.L. Schilling O. The stromal cell-surface protease fibroblast activation protein-alpha localizes to lipid rafts and is recruited to invadopodia.Biochim. Biophys. Acta. 2015; 1853: 2515-2525Crossref PubMed Scopus (15) Google Scholar). Briefly, CCM was concentrated then precipitated with trichloroacetic acid. The protein pellet was re-solubilized in ice-cold 2 m guanidine hydrochloride using ultrasonication. After adjusting the pH to 7.5 using HEPES, the samples were reduced and alkylated again as described above. Protein was then differentially labeled with 40 mm heavy [d(2)13C] or light [d(0)12C] formaldehyde (Cambridge Isotope Laboratories, Andover, MA) in the presence of 40 mm sodium borohydride (for 18 h at 37 °C). After quenching excess reagents with 50 mm Tris (for 2 h at 37 °C), samples were mixed at equal amounts, acetone precipitated and resolubilized with 100 mm ice-cold NaOH. The pH was adjusted to 8 with HEPES-free acid and samples were digested overnight with mass spectrometry grade trypsin (Worthington, Lakewood, NY) at 37 °C. Labeled peptides were enriched by a negative selection step using a dendritic polyglycerol aldehyde polymer, as described (32Kleifeld O. Doucet A. auf dem Keller U. Prudova A. Schilling O. Kainthan R.K. Starr A.E. Foster L.J. Kizhakkedathu J.N. Overall C.M. Isotopic labeling of terminal amines in complex samples identifies protein N-termini and protease cleavage products.Nat. Biotechnol. 2010; 28: 281-288Crossref PubMed Scopus (405) Google Scholar). Blocked N-terminal peptides (unbound) were physically separated from the polymer-captured peptides via a 10 kDa molecular weight cut-off filter (Amicon; EMD Millipore). Flow through and filtrate were combined and desalted using a reverse phase C18 column (Sep-Pak, Waters, Milford, MA). The SILAC protocol was performed as described (33Kern U. Wischnewski V. Biniossek M.L. Schilling O. Reinheckel T. Lysosomal protein turnover contributes to the acquisition of TGFbeta-1 induced invasive properties of mammary cancer cells.Mol. Cancer. 2015; 14: 39Crossref PubMed Scopus (28) Google Scholar). Briefly, MEFs were grown in heavy or light labeled DMEM (minus l-Arg and l-Lys; Silantes, Munich, Germany) supplemented with 10% dialyzed FCS and glutamine containing either l-arginine (Arg0) and l-lysine (Lys0) "light" or 13C6 15N4 l-arginine (Arg10) and 13C6 15N2 l-lysine (Lys8) "heavy" for at least five cell population doublings. After 16 h of seeding cells in heavy or light medium, CCM was collected as described above. Differentially labeled CCM was mixed at a ratio of 1:1 and concentrated to ∼200 μl using Amicon® Ultra 50 ml 5 kDa centrifugal filters (EMD Millipore), 4000 g, 4 °C. Samples were run on 12–16% SDS-PAGE (Criterion Tris-HCL gel, Bio-Rad and NuPage® buffers, Thermo Scientific) and proteins visualized using a Colloidal Blue staining kit (Thermo Scientific). After destaining, gels were cut into 1 mm cubes and further destained with three rounds of 0.1 m ammonium bicarbonate, 50% acetonitrile and 50 mm ammonium bicarbonate then 100% acetonitrile, sonicating on ice. Gel pieces were digested with sequencing grade trypsin (Worthington) at pH 7.5, overnight at 37 °C. After digestion, peptides were eluted from the gel with 100% ethanol, samples were then concentrated using a Speedvac concentrator (model 5301, Eppendorf, Hamburg, Germany) and desalted using a reversed phase C18 column (GRACE-VYDAC, Deerfield, IL). TAILS samples were then prefractionated by high performance liquid chromatography (HPLC) using a strong cation exchange column (SCX; PolyLC, Columbia, MD). Peptides were eluted in a linear gradient with increasing concentration of 5 mm KH2PO 1 m KCl 25% acetonitrile pH 2.7; 10–14 fractions were collected and desalted using self-packed C18 STAGE tips (Empore; 3 m, St Paul, MN) (34Rappsilber J. Ishihama Y. Mann M. Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics.Anal. Chem. 2003; 75: 663-670Crossref PubMed Scopus (1804) Google Scholar). Differential proteomics samples were pre-fractionated by SDS-PAGE as described previously (31Knopf J.D. Tholen S. Koczorowska M.M. De Wever O. Biniossek M.L. Schilling O. The stromal cell-surface protease fibroblast activation protein-alpha localizes to lipid rafts and is recruited to invadopodia.Biochim. Biophys. Acta. 2015; 1853: 2515-2525Crossref PubMed Scopus (15) Google Scholar). For nanoflow-LC-MS/MS analysis, samples for quantitative proteomic comparison were analyzed on a Qstar Elite (AB Sciex, Framingham, MA) and TAILS samples on an Orbitrap XL (Thermo Scientific GmbH, Bremen, Germany) mass spectrometer. Both instruments were coupled to an Ultimate3000 micro pump (Thermo Scientific) with a flow rate of 300 nL/min each. Buffer A was 0.5% (v/v) acetic acid, and buffer B was 0.5% (v/v) acetic acid in 80% (v/v) acetonitrile (water and acetonitrile were at least HPLC gradient grade quality). A gradient of increasing organic proportion was used for peptide separation. Column-tips with 75 μm inner diameter and a length of 11 cm were self-packed with Reprosil-Pur 120 ODS-3 (Dr. Maisch, Ammerbuch, Germany). The MS operated in data dependent mode, with the SMART being used for the QSTAR Elite. For the Orbitrap XL, each MS scan was followed by a maximum of five MS/MS scans; and for the Qstar Elite three MS/MS scans. Raw QSTAR LC-MS/MS data in wiff format (quantitative proteomic comparison, SILAC) was converted to the mzXML (35Pedrioli P.G. Eng J.K. Hubley R. Vogelzang M. Deutsch E.W. Raught B. Pratt B. Nilsson E. Angeletti R.H. Apweiler R. Cheung K. Costello C.E. Hermjakob H. Huang S. Julian R.K. Kapp E. McComb M.E. Oliver S.G. Omenn G. Paton N.W. Simpson R. Smith R. Taylor C.F. Zhu W. Aebersold R. A common open representation of mass spectrometry data and its application to proteomics research.Nat. Biotechnol. 2004; 22: 1459-1466Crossref PubMed Scopus (652) Google Scholar) format, using mzWiff (version 4.3.1, http://sourceforge.net/projects/sashimi/files/mzWiff%20(Analyst%20converter), with c
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