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• W2327151570 abstract "Familial hypertrophic cardiomyopathy (FHC) is associated with mild to severe cardiac problems and is the leading cause of sudden death in young people and athletes. Although the genetic basis for FHC is well-established, the molecular mechanisms that ultimately lead to cardiac dysfunction are not well understood. To obtain important insights into the molecular mechanism(s) involved in FHC, hearts from two FHC troponin T models (Ile79Asn [I79N] and Arg278Cys [R278C]) were investigated using label-free proteomics and metabolomics. Mutations in troponin T are the third most common cause of FHC, and the I79N mutation is associated with a high risk of sudden cardiac death. Most FHC-causing mutations, including I79N, increase the Ca2+ sensitivity of the myofilament; however, the R278C mutation does not alter Ca2+ sensitivity and is associated with a better prognosis than most FHC mutations. Out of more than 1200 identified proteins, 53 and 76 proteins were differentially expressed in I79N and R278C hearts, respectively, when compared with wild-type hearts. Interestingly, more than 400 proteins were differentially expressed when the I79N and R278C hearts were directly compared. The three major pathways affected in I79N hearts relative to R278C and wild-type hearts were the ubiquitin-proteasome system, antioxidant systems, and energy production pathways. Further investigation of the proteasome system using Western blotting and activity assays showed that proteasome dysfunction occurs in I79N hearts. Metabolomic results corroborate the proteomic data and suggest the glycolytic, citric acid, and electron transport chain pathways are important pathways that are altered in I79N hearts relative to R278C or wild-type hearts. Our findings suggest that impaired energy production and protein degradation dysfunction are important mechanisms in FHCs associated with poor prognosis and that cardiac hypertrophy is not likely needed for a switch from fatty acid to glucose metabolism. Familial hypertrophic cardiomyopathy (FHC) is associated with mild to severe cardiac problems and is the leading cause of sudden death in young people and athletes. Although the genetic basis for FHC is well-established, the molecular mechanisms that ultimately lead to cardiac dysfunction are not well understood. To obtain important insights into the molecular mechanism(s) involved in FHC, hearts from two FHC troponin T models (Ile79Asn [I79N] and Arg278Cys [R278C]) were investigated using label-free proteomics and metabolomics. Mutations in troponin T are the third most common cause of FHC, and the I79N mutation is associated with a high risk of sudden cardiac death. Most FHC-causing mutations, including I79N, increase the Ca2+ sensitivity of the myofilament; however, the R278C mutation does not alter Ca2+ sensitivity and is associated with a better prognosis than most FHC mutations. Out of more than 1200 identified proteins, 53 and 76 proteins were differentially expressed in I79N and R278C hearts, respectively, when compared with wild-type hearts. Interestingly, more than 400 proteins were differentially expressed when the I79N and R278C hearts were directly compared. The three major pathways affected in I79N hearts relative to R278C and wild-type hearts were the ubiquitin-proteasome system, antioxidant systems, and energy production pathways. Further investigation of the proteasome system using Western blotting and activity assays showed that proteasome dysfunction occurs in I79N hearts. Metabolomic results corroborate the proteomic data and suggest the glycolytic, citric acid, and electron transport chain pathways are important pathways that are altered in I79N hearts relative to R278C or wild-type hearts. Our findings suggest that impaired energy production and protein degradation dysfunction are important mechanisms in FHCs associated with poor prognosis and that cardiac hypertrophy is not likely needed for a switch from fatty acid to glucose metabolism. Familial hypertrophic cardiomyopathy (FHC) 1The abbreviations used are:FHCfamilial hypertrophic cardiomyopathyAMPadenosine monophosphateGC-TOFgas chromatography - time of flight mass spectrometryHCMhypertrophic cardiomyopathyLC-MS/MStandem mass spectrometryMyBPCMyosin binding protein CSCDsudden cardiac deathTnItroponin ITnTtroponin TIle79AsnI79NArg278CysR278C. is the most common monogenically inherited heart disease, estimated to affect one in 500 people (1.Frey N. Luedde M. Katus H.A. Mechanisms of disease: hypertrophic cardiomyopathy.Nat. Rev. Cardiology. 2012; 9: 91-100Crossref Scopus (152) Google Scholar, 2.Maron B.J. Hypertrophic cardiomyopathy: a systematic review.JAMA. 2002; 287: 1308-1320Crossref PubMed Scopus (0) Google Scholar). It is characterized by thickening of the left ventricle, contractile dysfunction, heart failure, and a high incidence of potentially lethal arrhythmias (3.Ashrafian H. McKenna W.J. Watkins H. Disease pathways and novel therapeutic targets in hypertrophic cardiomyopathy.Circulation Res. 2011; 109: 86-96Crossref PubMed Scopus (128) Google Scholar). Over a thousand mutations in more than twenty genes have been identified that cause FHC, most of which encode proteins of the sarcomere; however, the pathological mechanisms of this disease are not well understood (1.Frey N. Luedde M. Katus H.A. Mechanisms of disease: hypertrophic cardiomyopathy.Nat. Rev. Cardiology. 2012; 9: 91-100Crossref Scopus (152) Google Scholar, 3.Ashrafian H. McKenna W.J. Watkins H. Disease pathways and novel therapeutic targets in hypertrophic cardiomyopathy.Circulation Res. 2011; 109: 86-96Crossref PubMed Scopus (128) Google Scholar, 4.Xu Q. Dewey S. Nguyen S. Gomes A.V. Malignant and benign mutations in familial cardiomyopathies: insights into mutations linked to complex cardiovascular phenotypes.J. Mol. Cell. Cardiol. 2010; 48: 899-909Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Mutations in cardiac troponin T (TnT), encoded by TNNT2, carry a particularly poor prognosis for the patient and are associated with a high risk of sudden cardiac death (SCD) (5.Gomes A.V. Barnes J.A. Harada K. Potter J.D. Role of troponin T in disease.Mol. Cell. Biochem. 2004; 263: 115-129Crossref PubMed Scopus (56) Google Scholar, 6.Moolman J.C. Corfield V.A. Posen B. Ngumbela K. Seidman C. Brink P.A. Watkins H. Sudden death due to troponin T mutations. J.Am. College Cardiol. 1997; 29: 549-555Crossref PubMed Scopus (303) Google Scholar, 7.Watkins H. McKenna W.J. Thierfelder L. Suk H.J. Anan R. O'Donoghue A. Spirito P. Matsumori A. Moravec C.S. Seidman J.G. et al.Mutations in the genes for cardiac troponin T and alpha-tropomyosin in hypertrophic cardiomyopathy.New Engl. J. Med. 1995; 332: 1058-1064Crossref PubMed Scopus (776) Google Scholar, 8.Pasquale F. Syrris P. Kaski J.P. Mogensen J. McKenna W.J. Elliott P. Long-term outcomes in hypertrophic cardiomyopathy caused by mutations in the cardiac troponin T gene.Circulation Cardiovascular Genetics. 2012; 5: 10-17Crossref PubMed Scopus (88) Google Scholar). Most FHC-causing mutations alter how the sarcomere responds to Ca2+ by increasing the myofilament Ca2+ sensitivity, which changes its contractile properties and affects Ca2+ handling in the cell (9.Wen Y. Pinto J.R. Gomes A.V. Xu Y. Wang Y. Potter J.D. Kerrick W.G. Functional consequences of the human cardiac troponin I hypertrophic cardiomyopathy mutation R145G in transgenic mice.J. Biol. Chem. 2008; 283: 20484-20494Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 10.Gomes A.V. Venkatraman G. Davis J.P. Tikunova S.B. Engel P. Solaro R.J. Potter J.D. Cardiac troponin T isoforms affect the Ca(2+) sensitivity of force development in the presence of slow skeletal troponin I: insights into the role of troponin T isoforms in the fetal heart.J. Biol. Chem. 2004; 279: 49579-49587Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Proposed pathological mechanisms include altered cardiac contractility, changes in Ca2+ handling, and altered energy homeostasis (1.Frey N. Luedde M. Katus H.A. Mechanisms of disease: hypertrophic cardiomyopathy.Nat. Rev. Cardiology. 2012; 9: 91-100Crossref Scopus (152) Google Scholar, 11.Gomes A.V. Potter J.D. Molecular and cellular aspects of troponin cardiomyopathies.Ann. N. Y. Acad. Sci. 2004; 1015: 214-224Crossref PubMed Scopus (91) Google Scholar). Current treatments include β-adrenergic receptor blockers, Ca2+ channel antagonists, myocardial reduction by surgical or septal ablation, and implanted defibrillators (2.Maron B.J. Hypertrophic cardiomyopathy: a systematic review.JAMA. 2002; 287: 1308-1320Crossref PubMed Scopus (0) Google Scholar). Currently available pharmacological treatments can provide relief from symptoms and improve patient quality of life, but they do not target FHC-specific pathways and have not been shown to slow disease progression (1.Frey N. Luedde M. Katus H.A. Mechanisms of disease: hypertrophic cardiomyopathy.Nat. Rev. Cardiology. 2012; 9: 91-100Crossref Scopus (152) Google Scholar). An improved understanding of the molecular mechanisms of FHC is needed to develop specific therapies that address the underlying causes of the disease (2.Maron B.J. Hypertrophic cardiomyopathy: a systematic review.JAMA. 2002; 287: 1308-1320Crossref PubMed Scopus (0) Google Scholar). familial hypertrophic cardiomyopathy adenosine monophosphate gas chromatography - time of flight mass spectrometry hypertrophic cardiomyopathy tandem mass spectrometry Myosin binding protein C sudden cardiac death troponin I troponin T I79N R278C. Alterations of proteins and pathways that are comorbid with heart diseases like dilated cardiomyopathy, congestive heart failure, and myocardial infarction have been identified by protein mass spectrometry (12.Cui Z. Dewey S. Gomes A.V. Cardioproteomics: advancing the discovery of signaling mechanisms involved in cardiovascular diseases.Am. J. Cardiovascular Dis. 2011; 1: 274-292PubMed Google Scholar). To gain a better understanding of the pathways affected in TnT-related FHC, we utilized a label-free proteomic approach to investigate how hearts from mice with two different TnT mutations (Ile79Asn [I79N] and Arg278Cys [R278C]) are affected at the protein level. These mice (wild-type (WT), R278C, and I79N) all express human TnT at ∼50% of the total TnT with the endogenous mouse TnT accounting for the other 50% (13.Hernandez O.M. Szczesna-Cordary D. Knollmann B.C. Miller T. Bell M. Zhao J. Sirenko S.G. Diaz Z. Guzman G. Xu Y. Wang Y. Kerrick W.G. Potter J.D. F110I and R278C troponin T mutations that cause familial hypertrophic cardiomyopathy affect muscle contraction in transgenic mice and reconstituted human cardiac fibers.J.Biol. Chem. 2005; 280: 37183-37194Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 14.Miller T. Szczesna D. Housmans P.R. Zhao J. de Freitas F. Gomes A.V. Culbreath L. McCue J. Wang Y. Xu Y. Kerrick W.G. Potter J.D. Abnormal contractile function in transgenic mice expressing a familial hypertrophic cardiomyopathy-linked troponin T (I79N) mutation.J. Biol. Chem. 2001; 276: 3743-3755Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Transgenic mice expressing human TnT are a useful tool for investigating the disease, as mice with the R278C or I79N mutation show similar features to humans; humans with the I79N mutation are at a high risk of sudden cardiac death, and those with the R278C mutation show a milder phenotype (7.Watkins H. McKenna W.J. Thierfelder L. Suk H.J. Anan R. O'Donoghue A. Spirito P. Matsumori A. Moravec C.S. Seidman J.G. et al.Mutations in the genes for cardiac troponin T and alpha-tropomyosin in hypertrophic cardiomyopathy.New Engl. J. Med. 1995; 332: 1058-1064Crossref PubMed Scopus (776) Google Scholar, 15.Varnava A.M. Elliott P.M. Baboonian C. Davison F. Davies M.J. McKenna W.J. Hypertrophic cardiomyopathy: histopathological features of sudden death in cardiac troponin T disease.Circulation. 2001; 104: 1380-1384Crossref PubMed Scopus (246) Google Scholar, 16.Elliott P.M. D'Cruz L. McKenna W.J. Late-onset hypertrophic cardiomyopathy caused by a mutation in the cardiac troponin T gene.N Engl. J. Med. 1999; 341: 1855-1856Crossref PubMed Scopus (21) Google Scholar, 17.Van Driest S.L. Ellsworth E.G. Ommen S.R. Tajik A.J. Gersh B.J. Ackerman M.J. Prevalence and spectrum of thin filament mutations in an outpatient referral population with hypertrophic cardiomyopathy.Circulation. 2003; 108: 445-451Crossref PubMed Scopus (196) Google Scholar). To determine changes at the metabolite level, a time-of-flight gas chromatography mass spectrometer system (GC-TOF) was utilized to determine metabolite levels in the different transgenic hearts. A major advantage of this investigation is the comparison of two different FHC models: hearts containing the myofilament Ca2+-sensitizing I79N mutation that is associated with poor prognosis and SCD, and the R278C mutation, which does not alter myofilament Ca2+ sensitivity and is associated with mild, late-onset cardiac effects and no SCD (7.Watkins H. McKenna W.J. Thierfelder L. Suk H.J. Anan R. O'Donoghue A. Spirito P. Matsumori A. Moravec C.S. Seidman J.G. et al.Mutations in the genes for cardiac troponin T and alpha-tropomyosin in hypertrophic cardiomyopathy.New Engl. J. Med. 1995; 332: 1058-1064Crossref PubMed Scopus (776) Google Scholar, 15.Varnava A.M. Elliott P.M. Baboonian C. Davison F. Davies M.J. McKenna W.J. Hypertrophic cardiomyopathy: histopathological features of sudden death in cardiac troponin T disease.Circulation. 2001; 104: 1380-1384Crossref PubMed Scopus (246) Google Scholar, 16.Elliott P.M. D'Cruz L. McKenna W.J. Late-onset hypertrophic cardiomyopathy caused by a mutation in the cardiac troponin T gene.N Engl. J. Med. 1999; 341: 1855-1856Crossref PubMed Scopus (21) Google Scholar, 17.Van Driest S.L. Ellsworth E.G. Ommen S.R. Tajik A.J. Gersh B.J. Ackerman M.J. Prevalence and spectrum of thin filament mutations in an outpatient referral population with hypertrophic cardiomyopathy.Circulation. 2003; 108: 445-451Crossref PubMed Scopus (196) Google Scholar). No proteomic or metabolomic comparisons between mild and severe models of FHC related to sarcomeric mutations have been reported. Our data reveal that although metabolic pathways and stress responses were affected in both R278C and I79N hearts, a major protein complex affected differently by the two mutations was the proteasome, a large, ∼2 MDa complex that is conserved in eukaryotes and responsible for degrading the majority of cytosolic, nuclear, and nascent membrane proteins following their modification with the small protein ubiquitin (18.Li J. Horak K.M. Su H. Sanbe A. Robbins J. Wang X. Enhancement of proteasomal function protects against cardiac proteinopathy and ischemia/reperfusion injury in mice.J. Clin. Invest. 2011; 121: 3689-3700Crossref PubMed Scopus (146) Google Scholar). Through targeted degradation of proteins that are no longer required by the cell, the proteasome plays a role in virtually all cellular processes (19.Wang X. Li J. Zheng H. Su H. Powell S.R. Proteasome functional insufficiency in cardiac pathogenesis.American journal of physiology. Heart Circulatory Physiol. 2011; 301: H2207-H2219Crossref PubMed Scopus (56) Google Scholar), and particularly in cardiomyocyte proteostasis, as these cells are prone to protein damage due to continual mechanical and metabolic stresses (20.Patterson C. Ike C. Willis P.W.t. Stouffer G.A. Willis M.S. The bitter end: the ubiquitin-proteasome system and cardiac dysfunction.Circulation. 2007; 115: 1456-1463Crossref PubMed Scopus (78) Google Scholar, 21.Wang X. Robbins J. Heart failure and protein quality control.Circulation Res. 2006; 99: 1315-1328Crossref PubMed Scopus (167) Google Scholar). The heart has very limited capacity for self-renewal, meaning that cell death, which may result from impaired proteasome function, can be highly detrimental to the health of the organ (20.Patterson C. Ike C. Willis P.W.t. Stouffer G.A. Willis M.S. The bitter end: the ubiquitin-proteasome system and cardiac dysfunction.Circulation. 2007; 115: 1456-1463Crossref PubMed Scopus (78) Google Scholar, 22.Wang X. Repeated intermittent administration of a ubiquitous proteasome inhibitor leads to restrictive cardiomyopathy.Eur. J. Heart Failure. 2013; 15: 597-598Crossref PubMed Scopus (3) Google Scholar). A growing body of evidence indicates proteasome dysfunction is an important contributor to the pathogenesis of many cardiac diseases. In FHC caused by certain mutations in the thick filament-associated protein cardiac myosin binding protein-C (MyBP-C), the mutated protein is preferentially degraded by the proteasome, competitively inhibiting breakdown of other proteasome substrates (23.Bahrudin U. Morisaki H. Morisaki T. Ninomiya H. Higaki K. Nanba E. Igawa O. Takashima S. Mizuta E. Miake J. Yamamoto Y. Shirayoshi Y. Kitakaze M. Carrier L. Hisatome I. Ubiquitin-proteasome system impairment caused by a missense cardiac myosin-binding protein C mutation and associated with cardiac dysfunction in hypertrophic cardiomyopathy.J. Mol. Biol. 2008; 384: 896-907Crossref PubMed Scopus (73) Google Scholar, 24.Sarikas A. Carrier L. Schenke C. Doll D. Flavigny J. Lindenberg K.S. Eschenhagen T. Zolk O. Impairment of the ubiquitin-proteasome system by truncated cardiac myosin binding protein C mutants.Cardiovascular Res. 2005; 66: 33-44Crossref PubMed Scopus (132) Google Scholar, 25.Schlossarek S. Englmann D.R. Sultan K.R. Sauer M. Eschenhagen T. Carrier L. Defective proteolytic systems in Mybpc3-targeted mice with cardiac hypertrophy.Basic Res. Cardiol. 2012; 107: 235Crossref PubMed Scopus (72) Google Scholar, 26.Vignier N. Schlossarek S. Fraysse B. Mearini G. Kramer E. Pointu H. Mougenot N. Guiard J. Reimer R. Hohenberg H. Schwartz K. Vernet M. Eschenhagen T. Carrier L. Nonsense-mediated mRNA decay and ubiquitin-proteasome system regulate cardiac myosin-binding protein C mutant levels in cardiomyopathic mice.Circulation Res. 2009; 105: 239-248Crossref PubMed Scopus (117) Google Scholar). However, some single residue MyBP-C mutants do not seem to affect ubiquitin-dependent proteasomal proteolysis, and the proteasome has not been implicated in any other non-MyBP-C FHC model (23.Bahrudin U. Morisaki H. Morisaki T. Ninomiya H. Higaki K. Nanba E. Igawa O. Takashima S. Mizuta E. Miake J. Yamamoto Y. Shirayoshi Y. Kitakaze M. Carrier L. Hisatome I. Ubiquitin-proteasome system impairment caused by a missense cardiac myosin-binding protein C mutation and associated with cardiac dysfunction in hypertrophic cardiomyopathy.J. Mol. Biol. 2008; 384: 896-907Crossref PubMed Scopus (73) Google Scholar). To investigate the effect of TnT mutations on proteasomal activity, expression and activity of the proteasome was measured, revealing increased proteasome subunit expression in I79N hearts relative to R278C hearts but decreased constitutive proteasome and immunoproteasome activities in I79N mice relative to WT and R278C hearts. This is the first report of immunoproteasome activity being measured in heart tissue from any animal. Combined proteomic, metabolomic, immunological, and biological results all suggest that pathways associated with energy metabolism, protein degradation, and oxidant defense are major pathways that are altered. These studies show increased levels of enzymes associated with energy metabolism in I79N hearts and suggest I79N hearts favor glucose as a substrate for energy metabolism whereas R278C hearts favor fatty acids. Thus our findings suggest altered metabolism and proteasome function are likely important molecular mechanisms involved in TnT-related FHC. Urea, DTT, triethylphosphine, iodoethanol, and ammonium bicarbonate (NH4HCO3) were purchased from Sigma-Aldrich (St. Louis, MO). LC-MS grade 0.1% formic acid in ACN and 0.1% formic acid in water (H2O) were purchased from Burdick & Jackson (Muskegon, MI). Modified sequencing grade porcine trypsin was obtained from Princeton Separations (Freehold, NJ). All experiments involving animals were approved by the University of California, Davis Institutional Animal Care and Use Committee. Transgenic TnT mouse lines generated using the background mouse strain BL6SJF1/J were used (13.Hernandez O.M. Szczesna-Cordary D. Knollmann B.C. Miller T. Bell M. Zhao J. Sirenko S.G. Diaz Z. Guzman G. Xu Y. Wang Y. Kerrick W.G. Potter J.D. F110I and R278C troponin T mutations that cause familial hypertrophic cardiomyopathy affect muscle contraction in transgenic mice and reconstituted human cardiac fibers.J.Biol. Chem. 2005; 280: 37183-37194Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 14.Miller T. Szczesna D. Housmans P.R. Zhao J. de Freitas F. Gomes A.V. Culbreath L. McCue J. Wang Y. Xu Y. Kerrick W.G. Potter J.D. Abnormal contractile function in transgenic mice expressing a familial hypertrophic cardiomyopathy-linked troponin T (I79N) mutation.J. Biol. Chem. 2001; 276: 3743-3755Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Transgenic male mice were bred with B6SJLF1/J female mice purchased from the Jackson Laboratory (Sacramento, CA). Mice were fed standard chow and water ad libitum and maintained on a 12 h dark/light cycle. Male offspring were used in all studies. DNA was isolated from tail snips taken from mice at the time of weaning or sacrifice for genotyping. Transgenic mice were identified by performing a PCR reaction with primers to the 630-base pair 3′-untranslated region from the human growth hormone transcript that is included in the transgene insert (forward primer CTC CTG GCC CTG GAA GTT; reverse primer CTG GCC AAT ATG GTG AAA CC) (13.Hernandez O.M. Szczesna-Cordary D. Knollmann B.C. Miller T. Bell M. Zhao J. Sirenko S.G. Diaz Z. Guzman G. Xu Y. Wang Y. Kerrick W.G. Potter J.D. F110I and R278C troponin T mutations that cause familial hypertrophic cardiomyopathy affect muscle contraction in transgenic mice and reconstituted human cardiac fibers.J.Biol. Chem. 2005; 280: 37183-37194Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 14.Miller T. Szczesna D. Housmans P.R. Zhao J. de Freitas F. Gomes A.V. Culbreath L. McCue J. Wang Y. Xu Y. Kerrick W.G. Potter J.D. Abnormal contractile function in transgenic mice expressing a familial hypertrophic cardiomyopathy-linked troponin T (I79N) mutation.J. Biol. Chem. 2001; 276: 3743-3755Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Three month old transgenic TnT mice were used in these studies, an age equivalent to young adulthood in humans. Mice were euthanized with isoflurane (3%), followed by cervical dislocation, and hearts were immediately exercised and briefly washed with ice-cold PBS, weighed, and flash frozen. Hearts prepared using this method has been previously shown to be excellent for subsequent proteomic investigation (27.Gomes A.V. Young G.W. Wang Y. Zong C. Eghbali M. Drews O. Lu H. Stefani E. Ping P. Contrasting proteome biology and functional heterogeneity of the 20 S proteasome complexes in mammalian tissues.Mol. Cell. Proteomics. 2009; 8: 302-315Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 28.Gomes A.V. Zong C. Edmondson R.D. Li X. Stefani E. Zhang J. Jones R.C. Thyparambil S. Wang G.W. Qiao X. Bardag-Gorce F. Ping P. Mapping the murine cardiac 26S proteasome complexes.Circulation Res. 2006; 99: 362-371Crossref PubMed Scopus (147) Google Scholar). To 20 mg of pulverized aged-matched heart muscle each from 3 WT, 4 R278C, and 4 I79N mice 300 μl of 8 m urea was added. As described by Sengupta et al. (29.Sengupta D. Byrum S.D. Avaritt N.L. Davis L. Shields B. Mahmoud F. Reynolds M. Orr L.M. Mackintosh S.G. Shalin S.C. Tackett A.J. Quantitative Histone Mass Spectrometry identifies elevated Histone H3 Lysine 27 Trimethylation in Melanoma.Mol. Cell. Proteomics. 2015; PubMed Google Scholar), a minimum of triplicate biological samples with a significance level of 0.05, a 30% standard deviation, and large effect size, is needed to give a power of 0.69. Each tissue was treated by light sonication and mixing for 1 h followed by centrifugation at 13,000 rpm for 10 min. The protein concentration was measured by Bradford assay (30.Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215632) Google Scholar). 20 μl (100 μg) was removed and 20 μl 100 mm ammonium carbonate, pH 10.8 and 1 ng chicken lysozyme (to serve as an internal standard) were added to the samples. 40 μl of reduction/alkylation mixture (97.5% ACN, 2% iodoethanol and 0.5% triethylphosphine) was added to each sample, and the samples were incubated in a 37 °C incubator for 1.5 h. The samples were speed vacuumed to dryness overnight, and the dry pellets were resuspended in 50 μl ammonium bicarbonate. 2.5 μg trypsin in 100 μl ammonium bicarbonate was added to each sample, and they were incubated at 37 °C for 4 h. 2.5 μg trypsin in 50 μl ammonium bicarbonate was then added to each sample, and they were incubated at 37 °C overnight. The digested samples were analyzed using a Thermo Scientific Orbitrap Velos Pro mass spectrometer coupled with a Surveyor autosampler and MS HPLC system (Thermo Fisher Scientific, Waltham, MA). Each biological sample was run two independent times. Tryptic peptides (K/R cleavages) were injected onto a C18 reversed phase column (TSKgel ODS-100V, 3 μm, 1.0 mm × 150 mm) at a flow rate of 50 μl/min. The mobile phases A and B were LC-MS grade H2O with 0.1% formic acid and ACN with 0.1% formic acid, respectively. The gradient elution profile was as follows: 5% B for 5 min, 10–35% B for 150 min, 35–80% B for 10 min, 80% B for 10 min, and 5% B for 5 min. The data were collected in the “Data dependent MS/MS” mode of FT-IT (MS-MS/MS) with the ESI interface using normalized collision energy of 35% (CID). Dynamic exclusion settings were set to repeat count 1, repeat duration 30 s, exclusion duration 120 s, and exclusion mass width 10 ppm (low) and 10 ppm (high). The acquired data were searched against the UniProt protein sequence database of MOUSE (released on 03/19/2014) using X!Tandem algorithms in the Trans-Proteomic Pipeline (TPP, v. 4.6.3) (http://tools.proteomecenter.org/software.php). The Uniprot database used contained 102,778 entries including the decoy sequences (51,389 entries without decoys). General parameters were set to: parent monoisotopic mass error set as 10 ppm, cleavage semi set as yes, missed cleavage sites set at 2, static modification set as + 44.026215 Da on cysteine and no variable modifications. Although not including modifications reduced the number of peptides detected it also prevents ambiguous modification site assignments. A mass tolerance of 0.8 Da for fragment ion was used and only trypsin as a known contaminant was excluded in the analysis. The threshold score/expectation value for accepting individual spectra was the default value in the X!Tandem program. The searched peptides and proteins were validated by PeptideProphet (31.Ma K. Vitek O. Nesvizhskii A.I. A statistical model-building perspective to identification of MS/MS spectra with PeptideProphet.BMC Bioinformatics. 2012; 13: S1Crossref PubMed Scopus (76) Google Scholar) and ProteinProphet (32.Nesvizhskii A.I. Keller A. Kolker E. Aebersold R. A statistical model for identifying proteins by tandem mass spectrometry.Anal. Chem. 2003; 75: 4646-4658Crossref PubMed Scopus (3621) Google Scholar) in the TPP. Only proteins and peptides with protein probability ≥0.9000 and peptide probability ≥0.8000 were reported. Protein quantification was performed using a label-free quantification software package, IdentiQuantXLTM (33.Lai X. Wang L. Tang H. Witzmann F.A. A novel alignment method and multiple filters for exclusion of unqualified peptides to enhance label-free quantification using peptide intensity in LC-MS/MS.J. Proteome Res. 2011; 10: 4799-4812Crossref PubMed Scopus (43) Google Scholar). Using this IdentiQuantXLTM method complex biological samples containing identical amounts of spiked protein standard showed a coefficient of variation (CV) of 8.6% across eight injections between two groups (33.Lai X. Wang L. Tang H. Witzmann F.A. A novel alignment method and multiple filters for exclusion of unqualified peptides to enhance label-free quantification using peptide intensity in LC-MS/MS.J. Proteome Res. 2011; 10: 4799-4812Crossref PubMed Scopus (43) Google Scholar). This program individually aligns the retention time of each peptide and applies multiple filters for exclusion of unqualified peptides to enhance label-free protein quantification. Briefly, the protein ID, sequence, charge, m/z, scan time, injection, sample, and group information are extracted and collected for each peptide, and peptides with identical sequence and charge are considered as a single peptide entry for further analysis (33.Lai X. Wang L. Tang H. Witzmann F.A. A novel alignment method and multiple filters for exclusion of unqualified peptides to enhance label-free quantification using peptide intensity in LC-MS/MS.J. Proteome Res. 2011; 10: 4799-4812Crossref PubMed Scopus (43) Google Scholar). After peptide frequency is calculated, the first filter removes peptides that were not identified in at least three different biological replicates. Alignment to determine peptide retention time was carried out using clustering. Peptide intensity extraction was carried out using “Acquisition time (minutes)” and “Limit the search to only custom m/z values (ignoring auto-fragmented m/z's)” under Custom SIC Options in the program MASIC (33.Lai X. Wang L. Tang H. Witzmann F.A. A novel alignment method and multiple filters for exclusion of unqualified peptides to enhance label-free quantification using peptide intensity in LC-MS/MS.J. Proteome Res. 2011; 10: 4799-48" @default.
• W2327151570 created "2016-06-24" @default.
• W2327151570 creator A5001715587 @default.
• W2327151570 creator A5028081064 @default.
• W2327151570 creator A5053680463 @default.
• W2327151570 creator A5072698091 @default.
• W2327151570 date "2016-06-01" @default.
• W2327151570 modified "2023-10-10" @default.
• W2327151570 title "Delineation of Molecular Pathways Involved in Cardiomyopathies Caused by Troponin T Mutations" @default.
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