Quantitative Non-canonical Amino Acid Tagging (QuaNCAT) Proteomics Identifies Distinct Patterns of Protein Synthesis Rapidly Induced by Hypertrophic Agents in Cardiomyocytes, Revealing New Aspects of Metabolic Remodeling
2016; Elsevier BV; Volume: 15; Issue: 10 Linguagem: Inglês
10.1074/mcp.m115.054312
ISSN1535-9484
AutoresRui Liu, Justin W. Kenney, Antigoni Manousopoulou, Harvey E. Johnston, Makoto Kamei, Christopher H. Woelk, Jianling Xie, Michael Schwarzer, Spiros D. Garbis, Christopher G. Proud,
Tópico(s)Viral Infectious Diseases and Gene Expression in Insects
ResumoCardiomyocytes undergo growth and remodeling in response to specific pathological or physiological conditions. In the former, myocardial growth is a risk factor for cardiac failure and faster protein synthesis is a major factor driving cardiomyocyte growth. Our goal was to quantify the rapid effects of different pro-hypertrophic stimuli on the synthesis of specific proteins in ARVC and to determine whether such effects are caused by alterations on mRNA abundance or the translation of specific mRNAs. Cardiomyocytes have very low rates of protein synthesis, posing a challenging problem in terms of studying changes in the synthesis of specific proteins, which also applies to other nondividing primary cells. To study the rates of accumulation of specific proteins in these cells, we developed an optimized version of the Quantitative Noncanonical Amino acid Tagging LC/MS proteomic method to label and selectively enrich newly synthesized proteins in these primary cells while eliminating the suppressive effects of pre-existing and highly abundant nonisotope-tagged polypeptides. Our data revealed that a classical pathologic (phenylephrine; PE) and the recently identified insulin stimulus that also contributes to the development of pathological cardiac hypertrophy (insulin), both increased the synthesis of proteins involved in, e.g. glycolysis, the Krebs cycle and beta-oxidation, and sarcomeric components. However, insulin increased synthesis of many metabolic enzymes to a greater extent than PE. Using a novel validation method, we confirmed that synthesis of selected candidates is indeed up-regulated by PE and insulin. Synthesis of all proteins studied was up-regulated by signaling through mammalian target of rapamycin complex 1 without changes in their mRNA levels, showing the key importance of translational control in the rapid effects of hypertrophic stimuli. Expression of PKM2 was up-regulated in rat hearts following TAC. This isoform possesses specific regulatory properties, so this finding indicates it may be involved in metabolic remodeling and also serve as a novel candidate biomarker. Levels of translation factor eEF1 also increased during TAC, likely contributing to faster cell mass accumulation. Interestingly those two candidates were not up-regulated in pregnancy or exercise induced CH, indicating PKM2 and eEF1 were pathological CH specific markers. We anticipate that the methodologies described here will be valuable for other researchers studying protein synthesis in primary cells. Cardiomyocytes undergo growth and remodeling in response to specific pathological or physiological conditions. In the former, myocardial growth is a risk factor for cardiac failure and faster protein synthesis is a major factor driving cardiomyocyte growth. Our goal was to quantify the rapid effects of different pro-hypertrophic stimuli on the synthesis of specific proteins in ARVC and to determine whether such effects are caused by alterations on mRNA abundance or the translation of specific mRNAs. Cardiomyocytes have very low rates of protein synthesis, posing a challenging problem in terms of studying changes in the synthesis of specific proteins, which also applies to other nondividing primary cells. To study the rates of accumulation of specific proteins in these cells, we developed an optimized version of the Quantitative Noncanonical Amino acid Tagging LC/MS proteomic method to label and selectively enrich newly synthesized proteins in these primary cells while eliminating the suppressive effects of pre-existing and highly abundant nonisotope-tagged polypeptides. Our data revealed that a classical pathologic (phenylephrine; PE) and the recently identified insulin stimulus that also contributes to the development of pathological cardiac hypertrophy (insulin), both increased the synthesis of proteins involved in, e.g. glycolysis, the Krebs cycle and beta-oxidation, and sarcomeric components. However, insulin increased synthesis of many metabolic enzymes to a greater extent than PE. Using a novel validation method, we confirmed that synthesis of selected candidates is indeed up-regulated by PE and insulin. Synthesis of all proteins studied was up-regulated by signaling through mammalian target of rapamycin complex 1 without changes in their mRNA levels, showing the key importance of translational control in the rapid effects of hypertrophic stimuli. Expression of PKM2 was up-regulated in rat hearts following TAC. This isoform possesses specific regulatory properties, so this finding indicates it may be involved in metabolic remodeling and also serve as a novel candidate biomarker. Levels of translation factor eEF1 also increased during TAC, likely contributing to faster cell mass accumulation. Interestingly those two candidates were not up-regulated in pregnancy or exercise induced CH, indicating PKM2 and eEF1 were pathological CH specific markers. We anticipate that the methodologies described here will be valuable for other researchers studying protein synthesis in primary cells. Cardiac hypertrophy (CH) 1The abbreviations used are: CHCardiac hypertrophy2-DETwo-dimensional electrophoresis4E-BP14E-binding protein 1AcadlAcyl-Coenzyme A dehydrogenase, long-chainACO2Aconitase 2AHAL-azidohomoalanineALDOAAldolase AARVCAdult rat ventricular cardiomyocytesCDSCoding DNA sequenceFDRFalse discovery ratehnRNPsHeterogeneous nuclear RNA-binding proteinsHSP60Heat shock 60kDa protein 1IGF1Insulin-like growth factor 1LmodLeiomodinMDH2Malate dehydrogenase 2mTORC1mammalian target of rapamycin complex 1NRAPNebulin-related anchoring proteinPEPhenylephrineQuaNCATQuantitative Noncanonical Amino acid TaggingpSILACpulsed stable isotope-labeling with amino acids in cell culturePTBP1Polypyrimidine tract-binding protein 1RPsRibosomal proteinsS6KsS6 kinasesTACTransverse aortic constrictionTOPTerminal oligopyrimidine tract. describes the enlargement of the myocardium and can be classified as either the 'physiological' or 'pathological' types. The latter is usually caused by conditions such as chronic hypertension, and is initially an adaptive response to help the heart to maintain normal function. However, sustained pressure overload leads to chronic hypertrophy and myocardial dysfunction. Indeed, pathological cardiac hypertrophy usually progresses into heart failure and is a major cause of death in young adults in developed countries. In contrast, physiological hypertrophy, as caused by pregnancy or exercise, is beneficial (1.Kavazis A.N. Pathological vs. physiological cardiac hypertrophy.J. Physiol. 2015; 593: 3767Crossref PubMed Scopus (10) Google Scholar, 2.Morgan H.E. Gordon E.E. Kira Y. Chua H.L. Russo L.A. Peterson C.J. McDermott P.J. Watson P.A. Biochemical mechanisms of cardiac hypertrophy.Annu. Rev. Physiol. 1987; 49: 533-543Crossref PubMed Google Scholar, 3.Eghbali M. Wang Y. Toro L. Stefani E. Heart hypertrophy during pregnancy: a better functioning heart?.Trends Cardiovasc. 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Cardiac hypertrophy Two-dimensional electrophoresis 4E-binding protein 1 Acyl-Coenzyme A dehydrogenase, long-chain Aconitase 2 L-azidohomoalanine Aldolase A Adult rat ventricular cardiomyocytes Coding DNA sequence False discovery rate Heterogeneous nuclear RNA-binding proteins Heat shock 60kDa protein 1 Insulin-like growth factor 1 Leiomodin Malate dehydrogenase 2 mammalian target of rapamycin complex 1 Nebulin-related anchoring protein Phenylephrine Quantitative Noncanonical Amino acid Tagging pulsed stable isotope-labeling with amino acids in cell culture Polypyrimidine tract-binding protein 1 Ribosomal proteins S6 kinases Transverse aortic constriction Terminal oligopyrimidine tract. The differences between pathological and physiological hypertrophy are profound; for example, fetal genes such as natriuretic peptides A and B are up-regulated in pathological, but not physiological, hypertrophy (5.Depre C. Shipley G.L. Chen W. Han Q. Doenst T. Moore M.L. Stepkowski S. Davies P.J. Taegtmeyer H. Unloaded heart in vivo replicates fetal gene expression of cardiac hypertrophy.Nat. Med. 1998; 4: 1269-1275Crossref PubMed Scopus (362) Google Scholar). In pathological hypertrophy, cardiac function usually becomes impaired, whereas in physiological hypertrophy, it is usually preserved or even enhanced. Metabolic remodeling is another significant change during cardiac hypertrophy. In pathological hypertrophy, fatty acid β-oxidation decreases whereas glucose utilization increases. In contrast, both fatty acid oxidation and glucose oxidation are up-regulated in physiological hypertrophy. Pathological hypertrophy is also associated with mitochondrial dysfunction, fibrosis and cell apoptosis; in physiological hypertrophy mitochondrial biogenesis is up-regulated, and neither fibrosis nor apoptosis are observed. Furthermore, distinct signaling pathways are activated in different types of hypertrophy: in physiological hypertrophy, insulin-like growth factor (IGF) 1 activates signaling via phosphoinositide 3-kinase signaling while well-established inducers of pathological hypertrophy, such as the α1-adrenergic agonist phenylephrine (PE), which stimulates signaling through Gαq and the classical mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (ERK) pathway (6.Bernardo B.C. Weeks K.L. Pretorius L. McMullen J.R. Molecular distinction between physiological and pathological cardiac hypertrophy: experimental findings and therapeutic strategies.Pharmacol. Ther. 2010; 128: 191-227Crossref PubMed Scopus (599) Google Scholar). In both cases, mTORC1 (mammalian target of rapamycin complex 1) signaling is activated. Insulin activates the IGF-1 signaling pathway at the supraphysiological concentration of 100 nmol/L, so was believed to promote the physiological hypertrophic growth of cardiomyocytes at this concentration similarly to IGF-1 (7.McMullen J.R. Shioi T. Huang W.Y. Zhang L. Tarnavski O. Bisping E. Schinke M. Kong S. Sherwood M.C. Brown J. Riggi L. Kang P.M. Izumo S. The insulin-like growth factor 1 receptor induces physiological heart growth via the phosphoinositide 3-kinase(p110alpha) pathway.J. Biol. Chem. 2004; 279: 4782-4793Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). However, a recent study showed that insulin signaling actually contributes to the development of pathological cardiac hypertrophy (8.Shimizu I. Minamino T. Toko H. Okada S. Ikeda H. Yasuda N. Tateno K. Moriya J. Yokoyama M. Nojima A. Koh G.Y. Akazawa H. Shiojima I. Kahn C.R. Abel E.D. Komuro I. Excessive cardiac insulin signaling exacerbates systolic dysfunction induced by pressure overload in rodents.J. Clin. Invest. 2010; 120: 1506-1514Crossref PubMed Scopus (167) Google Scholar). The relationship between pathological CH induced by α1-adrenergic stimulation or by insulin has not previously been explored, and doing this is one goal of the present study. Both pathological and physiological types of CH are a consequence of cardiomyocyte growth. As most of a cell's dry mass is protein, elevated protein synthesis plays a central role in cell growth (9.Hannan R.D. Jenkins A. Jenkins A.K. Brandenburger Y. Cardiac hypertrophy: a matter of translation.Clin. Exp. Pharmacol. Physiol. 2003; 30: 517-527Crossref PubMed Scopus (124) Google Scholar), and mTORC1 is a key positive regulator of protein synthesis and cell growth. Activation of mTORC1 was observed in exercise-induced (10.McMullen J.R. Shioi T. Zhang L. Tarnavski O. Sherwood M.C. Kang P.M. Izumo S. Phosphoinositide 3-kinase (p110alpha) plays a critical role for the induction of physiological, but not pathological, cardiac hypertrophy.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 12355-12360Crossref PubMed Scopus (450) Google Scholar) cardiac hypertrophy. Similarly, in TAC mice, mTORC1 is initially rapidly activated, but subsequently inactivated. Indeed, administration of the mTORC1-specific inhibitor rapamycin prior to or after TAC can attenuate or reverse TAC-induced hypertrophy and heart dysfunction in mice (11.Shioi T. McMullen J.R. Tarnavski O. Converso K. Sherwood M.C. Manning W.J. Izumo S. Rapamycin attenuates load-induced cardiac hypertrophy in mice.Circulation. 2003; 107: 1664-1670Crossref PubMed Scopus (393) Google Scholar, 12.McMullen J.R. Sherwood M.C. Tarnavski O. Zhang L. Dorfman A.L. Shioi T. Izumo S. Inhibition of mTOR signaling with rapamycin regresses established cardiac hypertrophy induced by pressure overload.Circulation. 2004; 109: 3050-3055Crossref PubMed Scopus (406) Google Scholar). Given the distinct signaling pathways activated α-adrenergic stimulation and insulin in ARVC, and their roles in cardiac hypertrophy, it was therefore important to compare the impact on the synthesis of specific proteins in ARVC. mTORC1 plays important roles in regulating protein synthesis in isolated adult rat ventricular cardiomyocytes (ARVC). Indeed, activating mTORC1 by over-expressing a small GTPase, Rheb, which positively regulates mTORC1 (13.Huang J. Manning B.D. The TSC1-TSC2 complex: a molecular switchboard controlling cell growth.Biochem. J. 2008; 412: 179-190Crossref PubMed Scopus (922) Google Scholar), is sufficient to drive rapid and marked growth of ARVC (14.Wang Y. Huang B.P. Luciani D.S. Wang X. Johnson J.D. Proud C.G. Rheb activates protein synthesis and growth in adult rat ventricular cardiomyocytes.J. Mol. Cell. Cardiol. 2008; 45: 812-820Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). PE substantially enhances global protein synthesis and ARVC growth, which is largely blocked by rapamycin (14.Wang Y. Huang B.P. Luciani D.S. Wang X. Johnson J.D. Proud C.G. Rheb activates protein synthesis and growth in adult rat ventricular cardiomyocytes.J. Mol. Cell. Cardiol. 2008; 45: 812-820Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), again indicating that mTORC1 signaling is important for CH. Insulin also stimulates protein synthesis in an mTORC1-dependent manner in ARVC (15.Wang L. Wang X. Proud C.G. Activation of mRNA translation in rat cardiac myocytes by insulin involves multiple rapamycin-sensitive steps.Am. J. Physiol. Heart Circ. Physiol. 2000; 278: H1056-H1068Crossref PubMed Google Scholar). This study aims to address several key questions: which specific proteins' synthesis is increased in response to different hypertrophic stimuli? Are those proteins also altered in pathological and physiological animal models? Are changes in the synthesis of specific proteins exerted at the level of transcription or translation? Does mTORC1 signaling play a role in regulating the translation of specific mRNAs? To address these questions, we optimized a recently developed method wherein stable-isotope labeled amino acids are used to tag newly synthesized proteins (pulsed SILAC; pSILAC). Such a pSILAC approach was used to study the regulation of protein accumulation rates by mTOR signaling in HeLa cells (16.Huo Y. Iadevaia V. Yao Z. Kelly I. Cosulich S. Guichard S. Foster L.J. Proud C.G. Stable isotope-labelling analysis of the impact of inhibition of the mammalian target of rapamycin on protein synthesis.Biochem. J. 2012; 444: 141-151Crossref PubMed Scopus (66) Google Scholar), a rapidly dividing cancer cell line, which synthesizes proteins at a much higher rate than primary cells such as ARVC. We subsequently improved the pSILAC approach to enhance its selectivity and sensitivity for the analysis of newly synthesized proteins by combining it with azidohomoalanine (AHA) labeling/click-chemistry and subsequent bottom-up LC-MS proteomic analysis (Fig. 1) (17.Genheden M. Kenney J.W. Johnston H.E. Manousopoulou A. Garbis S.D. Proud C.G. BDNF stimulation of protein synthesis in cortical neurons requires the MAP kinase-interacting kinase MNK1.J. Neurosci. 2015; 35: 972-984Crossref PubMed Scopus (56) Google Scholar). During incubation in cell culture, AHA (an analog of methionine) becomes incorporated into, and covalently tags, newly synthesized proteins. The resulting azide-derivatized proteins can then be selectively coupled and enriched to biotin-alkyne beads for visualization using fluorescently labeled streptavidin. Alternatively, the azide-derivatized proteins are selectively isolated and enriched with alkyne-functionalized agarose beads and subjected to trypsin proteolysis followed by liquid chromatography-mass spectrometry (LC-MS/MS). The collective analytical attributes of this proteomics approach, referred to as quantitative noncanonical amino acid tagging (QuaNCAT), permits the sensitive and selective measurement of the synthesis rates of specific proteins in cells (18.Eichelbaum K. Winter M. Berriel Diaz M. Herzig S. Krijgsveld J. Selective of newly synthesized proteins for quantitative secretome analysis.Nat. Biotechnol. 2012; 30: 984-990Crossref PubMed Scopus (185) Google Scholar, 19.Howden A.J. Geoghegan V. Katsch K. Efstathiou G. Bhushan B. Boutureira O. Thomas B. Trudgian D.C. Kessler B.M. Dieterich D.C. Davis B.G. Acuto O. QuaNCAT: quantitating proteome dynamics in primary cells.Nat. Methods. 2013; 10: 343-346Crossref PubMed Scopus (120) Google Scholar). Other quantitative proteomic approaches that rely on the use of label-free and isobaric stable isotope labeling to examine the differential expression of proteins in hypertrophic or diseased heart (20.Dai D.F. Hsieh E.J. Chen T. Menendez L.G. Basisty N.B. Tsai L. Beyer R.P. Crispin D.A. Shulman N.J. Szeto H.H. Tian R. MacCoss M.J. Rabinovitch P.S. Global proteomics and pathway analysis of pressure-overload-induced heart failure and its attenuation by mitochondrial-targeted peptides.Circ. Heart Fail. 2013; 6: 1067-1076Crossref PubMed Scopus (108) Google Scholar, 21.Kocher T. Pichler P. Schutzbier M. Stingl C. Kaul A. Teucher N. Hasenfuss G. Penninger J.M. Mechtler K. High precision quantitative proteomics using iTRAQ on an LTQ Orbitrap: a new mass spectrometric method combining the benefits of all.J. Proteome Res. 2009; 8: 4743-4752Crossref PubMed Scopus (138) Google Scholar, 22.Gallego-Delgado J. Lazaro A. Osende J.I. Esteban V. Barderas M.G. Gomez-Guerrero C. Vega R. Vivanco F. Egido J. Proteomic analysis of early left ventricular hypertrophy secondary to hypertension: modulation by antihypertensive therapies.J. Am. Soc. Nephrol. 2006; 17: S159-S164Crossref PubMed Scopus (21) Google Scholar) only capture relative steady-state protein concentration levels rather than assessing rates of de novo synthesis. They are thus unsuitable for addressing the aims of the current study. We have further optimized and applied the QuaNCAT approach to measure the regulated synthesis of specific proteins in ARVC cells observed to occur at a very low rate. Our results revealed several key new features in the response of cardiomyocytes to hypertrophic stimuli. These include: (1) both insulin and PE increased the protein synthesis rate to a similar set of proteins. Additionally, marked differences were observed in the patterns of induction of metabolic enzymes and other proteins in response to PE and insulin; (2) increased synthesis of pyruvate kinase M2, a protein involved in anabolic remodeling of cellular metabolism, which may also represent a new early marker for cardiac hypertrophy; (3) the widespread importance of increased translation (rather than transcription) in the rapid effects of hypertrophic agents; and (4) a key role for mTORC1 signaling in the effects of hypertrophic agents of the translation of specific mRNAs, including many mRNAs that are not members of the subset of TOP (terminal oligo-pyrimidine) mRNAs, which are translationally regulated by mTORC1. ARVCs derived from wild-type Sprague-Dawley male rats were treated with phenylephrine (10 μmol/L), insulin (100 nmol/L), and rapamycin (100 nmol/L). AHA-pSILAC was combined with mass spectrometry to identify newly synthesized proteins as a result of each treatment. Replicate experiments were performed, three in the case of insulin and four for phenylephrine. Identified proteins were validated in ARVCs using two similar but independent methods. We determined the total mRNA levels of selected candidate proteins, to examine whether increased synthesis of these proteins reflects increased translation of their mRNAs or increased mRNA levels. To test the role of mTORC1 signaling in regulating the synthesis of specific proteins in ARVCs, we repeated the AHA-pSILAC labeling experiments using ARVCs that were treated with PE or insulin in the presence of the mTORC1 inhibitor rapamycin. Finally, key proteomic results were studied using in vivo rodent models of pathologic and physiologic cardiac hypertrophy. Data from independent experimental replicates were analyzed using Student's t test, Significance was set to p = 0.05. Animals used for isolating ARVC were wild-type Sprague-Dawley male rats from Charles River, Oxford, UK. Animals were reared and sacrificed in line with the United Kingdom Animals (Scientific Procedures) Act, 1986. The method of sacrifice employed here was cervical dislocation according to the UK Home Office regulations Schedule 1. Animals used in the TAC study were as described earlier (23.Schwarzer M. Schrepper A. Amorim P.A. Osterholt M. Doenst T. Pressure overload differentially affects respiratory capacity in interfibrillar and subsarcolemmal mitochondria.Am. J. Physiol. Heart Circ Physiol. 2013; 304: H529-H537Crossref PubMed Scopus (37) Google Scholar). Isolation and maintenance of adult rat ventricular cardiomyocytes (ARVC) were described earlier (15.Wang L. Wang X. Proud C.G. Activation of mRNA translation in rat cardiac myocytes by insulin involves multiple rapamycin-sensitive steps.Am. J. Physiol. Heart Circ. Physiol. 2000; 278: H1056-H1068Crossref PubMed Google Scholar, 24.Wang X. Levi A.J. Halestrap A.P. Kinetics of the sarcolemmal lactate carrier in single heart cells using BCECF to measure pHi.Am. J. Physiol. 1994; 267: H1759-H1769PubMed Google Scholar). ARVC were treated with following compounds at the indicated concentrations: phenylephrine (10 μmol/L), insulin (100 nmol/L), and rapamycin (100 nmol/L). ARVCs were cultured in complete M199 medium (M199 medium containing 5.55 mmol/L glucose, 0.68 mmol/L glutamine, 5 mmol/L creatine, 2 mmol/l-carnitine, and 5 mmol/L taurine) after isolation; 30 min prior to any treatments, ARVCs were transferred into Customized M199 media (Dundee Cell Products, Dundee, Scotland) containing 0.41 mmol/L heavy lysine and 0.71 mmol/L heavy arginine, or 0.41 mmol/L medium lysine and 0.71 mmol/L medium arginine. Both media contain low levels of methionine (25.13 μmol/L, about 25% of that in the standard M199 medium). After 30 min, ARVCs were treated with various inhibitors in the presence of 2 mmol/L azidohomoalanine (AHA). Forty-eight hours later, cells were washed twice with cold PBS and lysed in pSILAC lysis buffer (8 mol/L urea, 300 mmol/L Tris-HCl pH 8, 4% (w/v) CHAPS, 1 mol/L NaCI) containing proteinase inhibitors. 400 μg of lysate from the control and treatment animals were mixed together. The mixed protein lysates were reacted with alkyne agarose resin beads. The click reactions were conducted according to the manufacturer's instructions Life Technologies KIT C-10416 Life Technologies, Loughborough, UK, after which, the newly synthesized proteins were covalently conjugated to the alkyne-agarose resin beads. They were then digested using 0.1 μg Lys-C for 4 h, followed by trypsin digestion (0.1 μg/μl X 1 μl 37 °C overnight) and subjected to mass spectrometry analysis, as reported by the authors (17.Genheden M. Kenney J.W. Johnston H.E. Manousopoulou A. Garbis S.D. Proud C.G. BDNF stimulation of protein synthesis in cortical neurons requires the MAP kinase-interacting kinase MNK1.J. Neurosci. 2015; 35: 972-984Crossref PubMed Scopus (56) Google Scholar). Replicate experiments were performed, three in the case of insulin and four for PE. This procedure is summarized in Fig. 1. Peptide mixtures were de-salted with 100 μl capacity C18 tips (Thermo Scientific) with five incremental iterations from 2 to 98% HPLC grade acetonitrile (Fisher Scientific) in 0.1% analytical grade formic acid (Fisher Scientific). Eluates were lyophilized to dryness and reconstituted in 20 μl of loading solution (2% acetonitrile, 0.5% formic acid). A sample of volume 14 μl was then injected onto a C18 μ-Precolumn (300 μm ID × 10 mm L, 5 μm particle; 100 Å pore size, Acclaim PepMap100, Thermo Scientific) at 20 μl/min for 6 min using the loading solution as the mobile phase to trap and de-salt its peptide content. The purified peptides were then loaded onto a high-capacity nano-capillary reverse phase C18 column (75 μm × 50 cm, 2 μm particle; 100 Å pore size; Acclaim PepMap 100 column, ThermoScientific) retrofitted to a PicoTip nESI emitter (New Objective, Woburn, MA) and gradient separated as reported by the authors (17.Genheden M. Kenney J.W. Johnston H.E. Manousopoulou A. Garbis S.D. Proud C.G. BDNF stimulation of protein synthesis in cortical neurons requires the MAP kinase-interacting kinase MNK1.J. Neurosci. 2015; 35: 972-984Crossref PubMed Scopus (56) Google Scholar). Nanospray ionization was conducted at 2.4 kV and ions were characterized with an FT-Orbitrap Elite (Thermo Scientific) at 240,000 mass resolution. The top 12, +2 and +3 precursor ions per mass spectrometry (MS) scan (minimum intensity 1000) were characterized by high-energy collisional dissociation (HCD; 15,000 mass resolution, 1.2 Da isolation window, 40 keV collision energy) and collision-induced dissociation (CID; ion trap MS, 2 Da isolation window, 35 keV) with a dynamic exclusion (5 ppm) of 200 s. Peptide spectrum matching and quantifications were performed with Proteome Discoverer 1.4 (Thermo Scientific) with SequestHT against the UniprotKB SwissProt Rattus norvegicus proteome (downloaded 04/2014; 28861 entries). For matching and quantitation, precursor tolerance was set at 10 and 3 ppm, respectively. Fragment matching was set at 0.02 and 0.5 Da for HCD and CID, respectively. Target-decoy searching allowed for 1 missed cleavage, a minimum length of 6 residues and a maximum of three variable (1 equal) modifications of Met-AHA (M), deamidation (Asn, Gln), or phosphorylation (Ser, Thr, or Tyr). Carbamidomethyl (Cys) was set as a fixed modification with Lys (+4 Da), Arg (+6 Da), and Lys (+8 Da), Arg (+10 Da) searched to determine medium and heavy labeled peptides, respectively. The false discovery rate (FDR) was estimated with Percolator at <5% peptide FDR to enable parallel protein grouping and quantitation. The mass spectrometric proteomic data have been deposited to the ProteomeXchange Consortium (25.Vizcaino J.A. Deutsch E.W. Wang R. Csordas A. Reisinger F. Rios D. Dianes J.A. Sun Z. Farrah T. Bandeira N. Binz P.A. Xenarios I. Eisenacher M. Mayer G. Gatto L. Campos A. Chalkley R.J. Kraus H.J. Albar J.P. Martinez-Bartolome S. Apweiler R. Omenn G.S. Martens L. Jones A.R. Hermjakob H. ProteomeXchange provides globally coordinated proteomics data submission and dissemination.Nat. Biotechnol. 2014; 32: 223-226Crossref PubMed Scopus (2071) Google Scholar) via the PRIDE partner repository (26.Vizcaino J.A. Cote R.G. Csordas A. Dianes J.A. Fabregat A. Foster J.M. Griss J. Alpi E. Birim M. Contell J. O'Kelly G. Schoenegger A. Ovelleiro D. Perez-Riverol Y. Reisinger F. Rios D. Wang R. Hermjakob H. The PRoteomics IDEntifications (PRIDE) database and associated tools: status in 2013.Nucleic Acids Res. 2013; 41: D1063-D1069Crossref PubMed Scopus (1595) Google Scholar, 27.Wang R. Fabregat A. Rios D. Ovelleiro D. Foster J.M. Cote R.G. Griss J. Csordas A. Perez-Riverol Y. Reisinger F. Hermjakob H. Martens L. Vizcaino J.A. PRIDE Inspector: a tool to visualize and validate MS proteomics data.Nat. Biotechnol. 2012; 30: 135-137Crossref PubMed Scopus (102) Google Scholar) with the data set identifier PXD004127. Heat-map construction of proteins analyzed in at least one replicate of all four conditions (i.e. Ins versus control, PE versus control, Ins+Rapa versus Ins, PE+Rapa versus PE) was generated using Cluster 3.0 (http://bonsai.hgc.jp/∼mdehoon/software/cluster/software.htm) and Java Treeview (http://jtreeview.sourceforge.net). MetaCore (GeneGo, St. Joseph, MI) was applied to identify over-represented biological processes and to identify direct protein interaction networks of proteins of interest. FDR-corrected p values < 0.05 were considered significant. ARVCs were cultured and treated as described above, and then lysed in cell lysis buffer (1% Triton® X100, 50 mmol/L β-glycerophosphate, 1 mmol/L EDTA, 1 mmol/L EGTA, 0.5 mmol/L Na3VO4, 1 mmol/l-dithiothreitol, and proteinase inhibitor mixture; Roche, catalog number 11873580001). Protein concentrations were determined by Bradford assay. Fifty to 200 μg newly synthesized proteins were reacted with biotin-alkyne, according to the procedure described in detail in the manufacturer's instructions (Life Technologies: C-10276). Forty microliters of click reaction products were subjected to SDS-PAGE, then transferred onto a nitrocellulose microporous membrane, which was blocked with 5% (w/v) fat-free powdered milk in PBS-0.02% (v/v) Tween. After three washes with PBS-0.02% (v/v) Tween, the membrane was incubated with streptavidin, Alexa Fluor® 680 (1:10,000 dilution; 45 min) and, after another three washes, the membrane was scanned using the Odyssey® Infrared Imaging System. One μg of total RNA from rat heart and a gene-specific primer were used to generate the first strand cDNA containing the 5′-UTR and part of the coding region of mRNA, then use RNase H to degrade the mRNA template. Afterward, the first-strand cDNA was ligated with the DNA oligo linker by T
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