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Detergent-insoluble Aggregates Associated with Amyotrophic Lateral Sclerosis in Transgenic Mice Contain Primarily Full-length, Unmodified Superoxide Dismutase-1

2008; Elsevier BV; Volume: 283; Issue: 13 Linguagem: Inglês

10.1074/jbc.m707751200

ISSN

1083-351X

Autores

Bryan F. Shaw, Herman Lelie, Armando Durazo, Aram M. Nersissian, Guillan Xu, Pik K. Chan, Edith B. Gralla, Ashutosh Tiwari, Lawrence J. Hayward, David Borchelt, Joan Selverstone Valentine, Julian P. Whitelegge,

Tópico(s)

Alzheimer's disease research and treatments

Resumo

Determining the composition of aggregated superoxide dismutase 1 (SOD1) species associated with amyotrophic lateral sclerosis (ALS), especially with respect to co-aggregated proteins and post-translational modifications, could identify cellular or biochemical factors involved in the formation of these aggregates and explain their apparent neurotoxicity. The results of mass spectrometric and shotgun-proteomic analyses of SOD1-containing aggregates isolated from spinal cords of symptomatic transgenic ALS mice using two different isolation strategies are presented, including 1) resistance to detergent extraction and 2) size exclusion-coupled anti-SOD1 immunoaffinity chromatography. Forty-eight spinal cords from three different ALS-SOD1 mutant mice were analyzed, namely G93A, G37R, and the unnatural double mutant H46R/H48Q. The analysis consistently revealed that the most abundant proteins recovered from aggregate species were full-length unmodified SOD1 polypeptides. Although aggregates from some spinal cord samples contained trace levels of highly abundant proteins, such as vimentin and neurofilament-3, no proteins were consistently found to co-purify with mutant SOD1 in stoichiometric quantities. The results demonstrate that the principal protein in the high molecular mass aggregates whose appearance correlates with symptoms of the disease is the unmodified, full-length SOD1 polypeptide. Determining the composition of aggregated superoxide dismutase 1 (SOD1) species associated with amyotrophic lateral sclerosis (ALS), especially with respect to co-aggregated proteins and post-translational modifications, could identify cellular or biochemical factors involved in the formation of these aggregates and explain their apparent neurotoxicity. The results of mass spectrometric and shotgun-proteomic analyses of SOD1-containing aggregates isolated from spinal cords of symptomatic transgenic ALS mice using two different isolation strategies are presented, including 1) resistance to detergent extraction and 2) size exclusion-coupled anti-SOD1 immunoaffinity chromatography. Forty-eight spinal cords from three different ALS-SOD1 mutant mice were analyzed, namely G93A, G37R, and the unnatural double mutant H46R/H48Q. The analysis consistently revealed that the most abundant proteins recovered from aggregate species were full-length unmodified SOD1 polypeptides. Although aggregates from some spinal cord samples contained trace levels of highly abundant proteins, such as vimentin and neurofilament-3, no proteins were consistently found to co-purify with mutant SOD1 in stoichiometric quantities. The results demonstrate that the principal protein in the high molecular mass aggregates whose appearance correlates with symptoms of the disease is the unmodified, full-length SOD1 polypeptide. Mutations in the gene encoding superoxide dismutase 1 (SOD1) 7The abbreviations used are:SOD1superoxide dismutase 1ALSamyotrophic lateral sclerosisSEsize exclusionLCliquid chromatographyIAimmunoaffinityESIelectrospray ionizationMSmass spectrometryMS/MStandem MSMALDI-TOFmatrix-assisted laser desorption time-of-flightDRPdetergent-resistant pelletDTTdithiothreitolHPLChigh-performance liquid chromatography. 7The abbreviations used are:SOD1superoxide dismutase 1ALSamyotrophic lateral sclerosisSEsize exclusionLCliquid chromatographyIAimmunoaffinityESIelectrospray ionizationMSmass spectrometryMS/MStandem MSMALDI-TOFmatrix-assisted laser desorption time-of-flightDRPdetergent-resistant pelletDTTdithiothreitolHPLChigh-performance liquid chromatography. induce familial amyotrophic lateral sclerosis (ALS), and aggregation of the mutant SOD1 protein is hypothesized to cause pathogenesis (1Shaw B.F. Valentine J.S. Trends Biochem. Sci. 2007; 32: 78-85Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Support for the aggregation hypothesis includes pathological, cell-culture, and biophysical data such as: 1) the appearance of fibrillar, spherical or irregularly shaped SOD1-containing aggregates in murine and human ALS spinal cord (2Wang J. Xu G. Gonzales V. Coonfield M. Fromholt D. Copeland N.G. Jenkins N.A. Borchelt D.R. Neurobiol. Dis. 2002; 10: 128-138Crossref PubMed Scopus (202) Google Scholar, 3Shibata N. Hirano A. Kobayashi M. Siddique T. Deng H.X. Hung W.Y. Kato T. Asayama K. J. Neuropathol. Exp. Neurol. 1996; 55: 481-490Crossref PubMed Scopus (252) Google Scholar, 4Kato S. Nakashima K. Horiuchi S. Nagai R. Cleveland D.W. Liu J. Hirano A. Takikawa M. Kato M. Nakano I. Sakoda S. Asayama K. Ohama E. Neuropathology. 2001; 21: 67-81PubMed Google Scholar, 5Kato S. Horiuchi S. Liu J. Cleveland D.W. Shibata N. Nakashima K. Nagai R. Hirano A. Takikawa M. Kato M. Nakano I. Ohama E. 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Biol. 2003; 332: 601-615Crossref PubMed Scopus (173) Google Scholar) that accelerate aggregation in vitro (1Shaw B.F. Valentine J.S. Trends Biochem. Sci. 2007; 32: 78-85Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). superoxide dismutase 1 amyotrophic lateral sclerosis size exclusion liquid chromatography immunoaffinity electrospray ionization mass spectrometry tandem MS matrix-assisted laser desorption time-of-flight detergent-resistant pellet dithiothreitol high-performance liquid chromatography. superoxide dismutase 1 amyotrophic lateral sclerosis size exclusion liquid chromatography immunoaffinity electrospray ionization mass spectrometry tandem MS matrix-assisted laser desorption time-of-flight detergent-resistant pellet dithiothreitol high-performance liquid chromatography. Although the aggregation of SOD1 is a conspicuous signature of SOD1-linked ALS, the protein composition of SOD1 aggregates remains unclear. Identifying proteins that might be co-aggregated with SOD1 could help explain how the aggregates are formed and why they are apparently toxic. For example, the expression of ALS-SOD1 variants in mice and cell cultures can induce: the slowing of axonal transport (19Stokin G.B. Lillo C. Falzone T.L. Brusch R.G. Rockenstein E. Mount S.L. Raman R. Davies P. Masliah E. Williams D.S. Goldstein L.S. Science. 2005; 307: 1282-1288Crossref PubMed Scopus (970) Google Scholar), glutamate excitotoxicity (20Vermeiren C. Hemptinne I. Vanhoutte N. Tilleux S. Maloteaux J.M. Hermans E. J. Neurochem. 2006; 96: 719-731Crossref PubMed Scopus (59) Google Scholar, 21Tortarolo M. Grignaschi G. Calvaresi N. Zennaro E. Spaltro G. Colovic M. Fracasso C. Guiso G. Elger B. Schneider H. Seilheimer B. Caccia S. Bendotti C. J. Neurosci. Res. 2006; 83: 134-146Crossref PubMed Scopus (99) Google Scholar), and proteasomal inhibition (22Urushitani M. Kurisu J. Tsukita K. Takahashi R. J. 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Therefore, the identification of binding interactions or co-aggregation between SOD1 and specific proteins involved in any of these processes could provide clues about the cellular processes involved in ALS pathogenesis. Another unanswered question pertains to whether the SOD1 polypeptide is pathogenic as it is expressed or if post-translational modifications to SOD1 trigger the polypeptide's pathogenicity. Immunohistochemical analysis of SOD1 inclusions in mouse spinal cord using antibodies that are specific for oxidized proteins have suggested that aggregated SOD1 proteins are extensively modified (e.g. carboxymethylation of lysine residues (4Kato S. Nakashima K. Horiuchi S. Nagai R. Cleveland D.W. Liu J. Hirano A. Takikawa M. Kato M. Nakano I. Sakoda S. Asayama K. Ohama E. Neuropathology. 2001; 21: 67-81PubMed Google Scholar)). Likewise, the self-oxidation of metal coordinating histidine residues by SOD1 has been shown to inactivate SOD1 in vitro (28Rakhit R. Cunningham P. Furtos-Matei A. Dahan S. Qi X.F. Crow J.P. Cashman N.R. Kondejewski L.H. Chakrabartty A. J. Biol. Chem. 2002; 277: 47551-47556Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 29Valentine J.S. Free Radic. Biol. Med. 2002; 33: 1314-1320Crossref PubMed Scopus (62) Google Scholar, 30Valentine J.S. Doucette P.A. Zittin Potter S. Annu. Rev. Biochem. 2005; 74: 563-593Crossref PubMed Scopus (606) Google Scholar, 31Elam J.S. Malek K. Rodriguez J.A. Doucette P.A. Taylor A.B. Hayward L.J. Cabelli D.E. Valentine J.S. Hart P.J. J. Biol. Chem. 2003; 278: 21032-21039Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Such inactivation would be expected to cause metal ion loss and structural destabilization of SOD1, which could promote SOD1 aggregation. Current information regarding the protein composition of aggregated SOD1 and the post-translational state of aggregated SOD1 is based upon immunohistochemical and Western blot analysis of tissue inclusions and detergent-resistant complexes that form in murine ALS spinal cord. For example, in addition to containing SOD1 (3Shibata N. Hirano A. Kobayashi M. Siddique T. Deng H.X. Hung W.Y. Kato T. Asayama K. J. Neuropathol. Exp. Neurol. 1996; 55: 481-490Crossref PubMed Scopus (252) Google Scholar, 6Bruijn L.I. Becher M.W. Lee M.K. Anderson K.L. Jenkins N.A. Copeland N.G. Sisodia S.S. Rothstein J.D. Borchelt D.R. Price D.L. Cleveland D.W. Neuron. 1997; 18: 327-338Abstract Full Text Full Text PDF PubMed Scopus (1113) Google Scholar), the inclusions that are visible by light microscopy in ALS-affected spinal cord tissue are recognized by antibodies to neurofilament components (3Shibata N. Hirano A. Kobayashi M. Siddique T. Deng H.X. Hung W.Y. Kato T. Asayama K. J. Neuropathol. Exp. Neurol. 1996; 55: 481-490Crossref PubMed Scopus (252) Google Scholar, 12Wang J. Xu G. Borchelt D.R. Neurobiol. Dis. 2002; 9: 139-148Crossref PubMed Scopus (175) Google Scholar), ubiquitin (3Shibata N. Hirano A. Kobayashi M. Siddique T. Deng H.X. Hung W.Y. Kato T. Asayama K. J. Neuropathol. Exp. Neurol. 1996; 55: 481-490Crossref PubMed Scopus (252) Google Scholar, 6Bruijn L.I. Becher M.W. Lee M.K. Anderson K.L. Jenkins N.A. Copeland N.G. Sisodia S.S. Rothstein J.D. Borchelt D.R. Price D.L. Cleveland D.W. Neuron. 1997; 18: 327-338Abstract Full Text Full Text PDF PubMed Scopus (1113) Google Scholar, 32Basso M. Massignan T. Samengo G. Cheroni C. De Biasi S. Salmona M. Bendotti C. Bonetto V. J. Biol. Chem. 2006; 281: 33325-33335Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), vimentin (9Johnston J.A. Dalton M.J. Gurney M.E. Kopito R.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12571-12576Crossref PubMed Scopus (511) Google Scholar), heat shock proteins (33Watanabe M. Dykes-Hoberg M. Culotta V.C. Price D.L. Wong P.C. Rothstein J.D. Neurobiol. Dis. 2001; 8: 933-941Crossref PubMed Scopus (344) Google Scholar), Dorfin (34Niwa J. Ishigaki S. Hishikawa N. Yamamoto M. Doyu M. Murata S. Tanaka K. Taniguchi N. Sobue G. J. Biol. Chem. 2002; 277: 36793-36798Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), and NEDL1 (35Miyazaki K. Fujita T. Ozaki T. Kato C. Kurose Y. Sakamoto M. Kato S. Goto T. Itoyama Y. Aoki M. Nakagawara A. J. Biol. Chem. 2004; 279: 11327-11335Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The immunostaining of detergent-insoluble SOD1 aggregates from the spinal cord of ALS mice has suggested that many of the same proteins detected in visible SOD1 inclusions are also contained in detergent-insoluble aggregates (11Wang J. Slunt H. Gonzales V. Fromholt D. Coonfield M. Copeland N.G. Jenkins N.A. Borchelt D.R. Hum. Mol. Genet. 2003; 12: 2753-2764Crossref PubMed Scopus (250) Google Scholar, 32Basso M. Massignan T. Samengo G. Cheroni C. De Biasi S. Salmona M. Bendotti C. Bonetto V. J. Biol. Chem. 2006; 281: 33325-33335Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Consequently, hypotheses have emerged regarding the role of some of these proteins in ALS pathogenesis. Other reports, however, suggest that the co-localization of some of these proteins with SOD1 in inclusion bodies is minimal, inconsistent, and rare, i.e. ubiquitin (9Johnston J.A. Dalton M.J. Gurney M.E. Kopito R.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12571-12576Crossref PubMed Scopus (511) Google Scholar, 32Basso M. Massignan T. Samengo G. Cheroni C. De Biasi S. Salmona M. Bendotti C. Bonetto V. J. Biol. Chem. 2006; 281: 33325-33335Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) and heat shock proteins (36Kabashi E. Durham H.D. Biochim. Biophys. Acta. 2006; 1762: 1038-1050Crossref PubMed Scopus (78) Google Scholar). Immunohistochemical analyses have provided invaluable information on the composition of various types of SOD1 aggregates; however, the limitations to immuno-based analyses involve: 1) the possibility of cross-reactivity or nonspecific binding that produces a false positive identification of proteins; 2) a protein bias; that is, the researcher must know which protein or chemical modification to assay; and 3) the inability to discern if proteins that co-sediment or are co-localized with SOD1 are in fact aggregated with SOD1. In an attempt to directly characterize the SOD1 proteins that aggregate in ALS spinal cord and to broadly identify proteins that might be co-aggregated with SOD1, we have isolated aggregate forms of SOD1 from the spinal cords of transgenic ALS mice and analyzed these aggregate species with mass spectrometry, a technique used recently to analyze SOD1 aggregates formed in vitro (37Banci L. Bertini I. Durazo A. Girotto S. Gralla E.B. Martinelli M. Valentine J.S. Vieru M. Whitelegge J.P. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 11263-11267Crossref PubMed Scopus (192) Google Scholar). Two different methods were used to isolate SOD1-containing aggregates from mouse spinal cord. One method involves detergent extraction and differential centrifugation and has been previously described (11Wang J. Slunt H. Gonzales V. Fromholt D. Coonfield M. Copeland N.G. Jenkins N.A. Borchelt D.R. Hum. Mol. Genet. 2003; 12: 2753-2764Crossref PubMed Scopus (250) Google Scholar, 38Wang J. Xu G. Li H. Gonzales V. Fromholt D. Karch C. Copeland N.G. Jenkins N.A. Borchelt D.R. Hum. Mol. Genet. 2005; 14: 2335-2347Crossref PubMed Scopus (107) Google Scholar). This method isolates aggregates based upon their high molecular mass and resistance to various detergents. It is very possible, however, that a detergent extraction could dissociate meta-stable oligomeric species that might play a role in ALS pathogenesis (39Plakoutsi G. Bemporad F. Calamai M. Taddei N. Dobson C.M. Chiti F. J. Mol. Biol. 2005; 351: 910-922Crossref PubMed Scopus (116) Google Scholar, 40Pieri L. Bucciantini M. Nosi D. Formigli L. Savistchenko J. Melki R. Stefani M. J. Biol. Chem. 2006; 281: 15337-15344Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Therefore we also isolated SOD1-aggregate species from mouse spinal cord using a less harsh, detergent-free method that employs size exclusion liquid chromatography (SE-LC) coupled with anti-SOD1 immunoaffinity liquid chromatography (IA-LC). Analysis of the isolated aggregate species with electrospray ionization mass spectrometry (ESI-MS) and matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry has revealed that the detergent resistant SOD1 aggregates are composed predominantly of the full-length, non-ubiquitinated SOD1 polypeptide that is bearing no covalent or oxidative modifications. With the exception of trace amounts of highly abundant proteins, there were no other proteins that were found to be consistently co-aggregated with SOD1. Transgenic Mice—Transgenic mice expressing hWT, G93A, and G37R SOD1 and the double mutant mouse, expressing H46R/H48Q ALS SOD1 mutations were sacrificed, and their spinal cords were harvested as previously described (2Wang J. Xu G. Gonzales V. Coonfield M. Fromholt D. Copeland N.G. Jenkins N.A. Borchelt D.R. Neurobiol. Dis. 2002; 10: 128-138Crossref PubMed Scopus (202) Google Scholar, 11Wang J. Slunt H. Gonzales V. Fromholt D. Coonfield M. Copeland N.G. Jenkins N.A. Borchelt D.R. Hum. Mol. Genet. 2003; 12: 2753-2764Crossref PubMed Scopus (250) Google Scholar). In short, mice were sacrificed at the end stage of motor neuron degeneration when hind limb paralysis was pronounced. Non-transgenic mice and hWT SOD1 transgenic mice were age-matched with ALS mice when sacrificed. A total of 75 murine spinal cords were analyzed in this study: 12 G93A, 27 H46R/H48Q, 18 G37R, 9 hWT, and 9 non-transgenic. Detergent Extraction and Sedimentation of Aggregates from ALS Murine Spinal Cord—Detergent extraction and differential centrifugation of high molecular mass, detergent-stable complexes was performed as previously described (11Wang J. Slunt H. Gonzales V. Fromholt D. Coonfield M. Copeland N.G. Jenkins N.A. Borchelt D.R. Hum. Mol. Genet. 2003; 12: 2753-2764Crossref PubMed Scopus (250) Google Scholar, 38Wang J. Xu G. Li H. Gonzales V. Fromholt D. Karch C. Copeland N.G. Jenkins N.A. Borchelt D.R. Hum. Mol. Genet. 2005; 14: 2335-2347Crossref PubMed Scopus (107) Google Scholar). Spinal cords were sonicated in 10 volumes of TEN buffer (10 mm Tris, pH 7.5, 1 mm EDTA, pH 8.0, 100 mm NaCl), and the homogenate was centrifuged at 800 × g for 10 min. This initial pellet (denoted P1) was discarded, and the remaining supernatant was treated with 1/10 volume of TEN buffer containing 10% of Nonidet P-40. This solution was mixed well, sonicated, and centrifuged at 100,000 × g. The resulting supernatant from this preparation is referred to as S1, and the pellet is referred to as P2. An additional, harsher detergent extraction was performed on the P2 pellet by resuspension of the P2 pellet in 1 ml of TEN buffer with 0.5% Nonidet P-40, 0.25% SDS, and 0.5% deoxycholate. This homogenate was sonicated and centrifuged at 100,000 × g for 30 min. The resulting pellet is denoted as the P3 pellet. The P3 pellet was also subjected to an additional washing step; the P3 pellet was resuspended in 1 ml of TEN with 0.5% Nonidet P-40, 0.25% SDS, and 0.5% deoxycholate, and then sonicated at centrifuged at 100,000 × g for 30 min. The supernatant was discarded. For storage and analysis, the washed P3 pellet was resuspended in 200 μl of water and kept at –80 °C. The number of spinal cords analyzed from mice expressing G93A, H46R/H48Q, G37R, and hWT SOD1 were 12, 9, 3, and 3, respectively. Dissolution of Detergent-resistant Pellets and Analysis with LC-ESI MS—Frozen detergent-resistant pellet (DRP) samples were thawed, and 100-μl aliquots were mixed with 400 μl of a denaturing solution consisting of 3 m guanidine thiocyanate and 0.5 m DTT. Frozen S1 samples were also thawed and 1/10 volume (∼100 μl) was mixed with 400 μl of 3 m guanidine thiocyanate and 0.5 m DTT. These mixtures were incubated at room temperature for 30 min. Two 200-μl aliquots were removed and placed in separate Eppendorf tubes and to each tube methanol (600 μl), chloroform (200 μl), and water (400 μl) was added. The tubes were mixed vigorously for 2 min, yielding a white suspension. The tubes were then centrifuged for 1 min, yielding a biphasic mixture with a white precipitate at the aqueous/organic interface. The upper methanol/aqueous layer was removed, and 600 μl of methanol was added to each tube. The tubes were then inverted gently to yield one organic phase. The tubes were centrifuged again for 1 min. The organic layer was removed, and the white solid was allowed to dry for 2 min at room temperature and pressure (41Wessel D. Flugge U.I. Anal. Biochem. 1984; 138: 141-143Crossref PubMed Scopus (3162) Google Scholar, 42Whitelegge J.P. Katz J.E. Pihakari K.A. Hale R. Aguilera R. Gomez S.M. Faull K.F. Vavilin D. Vermaas W. Phytochemistry. 2004; 65: 1507-1515Crossref PubMed Scopus (52) Google Scholar). The white solid was dissolved in 88% formic acid (total volume: 90 μl), and the entire volume was immediately injected onto the HPLC column and separated using a two-solvent gradient. The solvents used were 0.1% trifluoroacetic acid in water (solvent “A”) and 49.9% isopropyl alcohol, 49.9% acetonitrile, 0.05% trifluoroacetic acid (solvent “B”). A compound linear gradient was ramped from 5% B at 5 min to 40% B at 30 min and to 100% B at 150 min. The chromatography was conducted at 40 °C using an PLRPS column, with a flow rate of 100 μl/min (43Whitelegge J.P. Zhang H. Aguilera R. Taylor R.M. Cramer W.A. Mol. Cell Proteomics. 2002; 1: 816-827Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). The output from the HPLC was outfitted with a splitter affording an equal flow of analyte to the mass spectrometer and to the fraction collector. The fraction collector collected 1-min (50 μl) fractions. The mass spectrometer (API III+, Sciex, Concord, Canada) was tuned and calibrated as described previously (44Whitelegge J.P. Gundersen C.B. Faull K.F. Protein Sci. 1998; 7: 1423-1430Crossref PubMed Scopus (167) Google Scholar) and scanned from 600 to 2300 m/z with a step size of 0.3 and a dwell of 1 ms. MS/MS Identification of Proteins Contained in DRPs—LC-ESI-MS provides a survey of the number of proteins present in the pellet, but mass alone is insufficient for an unambiguous identification of proteins. To identify proteins detected with LC-ESI-MS, they must be trypsinized and sequenced with tandem mass spectrometry (LC-ESI-MS/MS). Therefore, the collected fractions (∼30 in number) from the LC-ESI-MS separation were reduced, alkylated, and treated with trypsin (Promega, sequencing grade) for analysis with LC-ESI-MS/MS. ESI-LC-MS/MS analysis of DRPs was performed on a Q-STAR XL mass spectrometer (Applied Biosystems, Foster City, CA). See supplemental information for additional experimental details of the trypsinization of DRP samples and analysis with ESI-LC-MS/MS. Size Exclusion and Anti-hSOD1 Immunoaffinity Isolation of Soluble and Aggregated SOD1—Spinal cord tissue was weighed and homogenized in 10 volumes of chilled phosphate-buffered saline, pH 7.4, using an ultrasonic disintegrator. During sonication, the homogenate was kept on ice. The homogenate was then centrifuged (800 × g, 10 min). This low speed centrifugation only pellets the major cellular debris and does not pellet smaller structures such as mitochondria or aggregates. To obtain SOD1 quantities sufficient for detection with mass spectrometry, it was determined that the SE/IA procedure had to be scaled up to include three spinal cords. The supernatants from three individual spinal cords were combined and then loaded onto the G-75 SE column. The numbers of spinal cords analyzed from mice expressing H46R/H48Q, G37R, and hWT SOD1 were 18, 9, and 6, respectively. The size exclusion/immunoaffinity method used here involved the separation of SOD1-containing complexes first with size exclusion chromatography followed by anti-SOD1 immunoaffinity chromatography. Sephadex G-75 medium was packed into glass columns (1.0 cm × 50 cm and 1.5 cm × 75 cm, respectively). The packed SE columns were connected to a Bio-Logic fraction collector (Bio-Rad) programmed to collect 10-min fractions. The running buffer was phosphate-buffered saline (the chromatography methods did not use detergent). Typically, all of these columns flowed at 200–300 μl/min. To identify SOD1-positive fractions, a 10-μl aliquot from each collected fraction was analyzed with SDS-PAGE and anti-hSOD1 Western blotting. The collected SOD1-positive fractions corresponding to the void volume were combined, concentrated with Centricon centrifugal filtration devices (molecular mass allowance of 3000 Da, Millipore), and subjected to an immunoaffinity (IA) purification using the anti-SOD1 antibody-Sepharose matrix. The resolving fractions were also combined, concentrated with 3-kDa centrifugation filtration devices, and subjected to the anti-SOD1 IA column. SOD1-positive fractions that eluted from the SE column were incubated with the anti-SOD1-Sepharose beads overnight at 4 °C. The bound species were eluted using 0.1 m glycine/HCl buffer, pH 2.8, according to the same procedure used for the purification of anti-SOD1 antibodies from the SOD1-Sepharose media (see supplemental information for experimental details). The fractions were then washed with 10 mm Tris (pH 8.0) using Centricon centrifugal filtration devices (molecular mass allowance of 3000 Da) to prepare the sample for digestion with trypsin. In short, samples were concentrated to ∼200 μl, followed by the addition of 1.8 ml 10 mm Tris, pH 8.0. This procedure was repeated three to four times with the samples being finally concentrated to ∼200 μl. Mass Spectrometry of Soluble and Aggregated SOD1 Isolated with Size Exclusion/Anti-SOD1 Immunoaffinity Chromatography—SE/IA purified samples were analyzed directly with MALDI-TOF mass spectrometry. To dissociate aggregated SOD1, the samples were heated at ∼80 °C for 2 min in the presence of 100 mm DTT. Standard protocols were followed for MALDI-TOF-MS analysis (42Whitelegge J.P. Katz J.E. Pihakari K.A. Hale R. Aguilera R. Gomez S.M. Faull K.F. Vavilin D. Vermaas W. Phytochemistry. 2004; 65: 1507-1515Crossref PubMed Scopus (52) Google Scholar). In short, 1 μl of matrix solution (1.0 mg of sinapic acid dissolved into 100 μl of 70% acetonitrile/0.1% trifluoroacetic acid) was spotted onto the MALDI plate along with 0.5 μl of protein sample and 0.5 μl of myoglobin (1 nmol/100 μl of H2O; added as an internal calibrant). Spots were allowed to dry at room temperature, and data were collected on a Voyager-DE STR instrument (Applied Biosystems) and analyzed with Data Explorer software (Version 5.10). MS/MS Identification of Proteins Co-eluting with SOD1 in Chromatographic Fractions—SE/IA samples were concentrated to a volume between 150 and 200 μl using centrifugal filtration devices (Millipore, molecular mass allowance of 3000 Da) and subjected to digestion with trypsin. An aqueous solution of DTT was added to the digest so that the final concentration was 1 mm. Tryptic digests were analyzed by μLC-MS/MS with data-dependent acquisition using an LCQ-DECA ion trapquadrupole ESI-MS (ThermoFinnigan, San Jose, CA). See supplemental information for additional experimental details on the trypsinization of samples and analysis with LC-ESI-MS/MS. Preparation of Oxidatively Modified ALS Variant SOD1—The ALS variant H48Q SOD1

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