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• W2047051758 abstract "We report the use of a proteomic strategy to identify hitherto unknown substrates for mammalian protein l-isoaspartate O-methyltransferase. This methyltransferase initiates the repair of isoaspartyl residues in aged or stress-damaged proteins in vivo. Tissues from mice lacking the methyltransferase (Pcmt1-/-) accumulate more isoaspartyl residues than their wild-type littermates, with the most “damaged” residues arising in the brain. To identify the proteins containing these residues, brain homogenates from Pcmt1-/- mice were methylated by exogenous repair enzyme and the radiolabeled methyl donor S-adenosyl-[methyl-3H]methionine. Methylated proteins in the homogenates were resolved by both one-dimensional and two-dimensional electrophoresis, and methyltransferase substrates were identified by their increased radiolabeling when isolated from Pcmt1-/- animals compared with Pcmt1+/+ littermates. Mass spectrometric analyses of these isolated brain proteins reveal for the first time that microtubule-associated protein-2, calreticulin, clathrin light chains a and b, ubiquitin carboxyl-terminal hydrolase L1, phosphatidylethanolamine-binding protein, stathmin, β-synuclein, and α-synuclein, are all substrates for the l-isoaspartate methyltransferase in vivo. Our methodology for methyltransferase substrate identification was further supplemented by demonstrating that one of these methyltransferase targets, microtubule-associated protein-2, could be radiolabeled within Pcmt1-/- brain extracts using radioactive methyl donor and exogenous methyltransferase enzyme and then specifically immunoprecipitated with microtubule-associated protein-2 antibodies to recover co-localized protein with radioactivity. We comment on the functional significance of accumulation of relatively high levels of isoaspartate within these methyltransferase targets in the context of the histological and phenotypical changes associated with the methyltransferase knock-out mice. We report the use of a proteomic strategy to identify hitherto unknown substrates for mammalian protein l-isoaspartate O-methyltransferase. This methyltransferase initiates the repair of isoaspartyl residues in aged or stress-damaged proteins in vivo. Tissues from mice lacking the methyltransferase (Pcmt1-/-) accumulate more isoaspartyl residues than their wild-type littermates, with the most “damaged” residues arising in the brain. To identify the proteins containing these residues, brain homogenates from Pcmt1-/- mice were methylated by exogenous repair enzyme and the radiolabeled methyl donor S-adenosyl-[methyl-3H]methionine. Methylated proteins in the homogenates were resolved by both one-dimensional and two-dimensional electrophoresis, and methyltransferase substrates were identified by their increased radiolabeling when isolated from Pcmt1-/- animals compared with Pcmt1+/+ littermates. Mass spectrometric analyses of these isolated brain proteins reveal for the first time that microtubule-associated protein-2, calreticulin, clathrin light chains a and b, ubiquitin carboxyl-terminal hydrolase L1, phosphatidylethanolamine-binding protein, stathmin, β-synuclein, and α-synuclein, are all substrates for the l-isoaspartate methyltransferase in vivo. Our methodology for methyltransferase substrate identification was further supplemented by demonstrating that one of these methyltransferase targets, microtubule-associated protein-2, could be radiolabeled within Pcmt1-/- brain extracts using radioactive methyl donor and exogenous methyltransferase enzyme and then specifically immunoprecipitated with microtubule-associated protein-2 antibodies to recover co-localized protein with radioactivity. We comment on the functional significance of accumulation of relatively high levels of isoaspartate within these methyltransferase targets in the context of the histological and phenotypical changes associated with the methyltransferase knock-out mice. There is a general decline in physiological processes with aging but the molecular changes that induce senescence or agerelated pathologies have yet to be completely defined. It is thought, however, that the slow accumulation of damaged macromolecules as an organism ages probably contributes to the aging phenotype. Proteins, for example, are susceptible to undesired internal and external chemical modifications, including oxidation, glycation, deamidation, and isomerization, all of which can result in an alteration of protein function and/or stability (1Lindner H. Helliger W. Exp. Gerontol. 2001; 36: 1551-1563Crossref PubMed Scopus (97) Google Scholar, 2Clarke S. Ageing Res. Rev. 2003; 2: 263-285Crossref PubMed Scopus (238) Google Scholar). Cells have several strategies for removing damaged proteins, including lysosomal- and proteasomal-dependent degradation. Additionally, protein repair mechanisms exist to restore protein function without the requirement for de novo synthesis. One such protein repair enzyme, termed protein l-isoaspartate (d-aspartate) O-methyltransferase, PIMT 2The abbreviations used are: PIMT, protein l-isoaspartate O-methyltransferase; CAPS, 3-(cyclohexylamino)propane-1-sulfonic acid; IPG, immobilized pH gradient; MAPK; mitogen-activated protein kinase; MES, 2(N-morpholino)ethanesulfonic acid; K-MES, potassium-MES; PVDF, polyvinylidene difluoride; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; LC MS/MS, liquid chromatography and tandem mass spectrometry; MAP-2, microtubule-associated protein-2; AdoMet, S-adenosylmethionine; 1D, one-dimensional; 2D, two-dimensional; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1, 3-diol; DTT, dithiothreitol; KO, knockout; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; IP, immunoprecipitation; UCHL1, ubiquitin carboxyl-terminal hydrolase L1; PEBP, phosphatidylethanolamine-binding protein; GABA, γ-aminobutyric acid; ER, endoplasmic reticulum. 2The abbreviations used are: PIMT, protein l-isoaspartate O-methyltransferase; CAPS, 3-(cyclohexylamino)propane-1-sulfonic acid; IPG, immobilized pH gradient; MAPK; mitogen-activated protein kinase; MES, 2(N-morpholino)ethanesulfonic acid; K-MES, potassium-MES; PVDF, polyvinylidene difluoride; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; LC MS/MS, liquid chromatography and tandem mass spectrometry; MAP-2, microtubule-associated protein-2; AdoMet, S-adenosylmethionine; 1D, one-dimensional; 2D, two-dimensional; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1, 3-diol; DTT, dithiothreitol; KO, knockout; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; IP, immunoprecipitation; UCHL1, ubiquitin carboxyl-terminal hydrolase L1; PEBP, phosphatidylethanolamine-binding protein; GABA, γ-aminobutyric acid; ER, endoplasmic reticulum. (EC 2.1.1.77, also known as protein carboxylmethyltransferase 1 (PCMT1)), initiates the repair of isoaspartate within peptide chains. Isoaspartyl residues within peptides and proteins arise spontaneously by either deamidation of asparagine or isomerization of aspartic acid residues. Both reactions involve the formation of an intermediary five-membered l-succinimidyl ring, which is readily hydrolyzed to yield a normal l-aspartyl residue or an atypical l-isoaspartyl residue in which the peptide backbone now contains an extra methylene group and proceeds through the β-carboxyl group. The asymmetric nature of the succinimide ring favors hydrolysis resulting in an aspartate:isoaspartate ratio of ∼1:3 (see Fig. 1). The rate of isoaspartate formation in proteins depends greatly on both amino acid sequence and conformation but is overall one of the most common forms of protein damage and reflects aging at the molecular level (2Clarke S. Ageing Res. Rev. 2003; 2: 263-285Crossref PubMed Scopus (238) Google Scholar, 3Reissner K.J. Aswad D.W. Cell Mol. Life Sci. 2003; 60: 1281-1295Crossref PubMed Scopus (213) Google Scholar, 4Robinson N.E. Robinson Z.W. Robinson B.R. Robinson A.L. Robinson J.A. Robinson M.L. Robinson A.B. J. Pept. Res. 2004; 63: 426-436Crossref PubMed Scopus (164) Google Scholar). Methylation of isoaspartate by PIMT significantly increases the rate at which these damaged residues return to the succinimide form. Subsequent hydrolysis to aspartate or isoaspartate produces either a repaired or as yet unrepaired peptide linkage, respectively (Fig. 1). Albeit somewhat inefficiently, several rounds of PIMT methylation will reconvert the isoaspartate to aspartate, a process that can be concomitant with restoration of enzymatic function in model proteins and peptides that have been aged in vitro or in vivo (5Johnson B.A. Murray E.D. Clarke S. Glass D.B. Aswad D.W. J. Biol. Chem. 1987; 262: 5622-5629Abstract Full Text PDF PubMed Google Scholar, 6Johnson B.A. Langmack E.L. Aswad D.W. J. Biol. Chem. 1987; 262: 12283-12287Abstract Full Text PDF PubMed Google Scholar, 7McFadden P.N. Clarke S. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2595-2599Crossref PubMed Scopus (167) Google Scholar, 8Brennan T.V. Anderson J.W. Jia Z.C. Waygood E.B. Clarke S. J. Biol. Chem. 1994; 269: 24586-24595Abstract Full Text PDF PubMed Google Scholar, 9Galletti P. Ingrosso D. Manna C. Clemente G. Zappia V. Biochem. J. 1995; 306: 313-325Crossref PubMed Scopus (74) Google Scholar, 10Ingrosso D. D'Angelo S. di Carlo E. Perna A.F. Zappia V. Galletti P. Eur. J. Biochem. 2000; 267: 4397-4405Crossref PubMed Scopus (79) Google Scholar, 11Lanthier J. Desrosiers R.R. Exp. Cell Res. 2004; 293: 96-105Crossref PubMed Scopus (50) Google Scholar, 12Shimizu T. Matsuoka Y. Shirasawa T. Biol. Pharm. Bull. 2005; 28: 1590-1596Crossref PubMed Scopus (158) Google Scholar). The generation of Pcmt1-/- mice has provided an invaluable insight into the physiological consequences of isoaspartate formation. These mice accumulate higher levels of isoaspartate, relative to their wild-type littermates, in all tissues that normally express the enzyme (13Kim E. Lowenson J.D. MacLaren D.C. Clarke S. Young S.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6132-6137Crossref PubMed Scopus (254) Google Scholar, 14Yamamoto A. Takagi H. Kitamura D. Tatsuoka H. Nakano H. Kawano H. Kuroyanagi H. Yahagi Y. Kobayashi S. Koizumi K. Sakai T. Saito K. Chiba T. Kawamura K. Suzuki K. Watanabe T. Mori H. Shirasawa T. J. Neurosci. 1998; 18: 2063-2074Crossref PubMed Google Scholar, 15Shimizu T. Ikegami T. Ogawara M. Suzuki Y. Takahashi M. Morio H. Shirasawa T. J. Neurosci. Res. 2002; 69: 341-352Crossref PubMed Scopus (26) Google Scholar). Pcmt1-/- mice have abnormal neuronal excitability, and most die from an epileptic seizure at less than 2 months of age (13Kim E. Lowenson J.D. MacLaren D.C. Clarke S. Young S.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6132-6137Crossref PubMed Scopus (254) Google Scholar, 14Yamamoto A. Takagi H. Kitamura D. Tatsuoka H. Nakano H. Kawano H. Kuroyanagi H. Yahagi Y. Kobayashi S. Koizumi K. Sakai T. Saito K. Chiba T. Kawamura K. Suzuki K. Watanabe T. Mori H. Shirasawa T. J. Neurosci. 1998; 18: 2063-2074Crossref PubMed Google Scholar, 16Kim E. Lowenson J.D. Clarke S. Young S.G. J. Biol. Chem. 1999; 274: 20671-20678Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). They experience aberrant mossy fiber-CA3 region synaptic neurotransmission, vacuolar degeneration at the axon hillock of dentate cells, and disorganized microtubules within the dendrites of pyramidal neurons (14Yamamoto A. Takagi H. Kitamura D. Tatsuoka H. Nakano H. Kawano H. Kuroyanagi H. Yahagi Y. Kobayashi S. Koizumi K. Sakai T. Saito K. Chiba T. Kawamura K. Suzuki K. Watanabe T. Mori H. Shirasawa T. J. Neurosci. 1998; 18: 2063-2074Crossref PubMed Google Scholar, 17Ikegaya Y. Yamada M. Fukuda T. Kuroyanagi H. Shirasawa T. Nishiyama N. Hippocampus. 2001; 11: 287-298Crossref PubMed Scopus (42) Google Scholar). Although smaller on average than their wild-type littermates, the Pcmt1-/- mice also have a progressive increase in brain size linked to alterations in the phosphatidyl-inositol 3-kinase/protein kinase B signaling pathway (14Yamamoto A. Takagi H. Kitamura D. Tatsuoka H. Nakano H. Kawano H. Kuroyanagi H. Yahagi Y. Kobayashi S. Koizumi K. Sakai T. Saito K. Chiba T. Kawamura K. Suzuki K. Watanabe T. Mori H. Shirasawa T. J. Neurosci. 1998; 18: 2063-2074Crossref PubMed Google Scholar, 16Kim E. Lowenson J.D. Clarke S. Young S.G. J. Biol. Chem. 1999; 274: 20671-20678Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 18Farrar C. Houser C.R. Clarke S. Aging Cell. 2005; 4: 1-12Crossref PubMed Scopus (42) Google Scholar, 19Farrar C.E. Huang C.S. Clarke S.G. Houser C.R. J. Comp. Neurol. 2005; 493: 524-537Crossref PubMed Scopus (23) Google Scholar). To better understand how isoaspartyl residues contribute to these defects, the primary in vivo substrates for PIMT must first be determined. Two main approaches have been used to limit isoaspartate repair and allow the substrates to accumulate to a detectable level: inhibition of PIMT (as well as other S-adenosylmethionine-dependent methyltransferases) in cultured cells using periodate-oxidized adenosine, and the generation of Pcmt1-/- mice. Both techniques produced unrepaired proteins that have been identified by subsequent methylation by exogenous PIMT and radioactive S-adenosylmethionine (AdoMet). In this way, histone H2B (20Young A.L. Carter W.G. Doyle H.A. Mamula M.J. Aswad D.W. J. Biol. Chem. 2001; 276: 37161-37165Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), synapsin 1 (15Shimizu T. Ikegami T. Ogawara M. Suzuki Y. Takahashi M. Morio H. Shirasawa T. J. Neurosci. Res. 2002; 69: 341-352Crossref PubMed Scopus (26) Google Scholar, 21Reissner K.J. Paranandi M.V. Luc T.M. Doyle H.A. Mamula M.J. Lowenson J.D. Aswad D.W. J. Biol. Chem. 2006; 281: 8389-8398Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), tau protein (14Yamamoto A. Takagi H. Kitamura D. Tatsuoka H. Nakano H. Kawano H. Kuroyanagi H. Yahagi Y. Kobayashi S. Koizumi K. Sakai T. Saito K. Chiba T. Kawamura K. Suzuki K. Watanabe T. Mori H. Shirasawa T. J. Neurosci. 1998; 18: 2063-2074Crossref PubMed Google Scholar), calmodulin (21Reissner K.J. Paranandi M.V. Luc T.M. Doyle H.A. Mamula M.J. Lowenson J.D. Aswad D.W. J. Biol. Chem. 2006; 281: 8389-8398Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 22O'Connor M.B. O'Connor C.M. J. Biol. Chem. 1998; 273: 12909-12913Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar), and tubulin (23Ohta K. Seo N. Yoshida T. Hiraga K. Tuboi S. Biochimie (Paris). 1987; 69: 1227-1234Crossref PubMed Scopus (14) Google Scholar, 24Najbauer J. Orpiszewski J. Aswad D.W. Biochemistry. 1996; 35: 5183-5190Crossref PubMed Scopus (88) Google Scholar, 25Lanthier J. Bouthillier A. Lapointe M. Demeule M. Beliveau R. Desrosiers R.R. J. Neurochem. 2002; 83: 581-591Crossref PubMed Scopus (46) Google Scholar) have thus far been shown to be physiological PIMT substrates. Identification of damaged proteins labeled with [3H]AdoMet has been hindered by the instability of the methyl esters and the low levels of methylatable residues even in tissues from Pcmt1-/- mice. To minimize these problems, we have used conditions that maintain base-labile methyl esters generated by exogenous PIMT on proteins from Pcmt1-/- tissues and employed a rapid and sensitive microchannel plate device to detect methylated proteins resolved by both 1D SDS-PAGE and 2D PAGE. We were able to identify nine new in vivo substrates for PIMT from brain tissue: microtubule-associated protein-2, calreticulin, clathrin light chains a and b, ubiquitin carboxylterminal hydrolase L1, phosphatidylethanolamine-binding protein, stathmin, β-synuclein, and α-synuclein. The methylation of these proteins suggests a role for PIMT in the maintenance of the neuronal cytoskeleton and regulation of neuronal signaling. Materials—IPG strips (pH 4-7; 7- and 17-cm lengths) were purchased from Bio-Rad, with all isoelectric focusing performed using a Bio-Rad Protean isoelectric focusing cell. NuPAGE Novex pre-cast gels (4-12% Bis-Tris gels for 1D SDS-PAGE and 4-12% Bis-Tris Zoom gels for 2D PAGE analysis), MES-SDS running buffer, transfer buffer, SeeBlue Plus2 prestained gel standards, and Safe stain were all obtained from Invitrogen. Dithiothreitol (DTT), iodoacetamide, MES, HEPES, wide range molecular weight markers, glycine, glutaraldehyde, and all SDS-PAGE reagents were purchased from Sigma. Isoquant isoaspartate detection kits were purchased from Promega. S-Adenosyl-l-[methyl-3H]methionine (37 MBq/ml), destreak reagent, and PlusOne silver staining kit were purchased from Amersham Biosciences. Bovine recombinant PIMT provided with the Isoquant kit was used for methylations of homogenates analyzed by 1D and 2D PAGE. This enzyme has a specific activity of ∼5000 pmol of methyl groups transferred/min/mg when assayed for trichloroacetic acid-precipitable counts into bovine γ-globulins (Sigma Cohn fraction II) (26Aswad D.W. Deight E.A. J. Neurochem. 1983; 40: 1718-1726Crossref PubMed Scopus (68) Google Scholar), an activity comparable to that produced by recombinant human PIMT (27MacLaren D.C. Clarke S. Protein Expr. Purif. 1995; 6: 99-108Crossref PubMed Scopus (35) Google Scholar) when assayed under identical conditions, and similar to that for PIMT purified directly from bovine brain (26Aswad D.W. Deight E.A. J. Neurochem. 1983; 40: 1718-1726Crossref PubMed Scopus (68) Google Scholar). Mice—Wild-type (Pcmt1+/+) and PIMT knock-out (KO) mice (Pcmt1-/-) were generated according to previously detailed procedures and breeding conditions (13Kim E. Lowenson J.D. MacLaren D.C. Clarke S. Young S.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6132-6137Crossref PubMed Scopus (254) Google Scholar, 16Kim E. Lowenson J.D. Clarke S. Young S.G. J. Biol. Chem. 1999; 274: 20671-20678Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Comparisons in all studies involved either male or female littermates. Preparation of Brain Extracts—Brains were removed immediately from sacrificed animals and weighed, and crude cytosolic extracts were prepared according to Lowenson et al. (28Lowenson J.D. Kim E. Young S.G. Clarke S. J. Biol. Chem. 2001; 276: 20695-20702Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Protein concentrations in all extracts were determined using the DC Protein Assay kit (Bio-Rad), with bovine serum albumin as a standard. Quantitation of Isoaspartate Levels—The levels of isoaspartate in tissue extracts were quantified by a methanol diffusion assay using an Isoquant kit according to the manufacturer's instructions. Briefly, extracts were methylated with recombinant bovine PIMT (Promega, 1.9 μm final concentration) utilizing 20 μm [3H]AdoMet (1 μCi/reaction, 2220 dpm/pmol final specific activity), in a buffer of 100 mm sodium phosphate, pH 6.8, containing 1 mm EGTA, 0.16% Triton X-100, and 0.004% sodium azide (final concentrations) for 30 min at 30 °C. Methylation was terminated, the methyl esters were hydrolyzed by CAPS (pH 10) (with 5% SDS, 2.2% methanol, 0.1% m-cresol purple), and the samples retained on ice for 10 min. 50 μl from each of the samples was then spotted onto a sponge inserted into the cap of a scintillation vial. The samples were incubated for 60 min at 40 °C to volatilize [3H]methanol from the labeled proteins into 10 ml of scintillation fluid, which was then counted for radioactivity. Each assay was performed in duplicate from which an average was determined. 50 pmol of isoaspartate-containing delta sleep-inducing peptide provided with the Isoquant kit was similarly methylated to provide a reference level of isoaspartate methylation from which methylation of the cytosolic proteins was quantified. 1D SDS-PAGE Separation of Proteins—Protein extracts were methylated in a final volume of 20 μl in a buffer of 50 mm K-MES, pH 6.2, containing 20 μm [3H]AdoMet (1.5 μCi/reaction, 8250 dpm/pmol final specific activity). Methylation was initiated by the addition of 5 μl of exogenous recombinant bovine PIMT (Promega, 2.4 μm final concentration) and incubated for 30 min at 30 °C. Methylation was terminated by the addition of a quarter of a volume of 5× concentrated reducing solution (10% SDS and 500 mm DTT), and the samples were heated for 10 min at 50 °C. One quarter of a volume of 5× Laemmli sample buffer was added (250 mm Tris/HCl, pH 6.8, 40% (v/v) glycerol, 5% (w/v) SDS, 0.005% (w/v) bromphenol) and proteins (typically 20 μg/gel lane) were resolved on 4-12% Bis-Tris gels for 2 h at 125V using MES running buffer (pH 7.3) run in an X Cell surelock gel tank (Invitrogen). Proteins were then electroblotted at 80 V for 2 h onto a PVDF membrane (Millipore) using NuPage Transfer buffer (pH 7.2). Membranebound protein was stained with Coomassie Blue (Safestain) for 30 min at room temperature and then further fixed by drying overnight. The membranes were destained with 50% methanol (v/v), 10% acetic acid (v/v), and then extensively washed with 20 mm K-MES, pH 6.2. Membrane-bound protein was crosslinked by incubation with 0.5% glutaraldehyde (v/v) in 20 mm K-MES pH 6.2 overnight. Membranes were again washed with 20 mm K-MES, pH 6.2, and then residual unreacted glutaraldehyde blocked by incubation with 0.2 m glycine in 20 mm K-MES pH6.2 for 4 h at room temperature. Blots were finally washed as before and left to dry before application of 14C markers onto the positions of the molecular weight standards. Blots were applied to a Microchannel Plate device and autoradiographed for 7 or 24 h (refer to Fig. 3) to visualize radioactive targets (29Lees J.E. Richards P.G. Electrophoresis. 1999; 20: 2139-2143Crossref PubMed Scopus (16) Google Scholar). Equivalent gels to those described above were stained with either colloidal Coomassie Brilliant Blue (Invitrogen) overnight or with silver (Amersham Biosciences PlusOne kit) according to the manufacturer's instructions to provide comparisons of protein expression in the Pcmt1+/+ and Pcmt1-/- tissues. Stained gels were either photographed using a Fuji digital camera or scanned using an Agfa Duoscan T1200 flatbead scanner. Radiolabeling and 1D SDS-PAGE were performed on five different pairs of littermates for brain tissue, and three different pairs of littermates for all other tissues. 2D PAGE Separation of Proteins—Methylation was performed similarly to that for 1D SDS-PAGE (but scaled up to 600 μg/analysis), terminated by the addition of 700 μl of protein precipitation solution (acetone:diethyl ether (2:1 (v/v)), and samples were placed on ice. Precipitated proteins were collected by centrifugation at 5000 rpm for 3 min. The precipitate was washed three times with ether:industrial methylated spirit: water (10:7:2, v/v) and air-dried, and then proteins were dissolved in a rehydration buffer (9.8 m urea, 2% (w/v) CHAPS, 0.5% IPG buffer) containing 12 μl/ml of the anti-oxidant destreak reagent, at room temperature for 1 h. The solubilized protein was actively rehydrated into either 7- or 17-cm, pH 4-7 IPG isoelectric focusing strips for 16 h at 50 V, and then focused for 16 h according to the manufacturer's guidelines for each strip. Focused strips were washed in an equilibration buffer of 0.375 m Tris/HCl, pH 6.8, 6 m urea, 2% SDS, 20% glycerol, containing 2% (w/v) DTT for 10 min, and then similarly washed with the same buffer except 2.5% (w/v) iodoacetamide replaced DTT. After this reduction and alkylation, strips were equilibrated in MES running buffer before protein separation by SDS-PAGE. Strips of 7-cm length were resolved in the second dimension using Zoom gels. Strips of 17 cm were layered onto freshly prepared 5-20% (w/v) acrylamide gradient gels, and proteins were resolved at a constant current of 22 mA for 20 h within a Protean II xi 2D cell (Bio-Rad). Proteins were then transferred to a PVDF membrane and autoradiographed for 16-24 h under conditions described for 1D SDS-PAGE. Equivalent 2D PAGE gels were stained with either colloidal Coomassie or silver, and an autoradiographic template was used to locate spots of interest for mass spectrometry. Brain tissue 2D maps were typically generated two or three times from each of five different pairs of littermates. MALDI-TOF Mass Spectrometry—Stained protein spots were excised and transferred into a 96-well plate using an automated MassPrep robotic system (ProteomeWorks-Bio-Rad). Gel pieces were destained, and then reduced and alkylated with DTT and iodoacetamide before tryptic digestion in situ. Tryptic peptides were desalted by binding and then elution from a C18 Zip-tip (Millipore), and then they were mixed with α-cyano-4-hydroxycinnamic acid (Sigma C-2020) matrix and analyzed by MALDI-TOF mass spectrometry (Micromass MALDI, Waters, UK). A number of intact singularly charged peptides were identified, and the masses were used in a search algorithm (MASCOT peptide mass fingerprint) to screen several protein databases, including SwissProt for peptide mass matches to enable protein identification. LC MS/MS—Stained protein spots were excised, reduced, and alkylated, and tryptic peptides were produced according to the MALDI method. Extracted tryptic peptides were then run on a Waters QTOF2 hybrid quadrupole mass spectrometer incorporating an integrated capillary LC system. Tryptic digestion products were initially loaded onto a C18 pre-column for desalting and then eluted onto an analytical capillary C18 column (100-mm × 0.75-mm internal diameter). The LC system incorporated a flow splitting device to give a final flow through the column of 200 nl/min. Typically, a solvent gradient was run over a total of 1 h to elute peptides from the capillary column and re-equilibrate prior to loading the next sample. Eluted peptides from the analytical column were directly submitted into the mass spectrometer via a nanosprayer device attached to the outflow from the LC system and operating at 3 kV. In addition, a reference solution containing a peptide of known mass was sprayed into the mass spectrometer from a separate sprayer. This ion source was sampled at regular intervals throughout the run to assist in maintaining accurate mass measurements of the ionized peptides from the analyte spray. Data-dependent switching was incorporated so that whenever a peptide with an associated charge of 2+ or 3+ was detected above a preset threshold signal, the mass spectrometer would automatically switch to MS/MS mode to generate fragmentation data from the detected peptide. Software was enabled to scan over multiple channels to simultaneously fragment up to three co-eluting peptides and collect the fragmentation data from each one individually. A preset range of collision voltages was set up in the method to fragment each peptide as efficiently as possible. Raw data files were analyzed using MassLynx 4.0 (incorporating BioLynx) and ProteinLynx Globalserver 2 (Waters) to assess the identities of proteins present in the digest. The peak list file generated from ProteinLynx Globalserver 2 analysis was also used in alternative search engines accepting this format of data file, including MASCOT. In addition, some of the fragmentation data were analyzed manually, and de novo sequencing was carried out on selected peptides. Immunoprecipitation of MAP-2—Methylation of protein extracts were performed similarly to that for 1D SDS-PAGE but with 160 μg of protein used from each genotype. After methylation, samples were placed on ice, and then precleared by rotation with normal rabbit IgG-agarose conjugate (sc-2345, Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at 4°C. MAP-2 polyclonal antibody was then added (0.6 μg of sc-20172, Santa Cruz Biotechnology) for 1 h at 4 °C. Protein A-agarose (sc-2001, Santa Cruz Biotechnology) was added for 45 min at 4 °C to capture immune complexes. The agarose beads were then pelleted by centrifugation and washed five times with an immunoprecipitation (IP) wash buffer of 20 mm K-MES, pH 6.2, containing 150 mm NaCl and 0.05% Tween 20. Washed beads were rehydrated in 20 μl of 20 mm K-MES, pH 6.2, and 5× concentrated reducing solution (10% SDS and 500 mm DTT) added, and the samples were heated for 10 min at 50 °C. One-quarter of a volume of 5× Laemmli sample buffer was added, and proteins which were liberated from the antibody separated by 1D SDS-PAGE as described previously. Proteins were then transferred to a PVDF membrane and autoradiographed for 24 h. Western Blotting—Blots prepared from the MAP-2 immunoprecipitation were washed with IP wash buffer and then incubated in a blocking buffer (IP wash buffer containing 5% (w/v) milk fat) for 1 h at room temperature. Blots were incubated with MAP-2 antibody (sc-20172), at a 1:2000 dilution in blocking buffer for 2 h at room temperature, washed with IP wash buffer, and then incubated with secondary antibody (polyclonal goat anti-rabbit immunoglobulins-horseradish peroxide-conjugated, Dako, P0448) for 1 h at room temperature. Blots were further washed with IP wash buffer, then MAP-2 immunolocalization visualized using ECL (Pierce), and captured on CL-Xposure x-ray film (Pierce). Films were scanned on an Agfa Duoscan T1200 flatbead scanner. PIMT Knock-out Mice Accumulate Higher Levels of Isoaspartate Than Wild-type Littermates—We determined the isoaspartate content of protein in seven tissues from both wild-type and PIMT KO mice using an Isoquant kit. In the absence of PIMT activity, all tissues examined, including brain, heart, spleen, liver, kidney, thymus, and skeletal muscle, accumulated higher levels of isoaspartyl residues than those of their wild-type littermates (Fig. 2). Brain homogenate from PIMT KO mice exhibited the highest levels of isoaspartate, having close to 2 pmol of isoaspartate/μg of protein, an ∼18-fold increase over wild type. By comparison, homogenates of the other assayed KO tissues ranged from 0.2 pmol of isoaspartate/μg of protein in liver to 0.8 pmol of isoaspartate/μg of protein i" @default.
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• W2047051758 title "Proteomic Identification of Novel Substrates of a Protein Isoaspartyl Methyltransferase Repair Enzyme" @default.
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