A Method of Mapping Protein Sumoylation Sites by Mass Spectrometry Using a Modified Small Ubiquitin-like Modifier 1 (SUMO-1) and a Computational Program*,S
Matthew Knuesel
,
Hiu Tom Cheung
,
,
Micah Hamady¶,
Kristen K. B. Barthel
,|| and
Xuedong Liu
,**
From the Departments of
Chemistry and Biochemistry and ¶ Computer Science, University of Colorado, Boulder, Colorado 80309
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ABSTRACT
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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 SUMO1 is emerging as an important regulatory event in many cellular processes (13). 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 (13). 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 (13). 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 (13). 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 (13).
Currently there are more than 60 proteins that have been shown to be capable of undergoing sumoylation (1). 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 (1). In general, SUMO modification targets the consensus sequence
KX(D/E) where
represents a hydrophobic residue and K is the acceptor lysine (1, 4, 5); however, nonconsensus sumoylation sites have also been reported for several SUMO targets (6, 7). 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 (6, 8). 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 (8). Detection of the signature peptide not only enables identification of the proteins undergoing ubiquitination but also allows precise determination of the ubiquitination sites (6, 8). 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) (9). Here we describe a method that enables application of the signature peptide approach to identify protein sumoylation sites in mammalian systems using mass spectrometry.

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FIG. 1. Sequence alignment of ubiquitin-like proteins. Alignment of several processed Ubls showing a common amino-terminal RGG motif, whereas SUMO family molecules terminate in a TGG sequence.
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EXPERIMENTAL PROCEDURES
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Protein Expression and Purification
pET11d-Uba2, pET28a-H-Aos1, pET23a-Ubc9, and pET11d-RanGAP-1 were gifts from Dr. Frauke Melchior. SUMO-1-(197) 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 (10). 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 (11). GST fusion proteins were purified according to standard procedures (GE Healthcare).
Fluorescent Labeling of SUMO-1
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 manufacturers protocol.
E1 Thioester Assays
Thioester assays, containing 50 ng of fluorescently labeled SUMO-1, 200 ng of GST-tagged E1, and 1x 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% ß-mercaptoethanol, and 10% glycerol as noted. Reduced samples were then boiled for 2 min. Proteins were separated on a 420% 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 Reaction and Sample Preparation for Mass Spectrometry Analysis
In vitro sumoylation assays were conducted essentially as described previously (11) 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, 1x 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 1x 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.
Generation of Inclusion List for Programmed Data Acquisition (PDA) Using the Web-based Ubl Finder Program
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.
HPLC and Mass Spectrometry Analysis
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 x 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): 020 min, 060% B; 2025 min, 60100% B; 2526 min, 100% B; 2627 min, 1000% B; 2728 min, 0100% B; 2829 min, 1000% 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 4502000 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 402000. 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.
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RESULTS
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A Sensitive and Quantitative Assay for Monitoring Protein Sumoylation in Vitro
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 (12, 13). 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 (11). 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.

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FIG. 2. Fluorescently labeled SUMO-1 can be easily detected both free and as high molecular weight conjugates. A, components of the sumoylation pathway can be purified to near homogeneity as detected by Coomassie Blue staining. Panel 1, purification of E1 heterodimer (His-Aos1, 40 kDa; Uba2, 70 kDa); Panel 2, purification of E2 Ubc9; Panel 3, purification of SUMO-1. B, fluorescently labeled SUMO-1 is incorporated as efficiently as unlabeled SUMO-1, and fluorescent SUMO-1 conjugates of RanGAP1 can be detected by scanning at 555 nm. A RanGAP1 sumoylation experiment is shown where lane 1 is 1 µg of recombinant mouse RanGAP1, lane 2 is two pooled in vitro sumoylation assays each using 150 ng of E1, 10 ng of E2, 150 ng of SUMO-1, an energy regeneration system, and 500 ng of RanGAP1 (30-min time point), and lane 3 is the same as the second except that 150 ng of fluorescently labeled SUMO-1 is used. C, fluorescently labeled SUMO-1 is conjugated to RanGAP1 with the same kinetics as unlabeled SUMO-1. A RanGAP1 sumoylation time course experiment is shown with conditions as in lane 3 of B. SUMO-1 conjugates were visualized by scanning the gel at 555 nm.
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RanGAP1 Sumoylation in Rabbit Reticulocyte Lysates
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.

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FIG. 3. Conjugation of the processed SUMO-1 but not ISG15 to RanGAP1 in rabbit reticulocyte lysate. cDNAs corresponding to processed SUMO-1 and ISG15 were cloned and epitope-tagged with FLAG in pREX-SP6-IRES-GFP vector. Recombinant SUMO-1 and ISG15 were synthesized in vitro using a coupled transcription/translation system (TNT system, Promega) in the presence of [35S]Met. The resulting products were analyzed by SDS-PAGE. The presence of the internal ribosome entry site (IRES) sequence allows translation of GFP independently of the upstream open reading frame. A, schematic diagrams of the relevant region of DNA encoding for SUMO-1 and ISG15. B, appearance of an 80-kDa [35S]Met-labeled protein, labeled with an asterisk, upon in vitro translation of SUMO-1 but not ISG15 in the presence of [35S]Met. The 30-kDa protein present in SUMO-1 and ISG15 is GFP. C, the 80-kDa protein is likely to be a SUMO-1 conjugate. Three labeled bands are produced upon in vitro translation of SUMO-1 (lane 1). The reaction products were subjected to FLAG affinity purification by passing the lysate through a column immobilized with anti-FLAG antibody. Proteins bound to the column were eluted with a FLAG peptide (1 mg/ml). Eluates and flow-through were analyzed by SDS-PAGE. D, time course analysis of the appearance of the 80-kDa protein in the reticulocyte lysate. Recombinant fluorescently labeled SUMO-1 was added to the reticulocyte lysates and incubated for the indicated times before reactions were stopped by adding SDS sample buffer. In lane 8, 0.5 µg of recombinant RanGAP1 was added to the rabbit reticulocyte lysate along with Alexa-SUMO-1 and incubated for 90 min. E, 150 ng of recombinant FLAG-SUMO-1 purified from E. coli was added to the reticulocyte lysates and incubated for 90 min (lane 1). Anti-FLAG-Sepharose beads were added and incubated for 1 h. The beads were recovered by a brief centrifugation and washed three times with TBS, and proteins collected on the beads were analyzed by SDS-PAGE. A portion of recombinant RanGAP1 incubated only with E1, E2, and in vitro sumoylated RanGAP1 were loaded on the gel as controls (lanes 2 and 3). Western blot analysis was performed with an anti-RanGAP1 antibody (Santa Cruz Biotechnology). IB, immunoblot.
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Conjugation of SUMO-1 Mutants with Arginine Substitutions near the Carboxyl-terminal Diglycine Motif
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 35). 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.

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FIG. 4. Conjugation of SUMO-1 mutants containing arginine substitutions near the carboxyl-terminal diglycine residues. A, wild type (WT) and SUMO-1 mutants were synthesized and labeled with [35S]Met by in vitro transcription/translation. Both wild type and various SUMO-1 substitution mutants can be conjugated to RanGAP1 in reticulocyte lysates. The amounts of input proteins were normalized by GFP. B, wild type and SUMO-1 (T95R) mutant can be conjugated to RanGAP1 with equal efficiencies in vitro. 100 ng of purified recombinant SUMO-1 and SUMO-1 (T95R) were incubated with 25 ng of in vitro translated RanGAP1 in the presence of 75 ng of SUMO E1 and 40 ng of Ubc9. Reactions were terminated by adding 2x SDS sample buffer after 90-min incubation at 37 °C.
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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.

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FIG. 5. E1 thioester assay shows specificity of SUMO-1 (T95R) to Aos1/Uba2 heterodimer. Upper panel, 50 ng of fluorescently labeled wild type (WT) SUMO-1 or SUMO-1 (T95R) mutant were incubated with 200 ng of SUMO E1 (GST-Aos1/GST-Uba2), ubiquitin E1 (GST-UbE1), or ISG15 E1 (GST-UBE1L) in the presence or absence of ATP. The reactions were quenched with SDS loading buffer either containing the reducing agent ß-mercaptoethanol or not as noted. A high molecular weight band that decreases in intensity when quenched with reducing buffer is consistent with a thioester bond between E1 and fluorescently labeled SUMO-1. Lower panel, after fluorescent analysis, the gels were stained with Coomassie Blue to show input E1 levels.
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Mapping the RanGAP1 Sumoylation Site Using SUMO-1 (T95R) by Tandem Mass Spectrometry
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 (9). 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 analysis were constrained by the parameters we defined prior to the survey run, we termed this approach for analyzing post-translational modifications in proteins PDA. Application of this approach led to identification of two peptides containing lysine modifications with a diglycine appendage (Fig. 7, AD). These two peptides, with Mascot ion scores of 38 and 43, correspond to amino acids 518530 and 518532 of RanGAP1 both with Lys-526 being the modification site. The latter peptide (518532) has a missed cleavage at Lys-530, suggesting that the isopeptide bond at Lys-526 may affect trypsin cleavage efficiency at Lys-530. CID spectra for both peptides clearly indicate a missed cleavage at Lys-526 and presence of the 114.06-Da appendage on this lysine residue. Therefore, the combination of PDA with SUMO-1 (T95R) enables us to clearly identify the sumoylation site in RanGAP1.

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FIG. 6. In vitro sumoylation of RanGAP1 with SUMO-1 (T95R) mutant for identification of sumoylation sites by mass spectrometry. Recombinant RanGAP1 purified from E. coli was incubated with an energy regeneration system, purified SUMO E1 (Aos1/Uba2), E2 (Ubc9), and SUMO-1 (T95R). The input proteins were analyzed by SDS-PAGE and stained with Coomassie Blue. Lane 1, E1 heterodimer; lane 2, Ubc9; lane 3, SUMO-1 T95R; lane 4, RanGAP1; lane 5, a portion of RanGAP1 sumoylation reaction prior to tryptic digestion.
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FIG. 7. A, schematic of the b- and y-ions of sumoylation site peptide in RanGAP1 detected from the 537.65 (+3) parent ion. ++ denotes a doubly charged fragment ion; * indicates loss of ammonia (17 Da) from the fragment ion. B, collision-induced dissociation spectrum in IDA mode of the triply charged parent ion at m/z 537.65 with b- and y-ions as well as their corresponding amino acid sequences labeled. C, schematic of b- and y-ions of sumoylation site peptide with a missed cleavage at Lys-530 in RanGAP1 detected from 926.52 (+2) parent ion. D, CID spectrum in IDA mode of the doubly charged parent ion at m/z 926.52.
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A Web-based Computation Program for Generating Input Lists for PDA
A full description of the Ubl Finder program and its utilities can be found under the tutorial section (bmf.colorado.edu/ublfinder/tutorial.html). Briefly Ubl Finder is a publicly available program developed to calculate the m/z values of the tryptic peptides for sumoylated (T95R), ubiquitinated, or unmodified proteins. When sumoylation or ubiquitination is chosen, the m/z of a diglycine tag is added to the m/z of each peptide that contains a potentially modified lysine. For sumoylation with SUMO-1 (T95R), a note is made in the output file as to whether the lysine residue falls into a consensus sumoylation sequence. Protein sequences can be typed into the input box in FASTA format, or the FASTA file can be directly input into the upload box (Fig. 8). Ion charge and m/z exclusion limits can be set to limit the size of the output m/z list. The maximum number of modified residues and missed cleavages can be set by the user. Note that one missed cleavage is expected due to the steric hindrance present from the isopeptide bond formed between the terminal glycine of the Ubl and the lysine in the target protein. Tab- or comma-delimited m/z list format can be chosen for ease of input into different mass spectrometry software. For use with the Analyst software package, tab-delimited output should be chosen.
The output of Ubl Finder (Supplemental Fig. 2A) enables the user to directly download the m/z list into a text file for input into the mass spectrometry software as well as displaying the data in a user-friendly table format. A coverage map is displayed at the end of the peptide list to show the percentage of potentially modified lysines present in the input protein list that are accounted for in the m/z list. The number of maximum missed cleavages and inclusion limits can be changed to maximize the coverage obtained.
To save the m/z list, right click on Download and select "save link as." Save the m/z list as a .txt file for direct input into the mass spectrometry software (Supplemental Fig. 2B). The list then can be used for PDA to increase the probability of sequencing sumoylated T95R peptides from a tryptic digestion.
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DISCUSSION
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Protein sumoylation is now emerging as an important post-translational modification mechanism in regulating protein functions in a variety of cellular processes. An in-depth understanding of the role of sumoylation requires the elucidation of sumoylation sites. Tandem mass spectrometry analysis of proteolyzed protein conjugates by trypsin is the most direct approach to identification of these modification sites. However, application of this method to identification of mammalian protein sumoylation sites is hampered by the absence of Arg or Lys in the carboxyl-terminal region of mammalian SUMO-1. Through systematic analysis of SUMO-1 variants with Arg substitutions in the carboxyl-terminal region of SUMO-1, we identified a SUMO-1 variant (T95R) that exhibits conjugation properties indistinguishable from the wild type SUMO-1 in vitro. We demonstrate that SUMO-1 (T95R) is particularly useful for identification of SUMO-1 modification sites by tandem mass spectrometry. This general method for sumoylation site identification works more effectively when a predetermined inclusion list generated by a web-based program is included during data acquisition.
One of the key differences between SUMO-1, -2, and -3 and other ubiquitin-like modifier proteins lies in the sequence near the diglycine motif. The three carboxyl-terminal residues in the SUMO family of proteins are TGG, instead of RGG found in ubiquitin, and are strictly evolutionarily conserved (Fig. 1A). Previous mutagenesis studies with ubiquitin indicated that Arg-74 mutation to Leu exhibited altered kinetics for E1-catalyzed ATP:PPi exchange compared with wild type ubiquitin, suggesting that the amino acid residue amino-terminal to the diglycine motif is functionally important for conjugation (14). Surprisingly mutation of TGG to RGG has little effect on SUMO-1 activation by the SUMO E1 and subsequent conjugation to target proteins such as RanGAP1 in vitro (Fig. 4A). Crystallographic studies indicate that Glu-93, Gln-94, Thr-95, Gly-96, and Gly-97 within SUMO-1 contact the SUMO-activating enzyme Sae1/Sae2 (15). It is rather unexpected that single Arg substitution at position Glu-93, Gln-94, or Thr-95 results in no substantial reduction in SUMO-1 conjugation in vitro. These results suggest that Glu-93, Gln-94, or Thr-95 in SUMO-1 are not obligatory for SUMO-1 activation and conjugation. Alternatively Thr-95 in SUMO is analogous to Arg-74 in ubiquitin that has been shown previously to be important for interaction with E1 and activation (14). The effect of this mutation is subtle and hardly discernable in the overall conjugation reaction sequence because E1 activation is a fast reaction in both ubiquitination and sumoylation and therefore not rate-limiting. Although these residues do not seem to be required for SUMO-1 conjugation, they may be important for deconjugation by isopeptidase. Previous studies indicate that mutation of Arg-74 in ubiquitin is permissive for ubiquitin conjugation but prevents removal of ubiquitin from conjugated substrate by deubiquitinating enzymes (16). It will be interesting to test whether SUMO-1 (T95R) conjugates are still sensitive to SUMO-1 isopeptidase.
Although SUMO-1 (T95R) is permissive for E1 and downstream sumoylation reactions, one question remains as to whether this mutant retains its specificity for E1 and substrate conjugation sites. It has been shown previously that the residues near the diglycine motif in Nedd8 are critical determinants for Nedd8 E1 discrimination between Nedd8 and ubiquitin (17). SUMO-1 (T95R) does not appear to alter the E1 specificity for cognate Ubl as only Aos1/Uba2 is able to be charged with SUMO-1 (T95R) (Fig. 5). Furthermore the identification of Lys-526 as the sumoylation site of RanGAP1 by SUMO-1 (T95R) further reinforces the notion that this SUMO-1 variant behaves very much like the wild type SUMO-1 at least in vitro. Conjugation of SUMO-1 (T95R) or wild type SUMO-1 to the endogenous RanGAP1 present in the rabbit reticulocyte lysate with similar efficiency is yet another indication that they are interchangeable without any apparent loss of specificity. Now that the specificity of SUMO-1 (T95R) has been validated, it can be used to map sumoylation sites for other SUMO targets in vitro as long as the sumoylated products are detectable by Coomassie Blue staining. Future studies are necessary to determine whether SUMO-1 (T95R) is also suitable for in vivo investigations.
Definitive identification of sumoylation sites by mass spectrometry is still a very challenging procedure. Protein sumoylation appears to be very dynamic in vivo, and the steady state levels of sumoylated proteins are often very low. Identification of the actual site(s) of modification with informative CID spectrum is the most persuasive evidence to implicate SUMO-1 modification of a candidate protein and to initiate investigation into the biological relevance of the modification. The yeast SUMO (SMT3) has an Arg at position 93, and trypsinolysis of sumoylated protein conjugates in yeast will yield a signature peptide with a five-amino acid remnant (EQIGG) on the modified lysine residue (6, 18). Such a feature has been used for identification of novel SUMO substrates in yeast (6, 18). Such a strategy may be very difficult to be applied to sumoylation site identification with the native mammalian SUMO proteins due to the absence of Arg or Lys adjacent to the diglycine motif. MALDI-TOF mass spectrometric analysis of SUMO-1-RanGAP1 conjugates identified two peptides with mass values of 3634 and 3877 Da (9). Fragmentation of these large branched peptides to delineate the sumoylation site was not possible with MALDI-TOF. Using LC-MS/MS and SUMO-1 (T95R), we obtained the +2 and +3 ions for peptides conjugated to Lys-526. Our results are in agreement with the MALDI-TOF data in that Lys-526 is the modification site, and Lys-530 is partially resistant to trypsin digestion resulting in a miscleavage at this site (9). The availability of +2 and +3 charged ions enables us to obtain informative CID spectra that clearly indicate the modification site. Therefore, the combination of SUMO-1 (T95R) and LC-MS/MS is an effective method to identify the authentic sumoylation site of RanGAP1.
Successful identification of sumoylation sites depends heavily on the sensitivity of the mass spectrometer and retrieval of the low abundance sumoylated peptides for fragmentation. The LTQ-FT mass spectrometer (Thermo Electron Corporation), which offers subfemtomole sensitivity and high mass accuracy, has been shown to be well suited for this kind of application (6). Another way to maximize the chance of catching the sumoylated signature peptide in a single LC-MS/MS run is by using IDA. However, normal IDA in the Analyst software automatically chooses high abundance parent ions for fragmentation. The low abundance sumoylated peptides may never exceed the threshold to be selected for fragmentation. It is possible to run manual LC-MS/MS to choose parent ions of interest. This requires a manual input list of appropriate candidate ions. With PDA, the input list is generated by the theoretical calculation of the m/z values of all possible modified peptides with different charge states using the Ubl Finder program. By directing the mass spectrometer to focus on collecting evidence for post-translational modifications in the protein, which usually represent minor components in the sample, PDA could significantly improve the content of relevant information collected from a single LC-MS/MS acquisition. This approach is applicable for any type of post-translational modification. PDA in combination with sensitive mass spectrometers such as LTQ-FT may offer a unique solution for the most demanding task of elucidating the site of post-translational modifications, particularly other sumoylation targets with unidentified acceptor lysines. By extension, one could immunoprecipitate SUMO-1 (T95R) and, using the combinatorial approach of LC-MS/MS with PDA, identify both known and unknown sumoylation targets while at the same time identifying the site of sumoylation.
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ACKNOWLEDGMENTS
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We thank Drs. Frauke Melchior, Jacob Seeler, Anne Dejean, Guntram Suske, and Stephan Müller for the generous supply of the cDNA clones and reagents used in this study and Drs. Ahn and Resing for help with mass spectrometry.
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FOOTNOTES |
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Received, May 20, 2005, and in revised form, July 11, 2005.
Published, MCP Papers in Press, July 14, 2005, DOI 10.1074/mcp.T500011-MCP200
1 The 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. 
* This work was supported in part by National Institutes of Health Grant CA107098-01 (to X. L.). 
S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material. 
Supported by NHLBI, National Institutes of Health Predoctoral Training Grant 5T32HL07851. 
|| Supported by NIGMS, National Institutes of Health Predoctoral Training Grant T32GM08759. 
** To whom correspondence should be addressed. Tel.: 303-735-6161; Fax: 303-735-6161; E-mail: Xuedong.Liu{at}Colorado.edu
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