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• W2122323877 abstract "Post-translational modification by small ubiquitin-like modifier 1 (SUMO-1) is a highly conserved process from yeast to humans and plays important regulatory roles in many cellular processes. Sumoylation occurs at certain internal lysine residues of target proteins via an isopeptide bond linkage. Unlike ubiquitin whose carboxyl-terminal sequence is RGG, the tripeptide at the carboxyl terminus of SUMO is TGG. The presence of the arginine residue at the carboxyl terminus of ubiquitin allows tryptic digestion of ubiquitin conjugates to yield a signature peptide containing a diglycine remnant attached to the target lysine residue and rapid identification of the ubiquitination site by mass spectrometry. The absence of lysine or arginine residues in the carboxyl terminus of mammalian SUMO makes it difficult to apply this approach to mapping sumoylation sites. We performed Arg scanning mutagenesis by systematically substituting amino acid residues surrounding the diglycine motif and found that a SUMO variant terminated with RGG can be conjugated efficiently to its target protein under normal sumoylation conditions. We developed a Programmed Data Acquisition (PDA) mass spectrometric approach to map target sumoylation sites using this SUMO variant. A web-based computational program designed for efficient identification of the modified peptides is described. Post-translational modification by small ubiquitin-like modifier 1 (SUMO-1) is a highly conserved process from yeast to humans and plays important regulatory roles in many cellular processes. Sumoylation occurs at certain internal lysine residues of target proteins via an isopeptide bond linkage. Unlike ubiquitin whose carboxyl-terminal sequence is RGG, the tripeptide at the carboxyl terminus of SUMO is TGG. The presence of the arginine residue at the carboxyl terminus of ubiquitin allows tryptic digestion of ubiquitin conjugates to yield a signature peptide containing a diglycine remnant attached to the target lysine residue and rapid identification of the ubiquitination site by mass spectrometry. The absence of lysine or arginine residues in the carboxyl terminus of mammalian SUMO makes it difficult to apply this approach to mapping sumoylation sites. We performed Arg scanning mutagenesis by systematically substituting amino acid residues surrounding the diglycine motif and found that a SUMO variant terminated with RGG can be conjugated efficiently to its target protein under normal sumoylation conditions. We developed a Programmed Data Acquisition (PDA) mass spectrometric approach to map target sumoylation sites using this SUMO variant. A web-based computational program designed for efficient identification of the modified peptides is described. Protein modification by SUMO 1The abbreviations used are: SUMO, small ubiquitin-like modifier; PDA, programmed data acquisition; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; IDA, information-dependent acquisition; GFP, green fluorescent protein; ISG15, interferon stimulated gene 15; PDA, Programmed Data Acquisition. 1The abbreviations used are: SUMO, small ubiquitin-like modifier; PDA, programmed data acquisition; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; IDA, information-dependent acquisition; GFP, green fluorescent protein; ISG15, interferon stimulated gene 15; PDA, Programmed Data Acquisition. is emerging as an important regulatory event in many cellular processes (1Melchior F. SUMO—nonclassical ubiquitin.Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Google Scholar, 2Schwartz D.C. Hochstrasser M. A superfamily of protein tags: ubiquitin, SUMO and related modifiers.Trends Biochem. Sci. 2003; 28: 321-328Google Scholar, 3Johnson E.S. Protein modification by SUMO.Annu. Rev. Biochem. 2004; 73: 355-382Google Scholar). Although SUMO-1 is only 18% identical to ubiquitin, they display high structural homology, and sumoylation occurs by a mechanism closely related to that of ubiquitination. As such, it involves an E1-activating enzyme, which in the case of SUMO is a heterodimer, Aos1/Uba2. Like the ubiquitination pathway, there is one E1 common to all SUMO substrates. The E1 is charged with SUMO in an ATP-dependent fashion via a thioester linkage between the active site Cys of Uba2 and the carboxyl-terminal Gly of SUMO. Subsequently SUMO is passed to an E2-conjugating enzyme where it is again covalently linked through a thioester bond, paralleling once more the ubiquitination mechanism (1Melchior F. SUMO—nonclassical ubiquitin.Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Google Scholar, 2Schwartz D.C. Hochstrasser M. A superfamily of protein tags: ubiquitin, SUMO and related modifiers.Trends Biochem. Sci. 2003; 28: 321-328Google Scholar, 3Johnson E.S. Protein modification by SUMO.Annu. Rev. Biochem. 2004; 73: 355-382Google Scholar). However, an interesting difference arises here; in the case of ubiquitination, there are dozens of E2s with known conjugating activity, whereas in the case of SUMO, there is only one known E2, Ubc9 (1Melchior F. SUMO—nonclassical ubiquitin.Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Google Scholar, 2Schwartz D.C. Hochstrasser M. A superfamily of protein tags: ubiquitin, SUMO and related modifiers.Trends Biochem. Sci. 2003; 28: 321-328Google Scholar, 3Johnson E.S. Protein modification by SUMO.Annu. Rev. Biochem. 2004; 73: 355-382Google Scholar). An additional intriguing divergence between the two pathways is that although ubiquitination requires an E3 ligase enzyme to complete the transfer of ubiquitin to the substrate protein, sumoylation of many substrates apparently does not (1Melchior F. SUMO—nonclassical ubiquitin.Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Google Scholar, 2Schwartz D.C. Hochstrasser M. A superfamily of protein tags: ubiquitin, SUMO and related modifiers.Trends Biochem. Sci. 2003; 28: 321-328Google Scholar, 3Johnson E.S. Protein modification by SUMO.Annu. Rev. Biochem. 2004; 73: 355-382Google Scholar). This has been ascribed to Ubc9 binding directly to many SUMO substrates and is displayed by the fact that sumoylation can occur in the absence of any E3 in a totally reconstituted in vitro system. Although a number of E3s specific to the sumoylation pathway have now been identified, they do not appear to be essential for the transfer of SUMO to the target molecule. However, when an E3 specific to the substrate is added, it generally enhances the rate and degree of sumoylation. To date, the SUMO E3 family is small and includes such members as RanBP2, Pc2, and the protein inhibitor of activated stat 1 proteins (1Melchior F. SUMO—nonclassical ubiquitin.Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Google Scholar, 2Schwartz D.C. Hochstrasser M. A superfamily of protein tags: ubiquitin, SUMO and related modifiers.Trends Biochem. Sci. 2003; 28: 321-328Google Scholar, 3Johnson E.S. Protein modification by SUMO.Annu. Rev. Biochem. 2004; 73: 355-382Google Scholar). Currently there are more than 60 proteins that have been shown to be capable of undergoing sumoylation (1Melchior F. SUMO—nonclassical ubiquitin.Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Google Scholar). Based on the limited number of proteins that have been identified as SUMO targets, SUMO has been implicated to be involved in protein transport, transcription regulation, protein stability, and localization to nuclear bodies (1Melchior F. SUMO—nonclassical ubiquitin.Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Google Scholar). In general, SUMO modification targets the consensus sequence ΨKX(D/E) where Ψ represents a hydrophobic residue and K is the acceptor lysine (1Melchior F. SUMO—nonclassical ubiquitin.Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Google Scholar, 4Muller S. Hoege C. Pyrowolakis G. Jentsch S. SUMO, ubiquitin’s mysterious cousin.Nat. Rev. Mol. Cell. Biol. 2001; 2: 202-210Google Scholar, 5Hochstrasser M. Biochemistry. All in the ubiquitin family.Science. 2000; 289: 563-564Google Scholar); however, nonconsensus sumoylation sites have also been reported for several SUMO targets (6Denison C. Rudner A.D. Gerber S.A. Bakalarski C.E. Moazed D. Gygi S.P. A proteomic strategy for gaining insights into protein sumoylation in yeast.Mol. Cell. Proteomics. 2005; 4: 246-254Google Scholar, 7Chung T.L. Hsiao H.H. Yeh Y.Y. Shia H.L. Chen Y.L. Liang P.H. Wang A.H. Khoo K.H. Shoei-Lung Li S. In vitro modification of human centromere protein CENP-C fragments by small ubiquitin-like modifier (SUMO) protein: definitive identification of the modification sites by tandem mass spectrometry analysis of the isopeptides.J. Biol. Chem. 2004; 279: 39653-39662Google Scholar). Thus, a precise sumoylation site has to be defined experimentally for each target protein. Tandem mass spectrometry amino acid sequencing is the most direct, unbiased, and sensitive approach to determine the site of post-translational modifications. This method has been successfully used to identify ubiquitination sites and sumoylation sites in yeast (6Denison C. Rudner A.D. Gerber S.A. Bakalarski C.E. Moazed D. Gygi S.P. A proteomic strategy for gaining insights into protein sumoylation in yeast.Mol. Cell. Proteomics. 2005; 4: 246-254Google Scholar, 8Peng J. Schwartz D. Elias J.E. Thoreen C.C. Cheng D. Marsischky G. Roelofs J. Finley D. Gygi S.P. A proteomics approach to understanding protein ubiquitination.Nat. Biotechnol. 2003; 21: 921-926Google Scholar). The isopeptide bond formed between the carboxyl-terminal glycine and the lysine residue targeted for ubiquitination renders this lysine resistant to trypsin cleavage. Thus proteolysis of ubiquitinated substrates by trypsin will yield a signature peptide showing a missed cleavage at the modified lysine residue and a 114.1-Da increase in peptide mass due to covalent attachment of the two glycines to the lysine (8Peng J. Schwartz D. Elias J.E. Thoreen C.C. Cheng D. Marsischky G. Roelofs J. Finley D. Gygi S.P. A proteomics approach to understanding protein ubiquitination.Nat. Biotechnol. 2003; 21: 921-926Google Scholar). Detection of the signature peptide not only enables identification of the proteins undergoing ubiquitination but also allows precise determination of the ubiquitination sites (6Denison C. Rudner A.D. Gerber S.A. Bakalarski C.E. Moazed D. Gygi S.P. A proteomic strategy for gaining insights into protein sumoylation in yeast.Mol. Cell. Proteomics. 2005; 4: 246-254Google Scholar, 8Peng J. Schwartz D. Elias J.E. Thoreen C.C. Cheng D. Marsischky G. Roelofs J. Finley D. Gygi S.P. A proteomics approach to understanding protein ubiquitination.Nat. Biotechnol. 2003; 21: 921-926Google Scholar). Unfortunately this signature peptide approach is difficult to apply to identification of sumoylation sites in mammalian cells because the carboxyl-terminal region of mammalian SUMO-1, -2, and -3 lacks Lys or Arg residues (Fig. 1) (9Mahajan R. Gerace L. Melchior F. Molecular characterization of the SUMO-1 modification of RanGAP1 and its role in nuclear envelope association.J. Cell Biol. 1998; 140: 259-270Google Scholar). Here we describe a method that enables application of the signature peptide approach to identify protein sumoylation sites in mammalian systems using mass spectrometry. pET11d-Uba2, pET28a-H-Aos1, pET23a-Ubc9, and pET11d-RanGAP-1 were gifts from Dr. Frauke Melchior. SUMO-1-(1–97) cDNA, kindly provided by Drs. Seeler and Dejean, was amplified by PCR and cloned into a modified pRAV-Flag vector in which the Protein A domains were deleted and replaced with FLAG and His6 tags (10Knuesel M. Wan Y. Xiao Z. Holinger E. Lowe N. Wang W. Liu X. Identification of novel protein-protein interactions using a versatile Mammalian tandem affinity purification expression system.Mol. Cell. Proteomics. 2003; 2: 1225-1233Google Scholar). Vector construction details are available upon request. SUMO-1 mutants with Arg substitution at various positions indicated were constructed using a QuikChange mutagenesis kit (Stratagene). GST-tagged Ubc9 was generated by subcloning Ubc9 into pGEX-4T-1 using PCR. Recombinant GST-tagged Uba2 and Aos1 were produced with a baculoviral expression system using the pFAST-Bac1 expression vector (Invitrogen). Recombinant mouse RanGAP1 and recombinant human Aos1/Uba2, Ubc9, and SUMO-1 were purified as described previously (11Pichler A. Gast A. Seeler J.S. Dejean A. Melchior F. The nucleoporin RanBP2 has SUMO1 E3 ligase activity.Cell. 2002; 108: 109-120Google Scholar). GST fusion proteins were purified according to standard procedures (GE Healthcare). Fluorescent labeling of the amino-terminal amine of purified SUMO-1 wild type and SUMO-1 (T95R) was performed using Alexa Fluor 555 carboxylic acid, succinimidyl ester (Molecular Probes A-20009) according to the manufacturer’s protocol. Thioester assays, containing 50 ng of fluorescently labeled SUMO-1, 200 ng of GST-tagged E1, and 1× ER (7.5 mm creatine phosphate, 1 mm ATP, 0.1 mm EGTA, and 1 mm MgCl2) in thioester buffer (20 mm Tris, pH 7.6, 50 mm NaCl, and 10 mm MgCl2) were incubated at 30 °C for 60 min and quenched with non-reducing SDS buffer to a final concentration of 50 mm Tris, pH 6.8, 2% SDS, 2 m urea, and 10% glycerol or with reducing SDS buffer to a final concentration of 50 mm Tris, pH 6.8, 2% SDS, 0.35% β-mercaptoethanol, and 10% glycerol as noted. Reduced samples were then boiled for 2 min. Proteins were separated on a 4–20% polyacrylamide gel and visualized by scanning at 555 nm with a Typhoon scanner (Amersham Biosciences). To confirm equal input of E1 proteins, the gels were stained with Coomassie Blue after fluorescent scanning. In vitro sumoylation assays were conducted essentially as described previously (11Pichler A. Gast A. Seeler J.S. Dejean A. Melchior F. The nucleoporin RanBP2 has SUMO1 E3 ligase activity.Cell. 2002; 108: 109-120Google Scholar) in a total volume of 20 μl in Transport Buffer (20 mm HEPES, 110 mm potassium acetate, 2 mm magnesium acetate, and 0.5 mm EGTA). Reactions containing ∼25 ng in vitro translated [35S]Met-labeled SUMO-1, 150 ng of recombinant RanGAP1, 75 ng of Aos1/Uba2 heterodimer, 40 ng of Ubc9, 1× ER, and 1 mm cycloheximide were incubated in transport buffer at 37 °C for 90 min. After completion, the reactions were quenched with SDS loading buffer and boiled for 2 min before separation on 12% polyacrylamide gels. The gels were dried under vacuum, and phosphorimaging was performed using a Typhoon scanner. For the reactions used to map the sumoylation site of RanGAP1, 12 μg of purified RanGAP1, 3 μg of GST-Aos1/Uba2 heterodimer, 1.75 μg of GST-Ubc9, and 6 μg of SUMO-1 (T95R) were incubated in transport buffer supplemented with 1× ER for 90 min at 37 °C. E1 and E2 proteins were subsequently depleted by incubation with glutathione-Sepharose 4B beads. Ten percent of the total reaction volumes were removed for SDS-PAGE analysis to determine sumoylation efficiencies. Disulfides were reduced in solution by incubating the completed reactions with 4 mm DTT at 50 °C for 10 min. Cysteine carbamidomethylation was performed with the addition of 14 mm iodoacetamide at room temperature for 30 min in the dark and was quenched with the addition of 3 mm DTT. Samples were then prepared for trypsin digestion by adding 20 mm Tris, pH 8.0, 2 m urea, and 1 mm CaCl2. Digestions were carried out by adding trypsin (Wako Chemical) at 1% total protein weight and incubating for 16 h at 37 °C. Digestion reactions were quenched with addition of 1% formic acid. Samples were frozen at −20 °C prior to analysis by mass spectrometry. Ubl Finder (Ubiquitin Like Molecule Site Finder) was written to calculate the m/z value of the +1, +2, +3, and +4 charges of the peptide for a given protein (bmf.colorado.edu/ublfinder/). At present, Ubl Finder can be used to calculate the m/z value of the peptides for T95R-sumoylated, -ubiquitinated, or unmodified proteins. In brief, for each peptide that contains a potential (T95R) sumoylation site on a lysine residue, the m/z value of the diglycine tag is added to the m/z value of the peptide. In the case where no modification is chosen, all m/z values for each of the peptides in the protein will be reported. All charges for each of the potential peptides (+1, +2, +3, and +4) are accounted for, and all m/z values are reported in a tab- or comma-delimited format for easy input into the mass spectrometer. Portions of peptides derived from trypsinolysis of SUMO-1 (T95R)-modified RanGAP1 were loaded onto a pre-equilibrated, in-house-packed fused silica 25 cm × 250 μm-inner diameter HPLC column (Polymicro Technologies) packed with Poros 20 R2 reverse phase HPLC resin (Applied Biosystems) at 10 μl/min in buffer A (1% formic acid in water) on an Agilent 1100 Series HPLC system. Samples were loaded manually with a 100-μl injection loop. Peptides were eluted at 5 μl/min with the following gradient of buffer B (80% acetonitrile and 1% formic acid in water): 0–20 min, 0–60% B; 20–25 min, 60–100% B; 25–26 min, 100% B; 26–27 min, 100–0% B; 27–28 min, 0–100% B; 28–29 min, 100–0% B. The HPLC system was coupled directly to an API Q-Star Pulsar system equipped with an Ion Sprayer electrospray source (Applied Biosystems). Data were collected using an information-dependent acquisition (IDA) method programmed with a dynamic exclusion of redundant m/z values for 60 s and a fixed inclusion list of the possible +2 and +3 SUMO-1 (T95R)-modified peptides of RanGAP1 as determined by the Ubl Finder program (input list of 130 values) ±100 ppm. For each 15-s cycle of the IDA, a 2-s TOF spectrum of m/z ranges 450–2000 was followed by three CID MS/MS spectra of the most abundant peaks meeting the inclusion and exclusion input parameters. The MS/MS spectra were collected for 3, 4, and 6 s, respectively, with the m/z range of 40–2000. Nitrogen was used as the collision gas with collision energies varying as a function of m/z and charge state of the precursor ion. Data from the IDA experiments were searched against the NCBInr database or an in-house database containing only RanGAP1 and SUMO-1 (T95R) protein sequences using a modified version of Mascot with the following parameters: trypsin digestion, two missed cleavages (one missed cleavage would be expected from the GG-tagged Lys, and one additional missed cleavage in case surrounding Lys residues are not surface-exposed), variable modifications of Cys carbamidomethylation and GG-tagged Lys (Lys +114.06 programmed into the in-house version of Mascot), mass tolerance of ±0.2 (0.5) Da, MS/MS tolerance of ±0.2 (0.5) Da, and peptide charges of +1, +2, and +3. To study the sumoylation reaction in vitro, we first purified all of the components that are involved in catalyzing SUMO transfer in vitro, namely E1 (Aos1/Uba2 heterodimer), E2 (Ubc9), and SUMO-1 (Fig. 2A). Next we developed a quantitative and direct visualization assay to analyze reconstituted in vitro sumoylation reactions. To this end, we chose to fluorescently label the amino terminus of purified recombinant human SUMO-1 with Alexa Fluor 555 succinimidyl ester, carboxylic acid. The labeled SUMO-1 was tested in two well characterized in vitro sumoylation reactions. RanGAP1 was the first discovered sumoylation substrate and remains one of the most efficiently and heavily sumoylated proteins known thus far (12Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. A small ubiquitin-related polypeptide involved in targeting RanGAP1 to nuclear pore complex protein RanBP2.Cell. 1997; 88: 97-107Google Scholar, 13Saitoh H. Pu R.T. Dasso M. SUMO-1: wrestling with a new ubiquitin-related modifier.Trends Biochem. Sci. 1997; 22: 374-376Google Scholar). It has been shown that RanGAP1 sumoylation can be fully reconstituted in vitro with only E1 (Aos1/Uba2 heterodimer), E2 (Ubc9), SUMO-1, and ATP (11Pichler A. Gast A. Seeler J.S. Dejean A. Melchior F. The nucleoporin RanBP2 has SUMO1 E3 ligase activity.Cell. 2002; 108: 109-120Google Scholar). Therefore, fluorescently labeled SUMO-1 was added to this reaction and compared with unlabeled SUMO-1. As demonstrated in the Coomassie Blue-stained gel in Fig. 2B, fluorescently labeled SUMO-1 is incorporated into RanGAP1-SUMO-1 conjugates to the same degree as unlabeled SUMO-1. Moreover a sumoylation time course experiment (Fig. 2C) using fluorescently labeled SUMO-1 shows that the kinetics of the reaction are also very similar when the fluorescence scan is compared with previously published results for RanGAP1 sumoylation. Therefore, it is apparent that this method is a reliable, specific, and efficient way to assay sumoylation in vitro and may be extended to a variety of other reconstituted systems. During the course of our study of SUMO-1 processing in reticulocyte lysates, we discovered that in vitro translation of the processed form of SUMO-1 in rabbit reticulocyte lysates in the presence of [35S]Met always produces a labeled protein at about 80 kDa (Fig. 3B). A labeled protein at this position is absent when another ubiquitin-related protein, ISG15, is produced by the same procedure (Fig. 3B). To determine whether the 80-kDa band is a SUMO conjugate, amino-terminal FLAG-tagged SUMO-1 was synthesized in the presence of [35S]Met in the reticulocyte lysate, and the resulting labeled proteins were immunoprecipitated with the anti-FLAG (M2) antibody. As a control, 35S-labeled GFP was synthesized in the same lysate. As shown in Fig. 3C, both SUMO-1 and the 80-kDa protein were immunoprecipitated by FLAG antibody, suggesting that the 80-kDa protein contains SUMO-1. If the 80-kDa protein is a SUMO-1 conjugate, we would expect that adding fluorescently labeled SUMO-1 to the rabbit reticulocyte lysate could also result in appearance of this conjugate. This is indeed the case as appearance of the 80-kDa protein upon addition of fluorescently labeled SUMO-1 to the reticulocyte lysates is time-dependent, and addition of recombinant RanGAP1, E1, and E2 further enhances the amount of the 80-kDa labeled protein, suggesting that the 80-kDa protein is most likely RanGAP1 (Fig. 3D). Finally to definitively demonstrate that the endogenous RanGAP1 in the rabbit reticulocyte lysate can be sumoylated by the processed form of recombinant SUMO-1, FLAG tagged SUMO-1, purified from bacteria, was incubated with the rabbit reticulocyte lysate for 1 h in the presence of ATP. The lysates were subsequently subjected to FLAG affinity purification, and proteins collected on the FLAG beads were separated by SDS-PAGE prior to immunoblotting with an anti-RanGAP1 antibody (Fig. 3E). Direct Western blot of reticulocyte lysate was not successful due to significant background from the antibody. Therefore, purified recombinant RanGAP1, E1, and E2 incubated in the presence or absence of FLAG-SUMO-1 were used as controls instead. Immunoprecipitation of FLAG-tagged SUMO-1 led to the recovery of sumoylated RanGAP1 from rabbit reticulocyte lysate, suggesting RanGAP1 is sumoylated upon incubation with recombinant SUMO-1 and the 80-kDa protein labeled by 35S or fluorescent SUMO is sumoylated RanGAP1. Conjugation of processed SUMO-1 to endogenous or exogenous RanGAP1 in the reticulocyte lysate indicates that the machinery of protein sumoylation is intact in this system, thus providing a simple and reliable assay for testing a mutant SUMO-1 for its conjugation activity. Mammalian SUMO-1 is devoid of Lys or Arg in the carboxyl-terminal region surrounding the diglycine motif. Given our interest in identifying SUMO-1 mutants that contain a suitable tryptic digestion residue for mapping sumoylation sites using mass spectrometry, we performed systematic site-specific mutagenesis by changing each amino acid residue in the carboxyl-terminal region of SUMO-1 near the conserved diglycine residues to arginine and subsequently testing whether these Arg substitutions affect protein sumoylation in vitro (Fig. 4). Surprisingly we found that Arg substitutions in a number of positions in the carboxyl-terminal region of SUMO-1 do not appear to affect the efficiency of SUMO conjugation in vitro (Fig. 4A). Mutation of SUMO-1 Thr-95 to Arg results in a SUMO-1 variant with an RGG terminus identical to that of ubiquitin. Upon in vitro translation in the reticulocyte lysate in the presence of [35S]Met, SUMO-1 (T95R) can be efficiently conjugated to the endogenous RanGAP1 (Fig. 4A, lane 2). Furthermore recombinant SUMO-1 (T95R) produced in Escherichia coli can be conjugated in vitro to RanGAP1 in a reconstituted sumoylation system as efficiently as the wild type SUMO-1 (Fig. 4B), suggesting that a mutation at this position of SUMO-1 does not affect protein sumoylation in vitro. Arginine substitutions in other positions of SUMO-1 were also tested. None of the substitutions appear to have any significant deleterious effects on SUMO-1 conjugation (Fig. 4A, lanes 3–5). Because the SUMO-1 (T95R) mutant is most suitable for mass spectrometric analysis due to its similarity to ubiquitin at the carboxyl terminus, we focused our efforts on characterizing this mutant and its application to mapping sumoylation sites. Mutation of Thr-95 of SUMO-1 could potentially affect its specificity and interaction with SUMO-1-conjugating enzymes. To test whether SUMO-1 (T95R) retains its specificity, wild type and mutant SUMO-1 were incubated with E1 enzymes of ubiquitin (UbE1), SUMO (Aos1/Uba2), and ISG15 (UBE1L), and thioester bond formation between the E1s and SUMO-1 (T95R) was analyzed. As shown in Fig. 5, both wild type and SUMO-1 (T95R) were activated efficiently by the SUMO-activating enzyme as visualized by the appearance of bands corresponding to the fluorescent labeled SUMO-1 or SUMO-1 (T95R) covalently attached to Uba2. In addition, the amounts of fluorescent SUMO-E1 conjugates are partially sensitive to reducing conditions, suggesting thioester linkage between E1 and SUMO. The residual SUMO-E1 conjugates in the reducing lane could be due to incomplete reduction of E1 thioester. Importantly neither the ubiquitin E1 nor the ISG15 E1 can form covalent linkages with wild type or the SUMO-1 (T95R) mutant under similar conditions. The amounts of input E1 protein used in this assay were shown by Coomassie Blue staining (Fig. 5). Taken together, these data indicate that mutation of Thr-95 of SUMO-1 does not appear to perturb its normal function, and it behaves very similarly to wild type SUMO-1 in vitro. The tryptic cleavage site nearest to the conserved diglycine residues in wild type SUMO-1 is Lys-78 (Supplemental Fig. 1A). Tryptic digestion of SUMO-1-conjugated peptide will yield a branched peptide with a predicted mass increase of more than 3634 Da due to the SUMO-1 addition. Such large and branched peptides are difficult to capture and accurately analyze by the current mass spectrometry technology. Substitution of Thr-95 of SUMO-1 with arginine would create a trypsin cleavage site next to the diglycine motif. Digestion of SUMO-1 (T95R) conjugates with trypsin will yield a branched peptide containing the diglycine tag, which can be easily analyzed by mass spectrometry. To test whether SUMO-1 (T95R) is indeed applicable to this type of analysis, an in vitro RanGAP1 sumoylation assay with recombinant SUMO-1 (T95R) was performed. A portion of the sumoylation reaction was analyzed by SDS-PAGE and Coomassie Blue staining analysis (Fig. 6). The reminder of the reaction was subjected to trypsin digestion and loaded onto a reverse phase HPLC column. The column effluent was analyzed by an API Q-Star Pulsar LC-MS/MS system equipped with an Ion Sprayer electrospray source (Applied Biosystems). Previous work indicated that lysine 526 of RanGAP1 is the acceptor site for SUMO-1 (9Mahajan R. Gerace L. Melchior F. Molecular characterization of the SUMO-1 modification of RanGAP1 and its role in nuclear envelope association.J. Cell Biol. 1998; 140: 259-270Google Scholar). Trypsinolysis of SUMO-1 (T95R)-modified RanGAP1 is predicted to produce a characteristic sumoylation site peptide featuring a missed tryptic cleavage at the site of modification and a diglycine (GG) appendage on this modified lysine (Supplemental Fig. 1B). The LC-MS/MS data were initially collected in an IDA mode on the mass spectrometer running Analyst® QS software. The IDA mode enables a full MS scan and then performs MS/MS on the three most intense peaks from the full scan every 15 s. The IDA spectra were searched against the Mascot database with an additional 114.06 Da, corresponding to diglycine appendage, added to each theoretical peptide within the database. CID spectra were compared with theoretical peptide fragments to deduce specific sumoylation sites. Surprisingly we did not readily detect the sumoylation site signature peptide of RanGAP1 with the initial analytical method outlined. Because the IDA method favors high intensity precursor ions, peptides such as the ones with an isopeptide bond may not have been efficiently captured by HPLC or the mass spectrometer. Being that we are only interested in possible lysine modifications by SUMO-1, we reasoned that capturing and identifying the sumoylation site peptide will be greatly improved if we can instruct the mass spectrometer to collect MS/MS data only on the relevant peptides. This can be achieved by incorporating an inclusion list of theoretically calculated m/z values of the +1, +2, +3, and +4 charges of ions predicted for possible sumoylation sites with a diglycine appendage. Using a web-based program described below, an inclusion list consisting of the m/z value for each peptide with a potential diglycine sumoylation T95R tag on a lysine residue was generated. The trypsin-digested sumoylated RanGAP1 sample was subjected to LC-MS/MS analysis again using the inclusion list to focus on molecular ions of m/z predicted for possible sumoylation sites. CID spectra were compared with theoretical peptide fragments to deduce specific sumoylation sites. Because the data acquisition and ana" @default.
• W2122323877 created "2016-06-24" @default.
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• W2122323877 creator A5089345224 @default.
• W2122323877 date "2005-10-01" @default.
• W2122323877 modified "2023-10-17" @default.
• W2122323877 title "A Method of Mapping Protein Sumoylation Sites by Mass Spectrometry Using a Modified Small Ubiquitin-like Modifier 1 (SUMO-1) and a Computational Program" @default.
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