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• W2113784998 abstract "Huntington disease (HD) is an autosomal dominant neurodegenerative disease that results from a CAG (glutamine) trinucleotide expansion in exon 1 of huntingtin (Htt). The aggregation of mutant Htt has been implicated in the progression of HD. The earliest degeneration occurs in the striatum. To identify proteins critical for the progression of HD, we applied acid-cleavable ICAT technology to quantitatively determine changes in protein expressions in the striatum of a transgenic HD mouse model (R6/2). The cysteine residues of striatal proteins from HD and wild-type mice were labeled, respectively, with the heavy and light forms of the ICAT reagents. Samples were trypsinized, uncovered by avidin affinity chromatography, and analyzed by nano-LC-MS/MS. Western blot analyses were used to confirm and to calibrate the ICAT ratios. Linear regression was used to uncover a group of proteins that exhibited consistent changes. In two independent ICAT experiments, we identified 427 cysteine-containing striatal proteins among which ∼66% (203 proteins) were detected in both ICAT experiments. Approximately two-thirds of proteins identified in each ICAT experiment were detected in both ICAT experiments. In total, 68 proteins with altered expressions in HD mice were identified. Elevated expressions of two down-regulated proteins (14-3-3ς and FKBP12) effectively reduced Htt aggregates in a striatal cell line, supporting the functional relevance of the above findings. Collectively by using a well defined protocol for data analysis, large scale comparisons of protein expressions by ICAT can be reliable and can provide valuable clues for identifying proteins critical for pathophysiological functions. Huntington disease (HD) is an autosomal dominant neurodegenerative disease that results from a CAG (glutamine) trinucleotide expansion in exon 1 of huntingtin (Htt). The aggregation of mutant Htt has been implicated in the progression of HD. The earliest degeneration occurs in the striatum. To identify proteins critical for the progression of HD, we applied acid-cleavable ICAT technology to quantitatively determine changes in protein expressions in the striatum of a transgenic HD mouse model (R6/2). The cysteine residues of striatal proteins from HD and wild-type mice were labeled, respectively, with the heavy and light forms of the ICAT reagents. Samples were trypsinized, uncovered by avidin affinity chromatography, and analyzed by nano-LC-MS/MS. Western blot analyses were used to confirm and to calibrate the ICAT ratios. Linear regression was used to uncover a group of proteins that exhibited consistent changes. In two independent ICAT experiments, we identified 427 cysteine-containing striatal proteins among which ∼66% (203 proteins) were detected in both ICAT experiments. Approximately two-thirds of proteins identified in each ICAT experiment were detected in both ICAT experiments. In total, 68 proteins with altered expressions in HD mice were identified. Elevated expressions of two down-regulated proteins (14-3-3ς and FKBP12) effectively reduced Htt aggregates in a striatal cell line, supporting the functional relevance of the above findings. Collectively by using a well defined protocol for data analysis, large scale comparisons of protein expressions by ICAT can be reliable and can provide valuable clues for identifying proteins critical for pathophysiological functions. Huntington disease (HD) 1The abbreviations used are: HD, Huntington disease; Htt, Huntingtin; PKC, protein kinase C; FKBP12, FK506-binding protein, 12 kDa; PrxV, peroxiredoxin 5; CSPG, chondroitin sulfate proteoglycan protein; CREB, cAMP-response element-binding protein; WT, wild-type; RSD, relative standard deviation; hrGFP, humanized Renilla green fluorescent protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; 2DGE, two-dimensional gel electrophoresis; PDE, phosphodiesterase. is a hereditary neurodegenerative disease characterized by dementia, chorea, and psychiatric symptoms. The causative mutation is a CAG (glutamine) trinucleotide expansion in exon 1 of the huntingtin (Htt) gene. The normal Htt gene has 35 or fewer repeats in the N-terminal region, whereas the appearance of neurological symptoms is associated with 36 or more CAG repeats (1The Huntington’s Disease Collaborative Research GroupA novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington’s disease chromosomes. The Huntington’s Disease Collaborative Research Group.Cell. 1993; 72: 971-983Abstract Full Text PDF PubMed Scopus (7118) Google Scholar). The major characteristic of HD is regional degeneration of neurons in the striatum and cortex that leads to movement disorders and dementia (2Martin J.B. Gusella J.F. Huntington’s disease. Pathogenesis and management.N. Engl. J. Med. 1986; 315: 1267-1276Crossref PubMed Scopus (17) Google Scholar, 3Vonsattel J.P. Myers R.H. Stevens T.J. Ferrante R.J. Bird E.D. Richardson Jr., E.P. Neuropathological classification of Huntington’s disease.J. Neuropathol. Exp. Neurol. 1985; 44: 559-577Crossref PubMed Scopus (2093) Google Scholar). The toxicity of Htt in specific neurons correlates with the length of polyglutamine expansion (4Davies S.W. Turmaine M. Cozens B.A. DiFiglia M. Sharp A.H. Ross C.A. Scherzinger E. Wanker E.E. Mangiarini L. Bates G.P. Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation.Cell. 1997; 90: 537Abstract Full Text Full Text PDF PubMed Scopus (1918) Google Scholar). The aggregate formation of mutant Htt with poly(Q) expansion causes a wide variety of dysfunctions (5Reddy P.H. Williams M. Tagle D.A. Recent advances in understanding the pathogenesis of Huntington’s disease.Trends Neurosci. 1999; 22: 248-255Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 6DiFiglia M. Sapp E. Chase K.O. Davies S.W. Bates G.P. Vonsattel J.P. Aronin N. Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain.Science. 1997; 277: 1990-1993Crossref PubMed Scopus (2331) Google Scholar). For example, insufficient protein degradation has been proposed as playing a major role (7Waelter S. Boeddrich A. Lurz R. Scherzinger E. Lueder G. Lehrach H. Wanker E.E. Accumulation of mutant huntingtin fragments in aggresome-like inclusion bodies as a result of insufficient protein degradation.Mol. Biol. Cell. 2001; 12: 1393-1407Crossref PubMed Scopus (532) Google Scholar). Htt aggregates were found to recruit components of protein folding and proteolytic pathways and therefore may suppress functions of the proteasome and heat shock proteins (8Jana N.R. Tanaka M. Wang G.-h. Nukina N. Polyglutamine length-dependent interaction of Hsp40 and Hsp70 family chaperones with truncated N-terminal huntingtin: their role in suppression of aggregation and cellular toxicity.Hum. Mol. Genet. 2000; 9: 2009-2018Crossref PubMed Scopus (364) Google Scholar, 9Wyttenbach A. Swartz J. Kita H. Thykjaer T. Carmichael J. Bradley J. Brown R. Maxwell M. Schapira A. Orntoft T.F. Kato K. Rubinsztein D.C. Polyglutamine expansions cause decreased CRE-mediated transcription and early gene expression changes prior to cell death in an inducible cell model of Huntington’s disease.Hum. Mol. Genet. 2001; 10: 1829-1845Crossref PubMed Google Scholar, 10Wyttenbach A. Carmichael J. Swartz J. Furlong R.A. Narain Y. Rankin J. Rubinsztein D.C. Effects of heat shock, heat shock protein 40 (HDJ-2), and proteasome inhibition on protein aggregation in cellular models of Huntington’s disease.Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2898-2903Crossref PubMed Scopus (306) Google Scholar, 11Suhr S.T. Senut M.-C. Whitelegge J.P. Faull K.F. Cuizon D.B. Gage F.H. Identities of sequestered proteins in aggregates from cells with induced polyglutamine expression.J. Cell Biol. 2001; 153: 283-294Crossref PubMed Scopus (189) Google Scholar). In addition, transcriptional dysfunction caused by mutant Htt is critical for polyglutamine diseases (12Okazawa H. Polyglutamine diseases: a transcription disorder?.Cell. Mol. Life Sci. 2003; 60: 1427-1439Crossref PubMed Scopus (87) Google Scholar, 13Okazawa H. Rich T. Chang A. Lin X. Waragai M. Kajikawa M. Enokido Y. Komuro A. Kato S. Shibata M. Interaction between mutant ataxin-1 and PQBP-1 affects transcription and cell death.Neuron. 2002; 34: 701Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Mutant Htt with poly(Q) expansion was shown to sequester and/or interfere with proteins important for the transcriptional machinery including p53, CREB, CREB-binding protein, TAFII130, and SP1 (14Steffan J.S. Kazantsev A. Spasic-Boskovic O. Greenwald M. Zhu Y.Z. Gohler H. Wanker E.E. Bates G.P. Housman D.E. Thompson L.M. The Huntington’s disease protein interacts with p53 and CREB-binding protein and represses transcription.Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6763-6768Crossref PubMed Scopus (880) Google Scholar, 15Nucifora Jr., F.C. Sasaki M. Peters M.F. Huang H. Cooper J.K. Yamada M. Takahashi H. Tsuji S. Troncoso J. Dawson V.L. Dawson T.M. Ross C.A. Interference by huntingtin and atrophin-1 with cbp-mediated transcription leading to cellular toxicity.Science. 2001; 291: 2423-2428Crossref PubMed Scopus (946) Google Scholar, 16Dunah A.W. Jeong H. Griffin A. Kim Y.M. Standaert D.G. Hersch S.M. Mouradian M.M. Young A.B. Tanese N. Krainc D. Sp1 and TAFII130 transcriptional activity disrupted in early Huntington’s disease.Science. 2002; 296: 2238-2243Crossref PubMed Scopus (588) Google Scholar, 17Kegel K.B. Meloni A.R. Yi Y. Kim Y.J. Doyle E. Cuiffo B.G. Sapp E. Wang Y. Qin Z.H. Chen J.D. Nevins J.R. Aronin N. DiFiglia M. Huntingtin is present in the nucleus, interacts with the transcriptional corepressor C-terminal binding protein, and represses transcription.J. Biol. Chem. 2002; 277: 7466-7476Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 18Li S.H. Cheng A.L. Zhou H. Lam S. Rao M. Li H. Li X.J. Interaction of Huntington disease protein with transcriptional activator Sp1.Mol. Cell. Biol. 2002; 22: 1277-1287Crossref PubMed Scopus (284) Google Scholar, 19Chiang M.-C. Lee Y.-C. Huang C.-L. Chern Y. cAMP-response element-binding protein contributes to suppression of the A2A adenosine receptor promoter by mutant huntingtin with expanded polyglutamine residues.J. Biol. Chem. 2005; 280: 14331-14340Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). These changes are specific because microarray analyses have revealed that expressions of a great number of other genes are not altered (20Luthi-Carter R. Strand A. Peters N.L. Solano S.M. Hollingsworth Z.R. Menon A.S. Frey A.S. Spektor B.S. Penney E.B. Schilling G. Ross C.A. Borchelt D.R. Tapscott S.J. Young A.B. Cha J.-H. J. Olson J.M. Decreased expression of striatal signaling genes in a mouse model of Huntington’s disease.Hum. Mol. Genet. 2000; 9: 1259-1271Crossref PubMed Scopus (625) Google Scholar). It should be noted that the mechanism underlying the toxicity caused by mutant Htt remains largely controversial (21Martindale D. Hackam A. Wieczorek A. Ellerby L. Wellington C. McCutcheon K. Singaraja R. Kazemi-Esfarjani P. Devon R. Kim S.U. Bredesen D.E. Tufaro F. Hayden M.R. Length of huntingtin and its polyglutamine tract influences localization and frequency of intracellular aggregates.Nat. Genet. 1998; 18: 150-154Crossref PubMed Scopus (422) Google Scholar). Although aggregation is correlated with HD pathogenesis, and many beneficial treatments reduce aggregate formation (22Tanaka M. Machida Y. Niu S. Ikeda T. Jana N.R. Doi H. Kurosawa M. Nekooki M. Nukina N. Trehalose alleviates polyglutamine-mediated pathology in a mouse model of Huntington disease.Nat. Med. 2004; 10: 148-154Crossref PubMed Scopus (650) Google Scholar, 23Chou S.Y. Lee Y.C. Chen H.M. Chiang M.C. Lai H.L. Chang H.H. Wu Y.C. Sun C.N. Chien C.L. Lin Y.S. Wang S.C. Tung Y.Y. Chang C. Chern Y. CGS21680 attenuates symptoms of Huntington’s disease in a transgenic mouse model.J. Neurochem. 2005; 93: 310-320Crossref PubMed Scopus (162) Google Scholar), evidence from different laboratories suggests that aggregate formation might confer protective effects against the toxicity induced by soluble mutant Htt with poly(Q) expansion (24Arrasate M. Mitra S. Schweitzer E.S. Segal M.R. Finkbeiner S. Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death.Nature. 2004; 431: 805-810Crossref PubMed Scopus (1627) Google Scholar, 25Gauthier L.R. Charrin B.C. Borrell-Pages M. Dompierre J.P. Rangone H. Cordelieres F.P. De Mey J. MacDonald M.E. Lessmann V. Humbert S. Saudou F. Huntingtin controls neurotrophic support and survival of neurons by enhancing BDNF vesicular transport along microtubules.Cell. 2004; 118: 127-138Abstract Full Text Full Text PDF PubMed Scopus (923) Google Scholar). Consistent with the above hypothesis, Schaffar et al. (26Schaffar G. Breuer P. Boteva R. Behrends C. Tzvetkov N. Strippel N. Sakahira H. Siegers K. Hayer-Hartl M. Hartl F.U. Cellular toxicity of polyglutamine expansion proteins: mechanism of transcription factor deactivation.Mol. Cell. 2004; 15: 95-105Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar) demonstrated that monomers or small soluble oligomers of poly(Q)-expanded mutant Htt were sufficient to inactivate TATA box-binding protein, an important transcription factor. Collectively the role of Htt aggregates at different stages of HD is complex and remains to be further elucidated. Present large scale analyses of biochemical changes in HD have mostly been focused at the level of genes. Only a handful of proteomics analyses of brain proteins using two-dimensional gel electrophoresis-based techniques have been conducted on HD (27Perluigi M. Poon H.F. Maragos W. Pierce W.M. Klein J.B. Calabrese V. Cini C. De Marco C. Butterfield D.A. Proteomic analysis of protein expression and oxidative modification in R6/2 transgenic mice: a model of Huntington disease.Mol. Cell. Proteomics. 2005; 4: 1849-1861Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). We thus set out to examine the global protein expression profiles in the striatum of HD mice using a quantitative proteomics approach (ICAT). This method uses two labeling reagents whose weights are only 8 Da apart for comparative studies (28Gygi 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 (4362) Google Scholar, 29Link A.J. Eng J. Schieltz D.M. Carmack E. Mize G.J. Morris D.R. Garvik B.M. Yates J.R. Direct analysis of protein complexes using mass spectrometry.Nat. Biotechnol. 1999; 17: 676-682Crossref PubMed Scopus (2075) Google Scholar). Recently an acid-cleavable ICAT reagent has begun to be used that avoids several shortcomings of the first generation ICAT reagent (30Hansen 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 (276) Google Scholar, 31Zhang R. Sioma C.S. Wang S. Regnier F.E. Fractionation of isotopically labeled peptides in quantitative proteomics.Anal. Chem. 2001; 73: 5142-5149Crossref PubMed Scopus (247) Google Scholar). In the present study, 427 cysteine-containing striatal proteins were detected from two independent ICAT experiments. Western blot analyses were used to confirm and to calibrate the ICAT data followed by a linear regression analysis to remove the outliers. In total, changes in the expressions of 68 proteins were found. From this it was determined that 6% (four proteins) were up-regulated, whereas 94% (64 proteins) were down-regulated. Among them, disease stage-dependent alterations of 10 proteins in the striatum of HD mice were investigated using Western blot analysis. The functional relevance of two down-regulated proteins (14-3-3ς and FKBP12) was also demonstrated by their abilities to reduce Htt aggregates. Our study shows that large scale proteomics analysis using ICAT is a reliable approach for systematically identifying proteins critical for the development of HD. All reagents were purchased from Sigma except where specified. Dulbecco's modified Eagle's medium and fetal bovine serum were obtained from Invitrogen. Male R6/2 mice and littermate controls were originally obtained from The Jackson Laboratory (Bar Harbor, ME) and were mated to female control mice (B6CBAFI/J). Offspring were identified by a PCR genotyping technique described elsewhere (23Chou S.Y. Lee Y.C. Chen H.M. Chiang M.C. Lai H.L. Chang H.H. Wu Y.C. Sun C.N. Chien C.L. Lin Y.S. Wang S.C. Tung Y.Y. Chang C. Chern Y. CGS21680 attenuates symptoms of Huntington’s disease in a transgenic mouse model.J. Neurochem. 2005; 93: 310-320Crossref PubMed Scopus (162) Google Scholar). In total, 27 R6/2 transgenic mice and 25 wild-type (WT) littermate control mice were used in this study. Animals were housed at the Institute of Biomedical Sciences Animal Care Facility under a 12-h light/dark cycle. Animal experiments were performed using protocols approved by the Academia Sinica Institutional Animal Care and Utilization Committee, Taiwan. In total, 27 R6/2 transgenic mice and 25 WT littermate mice were used in this study. For each preparation, striatal tissues from two to six mice were removed, resuspended in 1–2 ml of ice-cold buffer A (10 mm Hepes (pH 8), 1.5 mm MgCl2, 10 mm KCl, 0.5 mm dithiothreitol, 1 mm Na3VO4, 20 mm NaF, 100 nm okadaic acid, and 0.5% Nonidet P-40), and homogenized (Dounce, 10 strokes) in buffer A. Lysates were first centrifuged at 367 × g for 1 min at 4 °C to remove the debris and then centrifuged at 2292 × g for 5 min at 4 °C to collect the pellets as the nucleus-enriched fractions and the supernatants as the cytosolic fractions. The pellets were resuspended in 0.5–1 ml of buffer B (20 mm Hepes (pH 8), 425 mm NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 0.5 mm dithiothreitol, 80 mg/ml phenylmethylsulfonyl fluoride, 1 mm Na3VO4, 20 mm NaF, 100 nm okadaic acid, and 25% glycerol) and incubated for 60 min on ice. Protein extracts were next collected by centrifugation at 19,275 × g for 5 min at 4 °C to remove the pellets. The protein concentration was measured using the Bio-Rad protein assay reagent. This protocol was adopted from earlier studies using the same mouse model of HD at similar disease stages (32Scherzinger E. Lurz R. Turmaine M. Mangiarini L. Hollenbach B. Hasenbank R. Bates G.P. Davies S.W. Lehrach H. Wanker E.E. Huntingtin-encoded polyglutamine expansions form amyloid-like protein aggregates in vitro and in vivo.Cell. 1997; 90: 549-558Abstract Full Text Full Text PDF PubMed Scopus (1090) Google Scholar) or cell models with large aggregates (19Chiang M.-C. Lee Y.-C. Huang C.-L. Chern Y. cAMP-response element-binding protein contributes to suppression of the A2A adenosine receptor promoter by mutant huntingtin with expanded polyglutamine residues.J. Biol. Chem. 2005; 280: 14331-14340Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The majority of Htt aggregates were detected in the nucleus-enriched fraction, not in the cytosolic fraction or the debris (Supplemental Figs. S2 and S3). In addition, Western blot analyses using antibodies against a nuclear marker (lamin A/C) and a cytosolic marker (α-tubulin) demonstrated the successful enrichment of the nuclear fraction. Only slight contamination of the cytosolic fraction was observed (Supplemental Fig. S1). From striatal tissues of one mouse, ∼0.4 mg of nucleus-enriched proteins at a concentration of 0.9–1 mg/ml was collected. In total, 12 R6/2 mice and 10 WT littermate control mice (at 10.5 weeks old) were used in two independent ICAT analyses. The nucleus-enriched protein fractions (1 mg) were prepared as described above and labeled with cleavable ICAT reagents (Applied Biosystems, Foster City, CA) following the manufacturer's protocol. Briefly striatal proteins collected from WT and HD mice were labeled, respectively, with isotopically light and heavy ICAT reagents. The labeled preparations were combined and digested with trypsin (Promega, Madison, WI) overnight at 37 °C using an enzyme-to-protein ratio of 1:50 (w/w). The resulting peptide mixture was fractionated by three-step chromatography. Samples were first separated using a Polysulfoethyl A column (Poly LC, Columbia, MD; 2.1 mm × 20 cm) at a flow rate of 200 μl/min. Peptides were eluted with 100% buffer A (5 mm KH2PO4 and 25% acetonitrile, pH 3.0) for 2 min followed by a linear gradient from buffer A to buffer B (5 mm KH2PO4, 350 mm KCl, and 25% acetonitrile, pH 3.0) over 48 min. Fractions were collected (one fraction/min) and subjected to affinity purification using a monomeric avidin cartridge (Applied Biosystems). The purified samples were then treated with acid to cleave the tag and separated by reverse-phase capillary liquid chromatography (Magic C18AQ, Michrom Bioresources, Auburn, CA; 75 μm × 11 cm) at a flow rate of 200 nl/min. The eluent was directly analyzed by ion trap mass spectrometry (LCQ Deca XP, Thermo Finnigan, San Jose, CA) under conditions described elsewhere (33Yi E.C. Lee H. Aebersold R. Goodlett D.R. A microcapillary trap cartridge-microcapillary high-performance liquid chromatography electrospray ionization emitter device capable of peptide tandem mass spectrometry at the attomole level on an ion trap mass spectrometer with automated routine operation.Rapid Commun. Mass Spectrom. 2003; 17: 2093-2098Crossref PubMed Scopus (95) Google Scholar). A survey scan followed by three CID events was used. The tolerances of the precursor ion and fragment ion were 2 and 1.5 amu, respectively. Peptide identification by CID was carried out in automated mode using the 3-min dynamic exclusion option. ICAT-labeled peptides were first analyzed using SEQUEST in Bioworks 3.1 (Thermo Finnigan) (34Eng J.K. McCormack A.L. Yates III, J.R. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database.J. Am. Soc. Mass Spectrom. 1994; 5: 976-989Crossref PubMed Scopus (5472) Google Scholar). The tolerances of the precursor ion and fragment ion were the same as described above. The database was downloaded from the website of the National Center for Biotechnology Information (NCBI; mouse; April 12, 2004; 84,599 entries) and then analyzed by Peptide Prophet Version 1.0 (35Keller A. Nesvizhskii A.I. Kolker E. Aebersold R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search.Anal. Chem. 2002; 74: 5383-5392Crossref PubMed Scopus (3912) Google Scholar), INTERACT_15-10-2004 (36Han D.K. Eng J. Zhou H. Aebersold R. Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry.Nat. Biotechnol. 2001; 19: 946-951Crossref PubMed Scopus (829) Google Scholar), and Protein Prophet.pl Version 2.0 (37Nesvizhskii 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 (3655) Google Scholar). Peptide prophet was used to increase the accuracy of peptide identification, and Protein Prophet was used to reduce the redundancy of the search results. The cutoff scores of Peptide Prophet and Protein Prophet were ≥0.9. The false-positive error rate for both ICAT experiments was 0.008. Relative quantification was performed using ASAPRatio 3.0 (38Li X.j. Zhang H. Ranish J.A. Aebersold R. Automated statistical analysis of protein abundance ratios from data generated by stable-isotope dilution and tandem mass spectrometry.Anal. Chem. 2003; 75: 6648-6657Crossref PubMed Scopus (318) Google Scholar). The above four software packages were kindly provided by Institute for Systems Biology Proteomics Windows Software Distribution. The resulting peptide spectra of proteins were manually checked for qualitative and quantitative results. For protein identification, the peptide sequences were first blasted through the NCBI databases to obtain the corresponding protein sequences, which were then transferred into UniProt to obtained the accession numbers using WU-Blast2 (a tool that effectively finds regions of sequence similarity; www.ebi.ac.uk/blast2). Through the process, 14 proteins were removed due to redundancy or changes in annotation. In three instances where the detected peptides were located in common domains of certain protein families, family names were assigned. The UniProt primary accession numbers of the remaining 203 proteins are listed in Table II and Supplemental Table S2 to minimize the redundancy. Note that as observed in quite a few published studies and summarized in a recent review (88Leitner A. Lindner W. Chemistry meets proteomics: the use of chemical tagging reactions for MS-based proteomics.Proteomics. 2006; 6: 5418-5434Crossref PubMed Scopus (106) Google Scholar), a low number of detected peptides per protein is commonly found in quantitative proteomics approaches using chemical tagging-enrichment strategies. In our ICAT study where cysteine was used to tag the proteins, 66 of 203 proteins (∼33%) were identified by only one peptide (sequence). This is consistent with other studies using chemical tagging-enrichment reactions and does not reflect the quality of our mass data. Names of the proteins for which only one peptide (sequence) was detected in each ICAT experiment are labeled in green (Table II and Supplemental Table S2). Identification of these proteins needs to be validated using orthogonal methods before reaching a conclusion (Table II and Supplemental Table S2).Table IILists of striatal proteins up-regulated or down-regulated in 10.5-week-old HD mice compared with WT mice revealed by ICAT Open table in a new tab Six protein ratios determined by Western blot analysis with a relative standard deviation (RSD) of ≤0.06 were used to calibrate the ICAT ratios as described in detail in the Supplemental Experimental Procedures. Correlations of different batches of samples were calculated using SAS/STAT, Version 8.0 (SAS Institute, Cary, NC). In total, 15 R6/2 mice and 15 WT littermates were used in the Western blot analyses. There were seven and eight mice in the groups that were 7 and 10.5 weeks old, respectively. Three to six independent preparations of nucleus-enriched proteins for each condition were prepared from two to three striatal tissues as described above and used for the Western blot analyses. Equal amounts of protein were separated by SDS-PAGE using 10% polyacrylamide gels according to the method of Laemmli (39Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). The resolved proteins were electroblotted onto Immobilon polyvinylidene difluoride membranes (Millipore, Bedford, MA). Membranes were blocked with 5% skim milk in PBS and incubated with an anti-β-actin antibody (1:2000 dilution; Santa Cruz Biotechnology), anti-PKCβ antibody (1:1000 dilution; Transduction Laboratories, Lexington, KY), anti-14-3-3ς antibody (1:1000 dilution; Santa Cruz Biotechnology), anti-FKBP12 antibody (1:1000 dilution; Santa Cruz Biotechnology), anti-adducin α antibody (1:1000 dilution; Santa Cruz Biotechnology), anti-casein kinase IIβ antibody (1:1000 dilution; Santa Cruz Biotechnology), anti-CSPG antibody (1:1000 dilution; Chemicon International, Temecula, CA), anti-Gβ1 antibody (1:1000 dilution; Santa Cruz Biotechnology), anti-Gγ2 antibody (1:1000 dilution; Santa Cruz Biotechnology), anti-PrxV antibody (1:1000 dilution; Santa Cruz Biotechnology), anti-lamin A/C antibody (1:2000 dilution; Santa Cruz Biotechnology), anti-α-tubulin antibody (1:3000 dilution; Sigma), anti-Htt antibody (1:1000 dilution; Chemicon International), and anti-V5 antibody (1:2500; Invitrogen) at 4 °C overnight followed by the corresponding secondary antibody for 1 h at room temperature. Immunoreactive bands were detected by enhanced chemiluminescence (Pierce) and recorded using Eastman Kodak Co. XAR-5 film. Values for the relative intensities were determined by normalizing the signal of the desired protein with that of the corresponding internal control (lamin A/C) and are expressed as multiples of that of R6/2 mice. Data were generated by quantitative computing densitometry from three to six independent experiments using the image analysis software package ImageQuant Version 3.15 (GE Healthcare). The pcDNA3.1-Htt-(Q)109-hrGFP constructs encoding an N-terminal fragment of Htt with the indicated number of poly(Q) residues fused to hrGFP were created as described elsewhere (19Chiang M.-C. Lee Y.-C. Huang C.-L. Chern Y. cAMP-response element-binding protein contributes to suppression of the A2A adenosine receptor promoter by mutant huntingtin with expanded polyglutamine residues.J. Biol. Chem. 2005; 280: 14331-14340Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Mouse cDNAs of 14-3-3ς (736 bp), FKBP12 (324 bp), and Lim (208 bp) were amplified from a mouse brain cDNA by PCR using primers listed in Supplemental Table S1 and subcloned into the pcDNA3.1/V5-His vector using a TOPO-TA expression kit (Invitrogen). The striatal progenitor cell line (ST14A) was a generous gift from Dr. E. Cattaneo (University of Milan, Milan, Italy) and was maintained in an incubation chamber gassed with 10% CO2 and 90% air at 33 °C as described previously (40Ehrlich M.E. Conti L. Toselli M. Taglietti L. Fiorillo E. Taglietti V. Ivkovic S. Guinea B. Tranberg A. Sipione S. ST14A cells have properties of a medium-size spiny neuron.Exp. Neuro" @default.
• W2113784998 created "2016-06-24" @default.
• W2113784998 creator A5000251736 @default.
• W2113784998 creator A5018776391 @default.
• W2113784998 creator A5038010731 @default.
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• W2113784998 date "2007-05-01" @default.
• W2113784998 modified "2023-10-16" @default.
• W2113784998 title "Systematic Uncovering of Multiple Pathways Underlying the Pathology of Huntington Disease by an Acid-cleavable Isotope-coded Affinity Tag Approach" @default.
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