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1 Institute of Molecular Biology, Academia Sinica, Taiwan, Republic of China
2 Institute of Atomic and Molecular Sciences, Academia Sinica, Taiwan, Republic of China
3 Division of Biotechnology, Animal Technology Institute Taiwan, Taiwan, Republic of China
4 Department of Animal Science, National Chung Hsing University, Taiwan, Republic of China
(RECEIVED November 16, 2006; FINAL REVISION February 1, 2007; ACCEPTED February 22, 2007)
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Abstract |
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-NG,NG-asymmetry dimethylarginines. A yeast type I arginine methyltransferase gene (HMT1) is put on a plasmid under the control of the Escherichia coli methionine aminopeptidase promoter for constitutive expression. The protein targeted for post-translational modification is put on the same plasmid behind a T7 promoter for inducible expression of His6-tagged proteins. Sbp1p and Stm1p were used as model proteins to examine this expression system. The 13 arginines within the arginine-glycine-rich motif of Sbp1p and the RGG sequence near the C terminus of Stm1p were methylated. Unexpectedly, the arginine residue on the thrombin cleavage site (LVPRGS) of the fusion proteins can also be methylated by Hmt1p. Sbp1p and Sbp1p/hmt1 were covalently attached to solid supports for the isolation of interacting proteins. The results indicate that arginine methylation on Sbp1p exerts both positive and negative effects on protein–protein interaction. Keywords: protein expression; protein arginine methylation; protein–protein interactions; Sbp1p; Stm1p
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Introduction |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsElectronic supplemental materialAcknowledgmentsReferences |
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-monomethylarginine (-MMA), -monomethylarginine (-MMA), asymmetric -NG,NG-dimethylarginine (aDMA), and symmetric -NG,NG’-dimethylarginine (sDMA). A family of isozymes, protein arginine N-methyltransferases (PRMTs), carries out these reactions (Gary and Clarke 1998; Niewmierzycka and Clarke 1999).
There are two major types of PRMTs (Gary and Clarke 1998). The type I enzymes catalyze the formation of -MMA and aDMA. The type II enzymes perform the conversion of arginine to -MMA and sDMA. In budding yeast, Hmt1p is a type I enzyme responsible for most of the arginine N-methylation reactions (Gary et al. 1996). Hsl7p was initially reported as a type II enzyme (Lee et al. 2000). A recent report suggests that it is a type III enzyme that synthesizes -MMA (Miranda et al. 2006). There is also a type IV enzyme (RMT2) in budding yeast that is accountable for the formation of -MMA (Niewmierzycka and Clarke 1999; Chern et al. 2002).
The substrates of PRMTs participate in various cellular functions such as RNA processing, transcriptional regulation, signal transduction, and DNA repair (Bedford and Richard 2005). Methylation of arginine residue(s) on these proteins often modulates protein–protein interactions. For instance, methylation of Sam68 inhibits its binding to the SH3 (Src homology 3) domains of p59fyn and phospholipase C
-1 (Bedford et al. 2000). In contrast, SMN (survival of motor neurons protein) preferentially interacts with methylated SmB and SmD (Friesen et al. 2001).
Purification of methylated proteins from natural sources for biochemical studies can be a difficult task. The target protein might not be abundantly expressed. Furthermore, the methyl groups on the protein might not exist in stoichiometric amounts (Chern et al. 2002; Ostareck-Lederer et al. 2006). The isolated proteins can be a mixture of methylated and unmodified polypeptides. Thus, characterization of a particular form of the protein is difficult.
We circumvent this difficulty by coexpressing the protein to be methylated with Hmt1p. Sbp1p and Stm1p were used as model proteins to test this expression system. Sbp1p is a 33-kDa, single-strand nucleic acid-binding protein. It has a large arginine-glycine-rich domain that includes 1 RGR, 5 RGG, and 3 RGRGG repeats (totaling 13 arginines from residues 125–167). It has been suggested as a substrate of Hmt1p (Frankel and Clarke 1999). Stm1p binds quadruplex and triple helical nucleic acids (Frantz and Gilbert 1995; Nelson et al. 2000). It has been implicated in telomere structure maintenance (Hayashi and Murakami 2002) and signaling of apoptosis-like cell death (Ligr et al. 2001). Stm1p has a single RGG motif near the C terminus of the protein (234NNRRGGRGARK244). We report here the characterization of the coexpressed proteins, Sbp1p/hmt1 and Stm1p/hmt1. Furthermore, Sbp1p and Sbp1p/hmt1 were used as baits in pull-down experiments. Results suggest Nop13p, Mas1p, and Rpl16Ap interact primarily with Sbp1p/hmt1.
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Results |
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32 kDa in size, but migrates close to 37 kDa on a denaturing polyacrylamide gel (Fig. 2A). Stm1p is prone to degradation during purification. Since the N-terminal His6-tagged Stm1p was purified with metal-affinity beads, polypeptides <28 kDa should not contain the methylation site. As shown on the fluorogram (exposed for 43 d), Stm1p was methylated, while Stm1p/hmt1 was not labeled (Fig. 2B). The result implies that Stm1p/hmt1 was methylated in E. coli cells. Therefore, the expression system we present here is not specific for Sbp1p; it can methylate proteins with RGG motif in general.
Mass spectrometric analysis of recombinant proteins
The purified recombinant proteins were analyzed with mass spectrometry. We obtained an average molecular mass of 34,998 ± 3 Da (calculated molecular mass 34,994 Da, Fig. 3A, peak a) for Sbp1p. The recombinant Sbp1p/hmt1 has an average mass of 35,365 ± 3, an increase of 367 Da (Fig. 3B, peak a'). The result implies an addition of 26 methyl groups onto Sbp1p. However, components with mass higher than that of the unmodified Sbp1p but lower than that represented by peak a' can be detected on the mass spectrum (Fig. 3B, denoted with asterisks). Probably, they represent Sbp1p/hmt1 carrying <26 methyl groups.
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The affinity-purified Stm1p and Stm1p/hmt1 were further separated from its degraded products on a C18 reverse-phase column. The full-length proteins were collected for mass spectrometric analysis. Unlike Sbp1p, the N-terminal of the recombinant Stm1p was not partially modified. We obtained an average molecular mass of 32,001 ± 2 Da (calculated average molecular mass 31,999 Da) for Stm1p. Stm1p/hmt1 has an average molecular mass of 32,027 ± 2 Da. The results imply an addition of two methyl groups onto Stm1p.
Amino acid analysis of Sbp1p/hmt1
To ensure the recombinant proteins have the expected arginine methylation, Sbp1p/hmt1 was subjected to amino acid analysis. The chromatograms of the phenylthiocarbamyl amino acids are presented in Figure 4. As expected, Sbp1p/hmt1 carries aDMA (Fig. 4B), while recombinant Sbp1p lacks this residue (Fig. 4C). A small amount of MMA can also be detected in the Sbp1p/hmt1 sample (Fig. 4B). Contrary to that reported by Lim et al. (1998), we observed consistently that aDMA was eluted from the HPLC column in front of sDMA. The discrepancy was possibly due to the different columns used in the amino acid separation.
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Proteins interact with Sbp1p or Sbp1p/hmt1
Sbp1p and Sbp1p/hmt1 were covalently linked to solid supports for pull-down assays. The interacting proteins were resolved on a denaturing polyacrylamide gel and presented in Figure 7. Noticeably, the Sbp1p sample has several bands (2, 3, 6, 11, 12, and 13) with higher dye-binding intensity than that of the Sbp1p/hmt1 sample, while band four of the Sbp1p/hmt1 sample is more intense than that of the Sbp1p sample. Apparently, methylation of Sbp1p changes its affinity, positively or negatively, toward several proteins. The protein patterns on Figure 7 are highly reproducible. The pull-down experiments have been performed four times, and we could not detect significant differences in protein patterns among the gels.
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Discussion |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsElectronic supplemental materialAcknowledgmentsReferences |
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The recombinant Sbp1p and Sbp1p/hmt1 were analyzed by mass spectrometry. According to the mass difference between Sbp1p and Sbp1p/hmt1, the modified proteins can carry up to 26 methyl groups (Fig. 3). Most of these methyl groups are added onto the 13 arginine residues within the arginine-glycine-rich domain of the protein, as demonstrated by the peptide maps of the chymotrypsin digests (Fig. 5; Table 1). Some of the His6-tags on the recombinant proteins have been modified with an addition of 178 Da (Fig. 3C). Geoghegan et al. (1999) have reported the isolation of modified His-tags from several recombinant proteins expressed in E. coli. The 
-amino group was blocked with a gluconoyl moiety and an addition of 178 Da. Possibly, some of the recombinant Sbp1p has an -N-D-gluconoyl-His6-tag.
Stm1p and Stm1p/hmt1 are unstable and fragmented easily during the purification process. The full-length proteins for mass measurement can be obtained with an additional chromatographic step on a reverse-phase column. The difference in mass between Stm1p (32,001 ± 3 Da) and Stm1p/hmt1 (32,027 ± 2 Da) implies an addition of two methyl groups. However, repeated peptide-mapping experiments on a MALDI-TOF instrument were unable to confirm the methylation site on Stm1p/hmt1.
Stm1p has a RGG motif near the C terminus of the protein (233FNNRRGGRGARKG245) and Arg237 within this motif is the most probable methylation site. We generated single mutants by replacing the arginines between residues 233 and 245 with lysine. The proteins were used as substrates in an in vitro methylation assay. The Stm1p R237K mutant is a poor substrate for Hmt1p (Fig. 6A,B, lanes 6). We can abolish the methylation reaction by replacing the arginine on the thrombin cleavage site of the R237K mutant with lysine (Fig. 6A,B, lanes 6). Therefore, Arg237 on Stm1p is the methylation site.
Even though the thrombin cleavage site (LVPRGS) does not contain an RGG or RXR sequence, it can be methylated by Hmt1p (Fig. 6B, lanes 3, 6). The same modification has been observed on recombinant Hmt1p with N-terminal His6-tag fusion (T.-C.S. Tam, L.-F. Liu, and M.F. Tam, unpubl.). The modified proteins are resistant to thrombin cleavage.
To affirm the Stm1p methylation site, the R236K, R237K, R240K, and R243K mutants were coexpressed with Hmt1p. The proteins were isolated for mass analysis. As expected, the R237K mutant (31,972 ± 2 Da) is 28 Da lighter than the other three mutants (32,000 ± 1 Da). Hence, Arg237 is the methylation site on Stm1p.
Sbp1p is a 33-kDa, single-strand nucleic acid-binding protein involved in RNA metabolism. Gavin et al. (2006) screened for protein complexes in Saccharomyces cerevisiae, and Sbp1p was detected in seven different complexes that are composed of at least 82 proteins. We identified 20 Sbp1p and Sbp1p/hmt1 interacting proteins, and 13 of them are among the list (Rpl4Ap, Rpl7Ap, Rpl8Bp, Rpl13Bp, Rpl16Ap, Rpl20Bp, Rps4Ap, Rps8Bp, Rps9Bp, Imd3p, Tef1p, Nop13p, and Nop1p) provided by Gavin et al. (2006).
Mam33p was detected only in the Sbp1p sample, together with Rps4Ap and Rpl2Bp (Fig. 6, band 9; Table 2). It is an acidic protein of the mitochondrial matrix and participates in oxidative phosphorylation (Seytter et al. 1998). Mam33p also binds Tef1p (Gavin et al. 2006), which has a higher affinity toward Sbp1p than Sbp1p/hmt1 (Fig. 7, band 2). We speculate that Mam33p does not interact directly with Sbp1p. Possibly, Mam33p was pulled down together with Tef1p.
Sbp1p is an in vivo substrate of Hmt1p. Sbp1p isolated from wild-type BY4741 cells carries arginine methylation. This modification is absent from Sbp1p isolated from BY4741 with HMT1 deletion (C.H. Hsieh, L.F. Liu, and M.F. Tam, in prep.). We observed a strong interaction between these two proteins (Fig. 7, band 6). Interestingly, Sbp1p/hmt1 is no longer a Hmt1p substrate, and this protein–protein interaction is abolished almost completely.
Nop13p is a nucleolar protein of unknown function (Wu et al. 2001) and detected only in the Sbp1p/hmt1 sample (Fig. 7, band 6; Supplemental Fig. S1). Nop13p can interact with Imd3p (Ho et al. 2002). Since the amount of Imd3p in the Sbp1p/hmt1 sample is significantly less than that in the Sbp1p sample (Fig. 7, band 6), we propose that Nop13p interacts with Sbp1p/hmt1 without the participation of Imd3p.
The extra protein band (band 4) in the Sbp1p/hmt1 sample represents the recombinant Sbp1p and Mas1p (Supplemental Fig. S2). Sbp1p and Sbp1p/hmt1 are dimeric in solution. Sbp1p/hmt1 dimers dissociate more readily than Sbp1p dimers under acid condition (data not shown). Probably, a minor population of Sbp1p/hmt1 did not have both subunits covalently bound to the solid supports. The dimers dissociated during the elution step in the presence of trifluoroacetic acid and mixed with the interacting proteins.
Mas1p is the smaller subunit of the mitochondrial processing protease (Witte et al. 1988). It also interacts with Rpl16Ap, another protein detected only in the Sbp1p/hmt1 sample (Fig. 7, band 13; Table 2). Further experiments are needed to determine whether Mas1p interacts directly with Sbp1p/hmt1.
There are 11 and 12 ribosomal proteins that interact with Sbp1p and Sbp1p/hmt1, respectively. Among them Rps8Bp, Rps9Bp, Rpl7Ap, and Rpl13Bp (Fig. 7, bands 11, 12, 13) have decreased affinity while Rpl16Ap (Fig. 6, band 13; Supplemental Fig. S3) can only be found in the Sbp1p/hmt1 sample. Additional experiments are needed to elucidate whether Sbp1p arginine methylation would affect ribosome assembly/processing.
Protein arginine methylation can exert either a positive or negative effect on protein–protein interactions. For example, arginine methylation inhibits the interaction of Npl3p with the TREX protein complex that participates in mRNA transport (Yu et al. 2004). Conversely, methylation of NIP45 promotes its interaction with NF-AT (nuclear factor of activated T cell), resulting in elevated cytokine production (Mowen et al. 2004).
Both positive and negative effects on protein interaction can be observed for the methylated Sbp1p (Fig. 7). For most proteins reported, the interacting proteins are regulated either positively or negatively by arginine methylation (Bedford et al. 2000; Friesen et al. 2001; Mowen et al. 2004; Yu et al. 2004). In this study, we used either completely unmodified or proteins highly enriched with arginine methylation in our experiments. Thus, our data are easier for interpretation.
In summary, we report here an expression system for the preparation of proteins with enriched methyl arginines. These recombinant proteins can be used as baits in pull-down experiments and identify proteins that interact preferentially with either the methylated or unmodified form of the query proteins. This simple expression system can be applied to other proteins with arginine methylation for protein–protein interaction or biochemical studies.
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Materials and Methods |
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HMT1 is available in the laboratory (Chern et al. 2002). It was inserted into pBS-MP (Hwang et al. 1999) to replace the coding region without changing the promoter sequence of the E. coli methionine aminopeptidase (MAP) gene (pBS-MAPp-hmt1). The HMT1 gene with the MAP promoter was then amplified from pBS-MAPp-hmt1 and inserted into the NotI site of a pGEX-STM1 plasmid, which is 11 nucleotides 3' to the stop codon of STM1. The STM1 and HMT1 genes in tandem were then amplified with 5' and 3' primers carrying NdeI and BamHI sites, respectively. The PCR product starts with the initiating codon of STM1 at the 5' end and inserted into the pET-15b vector to generate pET-STM1-(MAPp-hmt1). The sequence on the pET-15b vector encoding the His6-tag is between the NcoI and NdeI sites. Hence, STM1 is expressed as an N-terminal His6-tag fusion protein under the control of a T7 promoter. Finally, the STM1 on pET-STM1-(MAPp-hmt1) was replaced with SBP1 for Sbp1p/hmt1 production (pET-SBP1-[MAPp-hmt1], Fig. 1). In addition, the arginine residue on the thrombin cleavage site of all of the expression plasmids mentioned above (pET-SBP1, pET-STM1, pET-STM1-[MAPp-hmt1] and pET-SBP1-[MAPp-hmt1]) was replaced with a lysine residue. Site-directed mutagenesis of the thrombin cleavage site and generation of the Stm1p R236K, R237K, R240K, and R243K mutants were carried out with QuickChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions.
E. coli BL21(DE3) cells were used as host for protein expression. Cells transformed with plasmids were grown in Lauia broth at 30°C until the culture reached an absorbance of 0.6 at 600 nm. Isopropyl 
-D-1-thiogalactopyranoside was then added (IPTG, 1 mM final concentration) and cells were cultured for 4 h before harvesting. After cell lysis, the recombinant proteins were affinity purified with Ni2 chelating beads (Novagen) according to the manufacturer's instructions.
Amino acid analysis
Purified proteins were hydrolyzed in the gas phase (1 h at 150°C) with 6 N HCl. The hydrolysates were derivatized with phenylisothiocyanate according to the Pico-Tag amino acid analysis handbook (Waters Corporation). The phenylthiocarbamyl amino acids were separated on a reverse-phase column as reported by Lim et al. (1998), with modifications. Briefly, a Pico-Tag column was maintained at 43°C and equilibrated with 2.5% (v/v) acetonitrile in 70 mM sodium acetate (pH 6.5), and 3.6 mM triethylamine (Solvent A) at a flow-rate of 1 mL/min. After sample loading, the column was washed sequentially with 10 mL of Solvent A and 10 mL of 3% Solvent B (15% [v/v] methanol and 45% acetonitrile [v/v] in water). The methylated arginines were then eluted from the column with a shallow gradient going from 3% to 4% Solvent B in 15 min.
In vitro methylation assay
Reactions were carried out in 50 mM sodium phosphate (pH 7.2), at a final volume of 20 µL. The reaction mixture contained 50 pmols of protein substrates and 0.65 µM (0.83 µCi) of S-Adenosyl-L-[methyl-3H]methionine ([3H]AdoMet; 63.3 Ci/mmol, 0.55 mCi/mL). The enzyme (Hmt1p) to substrate ratio (mole/mole) was set at 1:2 for Stm1p or Stm1p/hmt1, and 1:20 for Sbp1p or Sbp1p/hmt1. Incubation was carried out at 30°C for 30 min. SDS-PAGE sample buffer was added to stop the methylation reaction and proteins were separated on precast Novex 4%–20% polyacrylamide gel (Invitrogen). Proteins were visualized with Blue-silver stain (Candiano et al. 2004), then dried and subjected to fluorography.
Preparation of yeast extracts
Yeast strain BY4742 with SBP1 deletion was obtained from Research Genetics. Cells were grown with constant shaking at 30°C in YPD medium (1% [w/v] yeast extract, 2% [w/v] bactopeptone, and 2% [w/v] D-glucose) to 1 O.D. at 600 nm. Cells were collected and resuspended in 50 mM sodium phosphate (pH 7.5), 0.1 M NaCl, and 10% glycerol. Cell breakage was induced by passing the suspension through an EmulsiFlex-C5 homogenizer (Avestin). IGEPAL CA-630, a nonionic detergent, was added to the cell lysates to a final concentration of 1%. The mixture was incubated at 4°C for 30 min with gentle swirling before cell debris and organelles were removed by ultracentrifugation (400,000g, 30 min). Proteins in the extracts were quantified by the dye-binding method of Bradford (1976) and used immediately for the pull-down assay.
Pull-down assay
Sbp1p and Sbp1p/hmt1 were covalently linked to Affi-Gel 10 (Bio-Rad) according to the manufacturer's instructions. The amount of proteins bound onto Affi-Gel 10 was estimated by quantifying the proteins remaining in the coupling solution after the reaction. Equal amounts of Affi-Gel 10 beads with the functional groups blocked were used as controls in the assays. Cell lysates at 50-fold (w/w) excess of linked proteins were incubated with the beads for 16 h at 4°C. The gel slurries were transferred into columns and washed with 10 column volumes of 50 mM sodium phosphate (pH 7.0) and 100 mM NaCl. The salts on the columns were then removed with two bed volumes of water and eluted immediately with 1% trifluoroacetic acid. The acid was removed in vacuo and the proteins were analyzed by SDS-PAGE.
Enzyme digestion
Sbp1p and Sbp1p/hmt1 were digested with Achromobacter protease 1 or chymotrypsin. Achromobacter protease 1 digestions were carried out overnight in 50 mM NH4HCO3 (pH 9.0), 10% (v/v) acetonitrile, and 10% (v/v) glycerol at 42°C, and an enzyme to substrate ratio (w/w) of 1:50. The protein N-terminal fragments were recovered with Ni2+ chelating beads and then desalted on ZipTipC18 pipette tips (Millipore Corp.) before MALDI-TOF mass spectrometric analysis.
Chymotrypsin digestions were carried out in 100 mM Tris-HCl (pH 7.8), and 10 mM CaCl2. The enzyme to substrate ratio was set at 1:40. Incubation was carried out at 25°C for 2.5 h. The reaction was stopped by lowering the pH of the mixture to four with the addition of 2% trifluoroacetic acid. The mass of the resulting peptides were determined on a MALDI-TOF mass spectrometer.
In-gel digestion of Sbp1p interacting proteins with trypsin was carried out essentially with the accelerated digestion protocol of Havlis et al. (2003), except that the digestion temperature was set at 50°C (Finehout et al. 2005).
Mass spectrometry
The molecular masses of recombinant proteins were determined on a Bruker (Bruker-Daltonics) Apex-IV 7.0e instrument with an Apollo electrospray source. The samples were desalted on ZipTipC4 pipette tips and then infused into the instrument at 1.5 µL/min and spectra were recorded in broad-band mode. The instrument was mass calibrated externally with an electrospray calibrant solution (G2421A) purchased from Agilent Technologies immediately prior to sample infusion. Spectra deconvolution was performed with the XMASS software package included with the instrument.
Peptide mass analysis of Sbp1p and Sbp1p/hmt1 chymotrypsin digests and mass determination of the protein N-terminal His6-tags were performed on a Bruker REFLEX II time-of-flight mass spectrometer equipped with a Scout source and delayed extraction. Samples were prepared on an AnchorChip with -cyano-4-hydroxycinnamic acid (CHCA) and nitrocellulose as matrix (Wu et al. 2006). Ions from Angiotensin II (1046.54) and CHCA ion clusters (855.07, 644.05, 568.14, and 379.09) or from a mixture of Angiotensin II and peptides generated from Lys-C digestion of horse heart myoglobin (1360.75, 1502.67, and 2859.49) were used as external calibration standards. Detection was set in positive reflector mode, with each mass determination being the average of 100 spectra.
Mass spectra for in-gel digests were acquired in reflector mode on a Bruker Autoflex time-of-flight instrument. Each sample was acquired separately with CHCA/nitrocellulose (Wu et al. 2006) and CHCA/pyridine as matrices. Data from both spectra were combined for database search. The CHCA/pyridine matrix is an acetonitrile solution containing 5 mM pyridine, 2 mg/mL of CHCA, and 0.1% (v/v) trifluoroacetic acid. The preparation is a modification of that reported by Zabet-Moghaddam (2006) for metal sample targets. Equal volumes of sample and matrix were mixed and deposited onto a 600-mm/384 well AnchorChip sample target for data acquisition. Spectra were acquired in positive-ion mode with ions from trypsin fragments (842.51, 1045.56, and 2211.10) as internal standards. Peptide masses obtained with signal to noise (S/N) ratio higher than seven were searched against the NCBInr database using the MASCOT program (http://www.matrixscience.com) for all taxonomy with 100 ppm peptide mass tolerance, one missed cleavage, and oxidized methionine as variable modification.
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Reprint requests to: Dr. Ming F. Tam, Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan 11529, Republic of China; e-mail: mftam{at}gate.sinica.edu.tw; fax: 886-2-2782-6085.
Abbreviations: PRMTs, protein arginine N-methyltransferases; aDMA, asymmetric -NG,NG-dimethylarginine; sDMA, symmetric -NG,NG’-dimethylarginine; -MMA, -monomethylarginine; [3H]AdoMet, S-Adenosyl-L-[methyl-3H]methionine; CHCA, -cyano-4-hydroxycinnamic acid; Sbp1p/hmt1, Sbp1p coexpressed with HMT1; Stm1p/hmt1, Stm1p coexpressed with HMT1.
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.062667407.
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