In Vitro Modification of Human Centromere Protein CENP-C Fragments by Small Ubiquitin-like Modifier (SUMO) Protein
2004; Elsevier BV; Volume: 279; Issue: 38 Linguagem: Inglês
10.1074/jbc.m405637200
ISSN1083-351X
AutoresTung‐Liang Chung, He‐Hsuan Hsiao, Yuh-Ying Yeh, Hui-Ling Shia, Yi‐Ling Chen, Po‐Huang Liang, Andrew H.‐J. Wang, Kay‐Hooi Khoo, Steven Shoei‐Lung Li,
Tópico(s)Peptidase Inhibition and Analysis
ResumoProtein sumoylation by small ubiquitin-like modifier (SUMO) proteins is an important post-translational regulatory modification. A role in the control of chromosome dynamics was first suggested when SUMO was identified as high-copy suppressor of the centromere protein CENP-C mutants. CENP-C itself contains a consensus sumoylation sequence motif that partially overlaps with its DNA binding and centromere localization domain. To ascertain whether CENP-C can be sumoylated, tandem mass spectrometry (MS) based strategy was developed for high sensitivity identification and sequencing of sumoylated isopeptides present among in-gel-digested tryptic peptides of SDS-PAGE fractionated target proteins. Without a predisposition to searching for the expected isopeptides based on calculated molecular mass and relying instead on the characteristic MS/MS fragmentation pattern to identify sumolylation, we demonstrate that several other lysine residues located not within the perfect consensus sumoylation motif ψKXE/D, where ψ represents a large hydrophobic amino acid, and X represnts any amino acid, can be sumolylated with a reconstituted in vitro system containing only the SUMO proteins, E1-activating enzyme and E2-conjugating enzyme (Ubc9). In all cases, target sites that can be sumoylated by SUMO-2 were shown to be equally susceptible to SUMO-1 attachments which include specific sites on SUMO-2 itself, Ubc9, and the recombinant CENP-C fragments. Two non-consensus sites on one of the CENP-C fragments were found to be sumoylated in addition to the predicted site on the other fragment. The developed methodologies should facilitate future studies in delineating the dynamics and substrate specificities of SUMO-1/2/3 modifications and the respective roles of E3 ligases in the process. Protein sumoylation by small ubiquitin-like modifier (SUMO) proteins is an important post-translational regulatory modification. A role in the control of chromosome dynamics was first suggested when SUMO was identified as high-copy suppressor of the centromere protein CENP-C mutants. CENP-C itself contains a consensus sumoylation sequence motif that partially overlaps with its DNA binding and centromere localization domain. To ascertain whether CENP-C can be sumoylated, tandem mass spectrometry (MS) based strategy was developed for high sensitivity identification and sequencing of sumoylated isopeptides present among in-gel-digested tryptic peptides of SDS-PAGE fractionated target proteins. Without a predisposition to searching for the expected isopeptides based on calculated molecular mass and relying instead on the characteristic MS/MS fragmentation pattern to identify sumolylation, we demonstrate that several other lysine residues located not within the perfect consensus sumoylation motif ψKXE/D, where ψ represents a large hydrophobic amino acid, and X represnts any amino acid, can be sumolylated with a reconstituted in vitro system containing only the SUMO proteins, E1-activating enzyme and E2-conjugating enzyme (Ubc9). In all cases, target sites that can be sumoylated by SUMO-2 were shown to be equally susceptible to SUMO-1 attachments which include specific sites on SUMO-2 itself, Ubc9, and the recombinant CENP-C fragments. Two non-consensus sites on one of the CENP-C fragments were found to be sumoylated in addition to the predicted site on the other fragment. The developed methodologies should facilitate future studies in delineating the dynamics and substrate specificities of SUMO-1/2/3 modifications and the respective roles of E3 ligases in the process. Protein modification by covalent attachment of SUMO 1The abbreviations used are: SUMO, small ubiquitin-like modifier; aa, amino acid; CID, collision-induced dissociation; ESI, electrospray ionization; MALDI, matrix-assisted laser desorption ionization; MS, mass spectrometry; MS/MS, tandem MS; nanoESI, nanospray; nanoLC, nanoflow liquid chromatography. 1The abbreviations used are: SUMO, small ubiquitin-like modifier; aa, amino acid; CID, collision-induced dissociation; ESI, electrospray ionization; MALDI, matrix-assisted laser desorption ionization; MS, mass spectrometry; MS/MS, tandem MS; nanoESI, nanospray; nanoLC, nanoflow liquid chromatography. (small ubiquitin-like modifier) proteins is emerging as an important regulatory mechanism in a diverse range of cellular processes (1Seeler J.S. Dejean A. Nat. Rev. Mol. Cell. Biol. 2003; 4: 690-699Crossref PubMed Scopus (575) Google Scholar, 2Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1369) Google Scholar). Through a three-step enzymatic pathway analogous to ubiquitylation, sumoylation is initiated by ATP-dependent formation of a thioester bond between the C-terminal glycine of a SUMO protein and a catalytic cysteine of the SUMO-specific E1-activating enzyme, known as SAE1/SAE2 and Uba2p/Aos1p for human and yeast, respectively. The activated SUMO protein is then trans-esterified from the E1 enzyme to the catalytic cysteine of an E2-conjugating enzyme, Ubc9, which in turn catalyzes the formation of an isopeptide bond between the C-terminal glycine of SUMO and the ∈-amino group of lysine residue in the substrate proteins. Ubc9 was reported to recognize and bind directly to a consensus sumoylation motif ψKXE/D, where ψ represents a large hydrophobic amino acid L, I, V, or F, and X represents any amino acid (3Melchior F. Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Crossref PubMed Scopus (649) Google Scholar, 4Sampson D.A. Wang M. Matunis M.J. J. Biol. Chem. 2001; 276: 21664-21669Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar, 5Bernier-Villamor V. Sampson D.A. Matunis M.J. Lima C.D. Cell. 2002; 108: 345-356Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar). Although sumoylation can be accomplished in vitro without an E3-like enzyme, several E3 ligases, e.g. PIAS, RanBP2, and PC2, have been identified which may aid Ubc9 in substrate selection and ligation efficiency in vivo (2Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1369) Google Scholar, 6Melchior F. Schergaut M. Pichler A. Trends Biochem. Sci. 2003; 28: 612-618Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar).Unlike yeast and other invertebrates, which contain only a single SUMO gene, vertebrates carry three. Human SUMO-1 protein exhibits 44% sequence identity with human SUMO-2 and SUMO-3 proteins, while SUMO-2 and SUMO-3 proteins share 86% sequence identity. All SUMO proteins from yeast to human share the conserved ubiquitin domain and the C-terminal diglycine cleavage/attachment site (3Melchior F. Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Crossref PubMed Scopus (649) Google Scholar, 7Mossessova E. Lima C.D. Mol. Cell. 2000; 5: 865-876Abstract Full Text Full Text PDF PubMed Scopus (559) Google Scholar). The most prominent difference between the SUMO proteins and ubiquitin is the presence of a highly variable N-terminal extension in all SUMO proteins, which is rich in charged amino acids, glycines, and prolines. This extension varies from 16 to 23 amino acids and is reasonably well conserved within, but not between, different SUMO-1/2/3 proteins. At present, human SUMO-1 and SUMO-2/3 are thought to be functionally non-overlapping and respond differently to stress signaling (2Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1369) Google Scholar). Although all three SUMO isoforms utilize the same E1-activating and E2-conjugating enzymes, the molecular basis for their substrates preference and the additional recruitment of E3 ligases, especially in relation to the selective use of sumoylation sites conforming and not conforming to the minimal consensus motif, remain unclear. SUMO-2/3, like yeast SUMO (Smt3), but not SUMO-1, contain a consensus ψKXE sumoylation site in their N-terminal extension. Accordingly, oligomerization of SUMO-2/3 or Smt3 chains can be demonstrated in vivo and effected with the reconstituted in vitro sumoylation system in the absence of E3 (8Tatham M.H. Jaffray E. Vaughan O.A. Desterro J.M. Botting C.H. Naismith J.H. Hay R.T. J. Biol. Chem. 2001; 276: 35368-35374Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar, 9Bencsath K.P. Podgorski M.S. Pagala V.R. Slaughter C.A. Schulman B.A. J. Biol. Chem. 2002; 277: 47938-47945Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). In contrast, formation of SUMO-1 chains with as yet unknown linkage apparently can only be demonstrated in the presence of an E3 ligase activity (10Pichler A. Gast A. Seeler J.S. Dejean A. Melchior F. Cell. 2002; 108: 109-120Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar).Sumoylation is a dynamic, reversible process, and often only little or no modified protein can be detected under physiological conditions (1Seeler J.S. Dejean A. Nat. Rev. Mol. Cell. Biol. 2003; 4: 690-699Crossref PubMed Scopus (575) Google Scholar). It should be noted that direct biochemical evidence for the implicated isopeptide or the sumoylation site has been generally lacking for most reported sumoylation cases due to its low abundance. To date, only a handful of isopeptides have been physically defined by mass spectrometry (MS) analysis (8Tatham M.H. Jaffray E. Vaughan O.A. Desterro J.M. Botting C.H. Naismith J.H. Hay R.T. J. Biol. Chem. 2001; 276: 35368-35374Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar, 9Bencsath K.P. Podgorski M.S. Pagala V.R. Slaughter C.A. Schulman B.A. J. Biol. Chem. 2002; 277: 47938-47945Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 11Mahajan R. Gerace L. Melchior F. J. Cell Biol. 1998; 140: 259-270Crossref PubMed Scopus (236) Google Scholar, 12Johnson E.S. Blobel G. J. Cell Biol. 1999; 147: 981-994Crossref PubMed Scopus (325) Google Scholar). The isopeptide formation and hence the sumoylation site were directly inferred from the molecular mass detected but in general not further verified by MS/MS sequencing analysis. This approach is problematic in view of the increasing probability in finding a non-consensus sumoylation site nor can it distinguish the exact sumoylated lysine residue in the event that the implicated endoproteinase-digested peptide carries more than one lysine residues. To better define the sumoylation site specificity and to delineate the respective roles of SUMO-1 versus SUMO-2/3, a high sensitivity MS/MS sequencing strategy for unambiguous identification of the isopeptide and sumoylation site is critically needed. By adopting an in vitro sumoylation system with defined components, we have thus undertaken to first develop the enabling analytical techniques while gearing toward addressing the role of sumoylation in regulating chromosome dynamics.The centromere is a highly ordered structure of the eukaryotic chromosomes which provides the site for microtubule (spindle) attachment during cell division (13Pluta A.F. Mackay A.M. Ainsztein A.M. Goldberg I.G. Earnshaw W.C. Science. 1995; 270: 1591-1594Crossref PubMed Scopus (307) Google Scholar). The first implication of sumoylation in centromere functions came from the original isolation of the yeast SUMO (Smt3) as a high copy suppressor of a mutation in the Mif2 gene, the yeast homologue of the gene for mammalian centromere protein CENP-C (14Meluh P.B. Koshland D. Mol. Biol. Cell. 1995; 6: 793-807Crossref PubMed Scopus (350) Google Scholar). CENP-C is an essential component of the kinetochore inner plate, which contributes to the formation of functional centromeres for correct chromosome segregation. Temperature-sensitive CENP-C mutants in vertebrate cells could likewise be suppressed by overexpression of SUMO-1 (15Fukagawa T. Regnier V. Ikemura T. Nucleic Acids Res. 2001; 29: 3796-3803Crossref PubMed Scopus (47) Google Scholar). Despite several studies, which demonstrated the localization of SUMO at or adjacent to the kinetochore and how sumoylation may affect centromeric chromosome cohesion (reviewed in Refs. 1Seeler J.S. Dejean A. Nat. Rev. Mol. Cell. Biol. 2003; 4: 690-699Crossref PubMed Scopus (575) Google Scholar and 2Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1369) Google Scholar), the mechanistic details remain largely unknown nor is it clear whether critical centromeric proteins like CENP-C are themselves sumoylated. Molecular dissection of CENP-C revealed that the protein is composed of several functional domains (16Brown M.T. Gene (Amst.). 1995; 160: 111-116Crossref PubMed Scopus (71) Google Scholar, 17Lanini L. McKeon F. Mol. Biol. Cell. 1995; 6: 1049-1059Crossref PubMed Scopus (41) Google Scholar, 18Yang C.H. Tomkiel J. Saitoh H. Johnson D.H. Earnshaw W.C. Mol. Cell. Biol. 1996; 16: 3576-3586Crossref PubMed Scopus (100) Google Scholar, 19Sugimoto K. Kuriyama K. Shibata A. Himeno M. Chromosome Res. 1997; 5: 132-141Crossref PubMed Scopus (51) Google Scholar, 20Song K. Gronemeyer B. Lu W. Eugster E. Tomkiel J.E. Exp. Cell Res. 2002; 275: 81-91Crossref PubMed Scopus (21) Google Scholar, 21Trazzi S. Bernardoni R. Diolaiti D. Politi V. Earnshaw W.C. Perini G. Della Valle G. J. Struct. Biol. 2002; 140: 39-48Crossref PubMed Scopus (31) Google Scholar) including a specific region of 12 amino acids at aa 522–533 that could confer DNA binding as well as centromere targeting functions (Fig. 1). Notably, its partial overlapping with a sumoylation consensus sequence of VKSE at aa 533–536 raises an intriguing possibility that regulated sumoylation of CENP-C may affect its functions.Using the methodologies developed, which were first validated against SUMO-2/SUMO-2 and SUMO-2/Ubc9 conjugation, we report here that a C28 fragment of CENP-C encoding aa 432–683 (Fig. 1), which includes the consensus sumoylation site, DNA binding and centromere localization regions, and two nuclear localization signals, could be in vitro sumoylated by both SUMO-1 and SUMO-2 at the expected site in the absence of any E3 ligases. In addition, another C10 fragment of CENP-C encoding aa 678–764, which comprises a Mif2 homology block at aa 737–759, could also be sumoylated in vitro by both SUMO-1 and SUMO-2 at two distinct sites, neither of which corresponds to the consensus sequence motif. We further demonstrate that SUMO-1 could be attached to SUMO-2 and the characteristic MS/MS fragmentation pattern associated with SUMO-2 isopeptides could be utilized as an effective fingerprint in identifying sumoylation site at high sensitivity.EXPERIMENTAL PROCEDUREScDNA Cloning, Protein Expression, and Purification—The C28 cDNA encoding aa 432–683 of human CENP-C was amplified by reverse transcriptase-PCR using poly(A)-containing RNAs from HeLa cells, as well as primers of 5′-CGGATCCAGAACACTTGATGTGGGAC-3′ and 5′-GTGCTCCAGTTCCTCTAAAACTGAAGT-3′, and then cloned into the BamHI-XhoI sites of bacterial expression vector pET-21b (His-tag at the C terminus). The C10 cDNA encoding aa 678–764 of human CENP-C was also amplified using primers of 5′-CGGATCCCACTTCAGTTTTAGAGGAA-3′ and 5′-GTGCTCCAGTCCTGATGGCCTTCCTTGATA-3′ and then cloned into the BamHI-XhoI sites of pET-21b. The human cDNA encoding the active form (exposing diglycine motif) of SUMO-1GG was obtained by PCR from the FLAG-SUMO-1 cDNA described previously (22Su H.L. Li S.S.L. Gene (Amst.). 2002; 296: 65-73Crossref PubMed Scopus (128) Google Scholar), and the cDNA encoding the active form of SUMO-2GG was obtained by PCR from the hSMT3 cDNA reported previously (23Mannen H. Tseng H.M. Cho C.L. Li S.S.L. Biochem. Biophys. Res. Commun. 1996; 222: 178-180Crossref PubMed Scopus (50) Google Scholar). Both SUMO-1GG and SUMO-2GG cDNAs were subsequently cloned into pET-32 Xa (both His-tag and S-tag at the N terminus). The human Ubc9 cDNA was amplified by reverse transcriptase-PCR using poly(A)-containing RNAs from HeLa cells and then cloned into pET-21b and pET-32 Xa. The Escherichia coli BL21 strain was used as host cells for protein expression. His-tag proteins were purified using nickel-nitrilotriacetic acid-agarose column (Qiagen). The purified proteins from pET-32 Xa were cleaved with Factor Xa to remove both His-tag and S-tag.In Vitro Sumoylation Assay—The in vitro sumoylation system employed in this investigation contains E1 enzyme (159 ng of SAE1/SAE2, LAE Biotechnology, Taichung, Taiwan), E2 enzyme (2 μg Ubc9), SUMO-1GG or SUMO-2GG protein (5 μg), and C28-His or C10-His protein substrate (5 μg) in sumoylation buffer (2 μl of 500 mm Tris, pH 7.5, 1 μl of 100 mm MgCl2, 1 μl of 100 mm ATP, 0.5 μl of 40 mm dithiothreitol, an ATP-regenerating system, including 0.5 μl of 400 mm creatine phosphate, 0.5 μl of 140 units/ml creatine kinase, 0.5 μl of 24 units/ml inorganic pyrophosphatase). The sumoylation reactions were incubated at 37 °C for 3 h. After termination with SDS buffer containing 1 mm dithiothreitol, reaction products were separated on 12% SDS-PAGE, and the gels were stained with Coomassie Blue.Identification of Sumoylation Sites by Tandem Mass Spectrometry Analyses—Following SDS-PAGE, Coomassie Blue-stained protein bands were excised from gels, reduced and alkylated with iodoacetamide, and in-gel-digested with sequencing grade, modified trypsin (Promega, Madison, WI). Peptides were extracted and subjected first to nanoLC-nanoESI-MS/MS analysis for protein identification as described previously (24Lee C.L. Hsiao H.H. Lin C.W. Wu S.P. Huang S.Y. Wu C.Y. Wang A.H. Khoo K.H. Proteomics. 2003; 3: 2472-2486Crossref PubMed Scopus (90) Google Scholar). Subsequently, MALDI-MS detection and MS/MS sequencing of isopeptides in reflectron mode were performed on an Applied Biosystems 4700 Proteomics Analyzer mass spectrometer (Applied Biosystems, Framingham, MA) equipped with an Nd:YAG laser (355 nm wavelength, <500-ps pulse, and 200 Hz repetition rate in both MS and MS/MS modes). 1000 and 10,000 shots were accumulated in positive ion mode MS and MS/MS modes, respectively. The tryptic digested peptide samples were dissolved in 50% acetonitrile with 0.1% formic acids and premixed with a 5 mg/ml matrix solution of α-cyano-4-hydroxycinnamic acid in 70% acetonitrile with 0.1% formic acid for spotting onto target plate. For collision-induced dissociation (CID) MS/MS operation, the indicated collision cell pressure was increased from 3.0 × 10–8 torr (no collision gas) to 5.0 × 10–7 torr, with the potential difference between the source acceleration voltage and the collision cell set at 1 kV. The resolution of timed ion selector for precursor ion was set at 100, which would allow in a mass window of about 50 Da for precursors at m/z 5000. Both MS and MS/MS data were acquired using the instrument default calibration. At a resolution above 10,000 in MS mode, accurate mass measurement (<50 ppm) of the monoisotopic isopeptide signals is possible when further adjusted against an internal reference peak at m/z 5557.8 derived from autolytic cleavage of the modified trypsin.RESULTSSumoylation of SUMO-2 and Ubc9 —The in vitro sumoylation system employed in this investigation contained E1 (SAE1/SAE2)-activating enzyme, E2 (Ubc9)-conjugating enzyme, the active SUMO proteins (SUMO-1GG or SUMO-2GG), with and without the centromere proteins (C28-His or C10-His), and an ATP-generating system, including creatine kinase. To better define the sumoylation reaction products as visualized by SDS-PAGE analysis, reactions of the active SUMO proteins with the E1/E2 enzymes were first investigated prior to the addition of the substrate proteins. Fig. 2 shows that in the absence of E1 enzyme, no reaction product could be detected (lanes 2 and 4), but one (lane 1) and two (lane 3) additional protein bands were clearly visible when E1 enzyme was added to the SUMO-1GG/E2 and SUMO-2GG/E2 reaction mixtures, respectively. By LC-ESI-MS/MS analysis of the respective in-gel tryptic digests, bands 2 and 3 were found to contain peptides derived from SUMO-1 and SUMO-2, respectively, in addition to those from Ubc9 (data not shown). Band 1 on the other hand only afforded peptides matching to SUMO-2. Since the tagged SUMO proteins and Ubc9 alone ran as protein bands close to 20 kDa (data not shown), the results indicated that the SUMO proteins can be stably conjugated to Ubc9 and ran as bands 2 and 3 and that only SUMO-2, but not SUMO-1, can itself be sumoylated to give a higher molecular weight product (band 1).Fig. 2SDS-PAGE analysis of sumoylation reaction products. The in vitro sumoylation reactions contained the E2 (Ubc9) and SUMO-1GG/2GG with and without the E1 enzyme, as well as creatine kinase (CK). The gel was stained with Coomassie Blue for visualization of the protein bands.View Large Image Figure ViewerDownload Hi-res image Download (PPT)MS/MS Characteristics of SUMO-2-conjugated Isopeptides— The monoisotopic mass of the SUMO-2-conjugated SUMO-2 isopeptide, in which the Gly93 of C-terminal tryptic peptide of aa 62–93 from one SUMO-2 was conjugated to the sumoylation site at Lys11 within the tryptic peptide of aa 8–21 from another SUMO-2, was calculated to be 5159.34. By nanoESI-MS analysis on a quadrupole/time-of-flight instrument of sufficiently high mass accuracy and resolution, a pair of well resolved, quadruply charged isotopic signal clusters could be detected with the respective monoisotopic molecular ions [M+4H]4+ occurring at m/z 1290.848 and 1294.847 (Fig. 3A, inset). The former corresponds exactly (within 10 ppm) to the expected isopeptide, while the latter at 16 mass units higher most likely was derived from the same peptide but containing an oxidized Met78 residue. The relative ratio of these two peaks varied from batch to batch, implying different degree of induced oxidation during sample preparation. To confirm the isopeptide linkage assignment predicted by previous MALDI-TOF MS study (8Tatham M.H. Jaffray E. Vaughan O.A. Desterro J.M. Botting C.H. Naismith J.H. Hay R.T. J. Biol. Chem. 2001; 276: 35368-35374Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar), the isopeptides detected here as singly charged [M+H]+ molecular ions on a MALDI-TOF/TOF instrument were selected as precursors for MALDI CID-MS/MS analysis (Fig. 3). Two well known fragmentation characteristics facilitated the spectral assignment. First, peptide fragmentation by MALDI-MS/MS is often dominated by facile cleavages at C terminus of an Asp residue (25Wattenberg A. Organ A.J. Schneider K. Tyldesley R. Bordoli R. Bateman R.H. J. Am. Soc. Mass Spectrom. 2002; 13: 772-783Crossref PubMed Scopus (76) Google Scholar). For tryptic peptides where the positive charge tends to be localized to C-terminal Lys or Arg residue, the MS/MS spectra will be dominated by a series of y ions, the most abundant of which are derived from cleavages at Asp. In the case of the SUMO-2 conjugated SUMO-2 isopeptide carrying an oxidized Met (m/z 5180.6, Fig. 3A), the major ions at m/z 624.5 (y5), 2456.5 (y22), 2785.7 (y25), 3029.7 (y27), 4060.1 (y36), and 4915.3 (y44) effectively and unambiguously confirmed the peptide sequence. Other y ions, y5–y10, followed by the apparent absence of y11–y13 and the reappearance of the y ion series at y14 clearly define the SUMO-2 sumoylation site at Lys11. Second, consistent with the presence of an oxidized Met residue, the major fragment ions y36 and y44 and the molecular ion that contains the oxidized Met were accompanied by facile neutral loss of HSOCH3 (64 mass units) (26Jiang X. Smith J.B. Abraham E.C. J. Mass Spectrom. 1996; 31: 1309-1310Crossref Google Scholar, 27Lagerwerf F.M. van de Weert M. Heerma W. Haverkamp J. Rapid Commun. Mass Spectrom. 1996; 10: 1905-1910Crossref PubMed Scopus (108) Google Scholar), giving rise to ions at m/z 3996.0, 4951.3, and 5116.9, respectively. These neutral losses were not observed in the MS/MS spectra of isopeptides with non-oxidized Met, e.g. that of the monoisotopic [M+H]+ molecular ion signal at m/z 5164.3 (data not shown).Fig. 3MALDI-MS/MS spectra of the SUMO-2-conjugated SUMO-2 isopeptides carrying an oxidized Met78 residue (A) or a trypsin missed cleavage site at Arg61 (B). In A, the majority of the fragment ions could be assigned to the y ion series as annotated, which were commonly accompanied by neutral loss of H2O (18 mass units) giving rise to pairs of signals. Ions that carry the oxidized Met also afforded neutral loss of HSOCH3 (64 mass units). The quadruply charged molecular ion signals corresponding to the isopeptides containing a non-oxidized and oxidized Met as detected by nanoESI-MS analysis are shown as the inset in A. The spectrum in B is normalized to the intensity of the b12 ion signal, but only the portion below 50% intensity is shown. Localization of an Arg residue near the N terminus contributed to the predominance of the b ion series except for a couple of y ions as annotated. Neutral loss of H2O (18 mass units) is also evident for the b21, b23, and b26 ions. The labeled m/z values for this and other mass spectra figures mostly correspond to monoisotopic accurate masses as originally annotated by the instrument except for signals at above m/z 3500, especially those of relatively weak intensity, which may not afford full isotopic resolution and may thus be labeled according to the detected peak top, usually at several mass units higher than the theoretical monoisotopic masses.View Large Image Figure ViewerDownload Hi-res image Download (PPT)It is important to note that, depending on the substrate proteins, the molecular weight of the SUMO-2-conjugated isopeptide will naturally be different. However, the tryptic fragment corresponding to the C-terminal peptide of SUMO-2 will remain the same. The four Asp residues, namely Asp71, Asp80, Asp82, and Asp85, will always induce four major fragment ions due to cleavage at their C termini with mass intervals corresponding to 1014 (TPAQLEMED) or 1030 (TPAQLEMoxED), 244 (ED), and 329 (TID) mass units. Detection of this characteristic MALDI-MS/MS pattern is thus a first indication that the parent ion signal is most probably a SUMO-2-sumoylated isopeptide. As reported by others (8Tatham M.H. Jaffray E. Vaughan O.A. Desterro J.M. Botting C.H. Naismith J.H. Hay R.T. J. Biol. Chem. 2001; 276: 35368-35374Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar), there is a tendency of missed cleavage by trypsin at the Arg61 located immediately upstream of the 62–93 C-terminal peptide. As a consequence of the additional two residues Phe60-Arg61 at the N terminus, strong fragment ion signals corresponding to the b ion series could be found to dominate the MS/MS spectrum in place of the y ions, a phenomenon best rationalized as charge localization at the N-terminal Arg in preference to the C-terminal Lys. Consequently, the mass intervals of 1014/244/329 are now defined by the b ions, namely b12 (m/z 1420.6), b21 (m/z 2435.0), b23 (m/z 2679.0), and b26 (m/z 3008.2) ions for all SUMO-2-conjugated isopeptides carrying the missed cleavage which, together with the prominent b4 ion at m/z 566.3, constitute the signature pattern (Fig. 3B). The complementary y ions could still be detected albeit at lower intensity, e.g. y44 and y36 at m/z 4900.4 and 4044.3, respectively, which can be further used as supporting evidence for confident sequence mapping. Interestingly, we noted that if the SUMO-2-sumoylated tryptic peptide from the substrate protein is itself terminating with an Arg residue instead of Lys, the presence of Arg at both the C and N termini of the isopeptide would lead to both y and b ion series as exemplified by the SUMO-2-sumoylated S-tag isopeptides (supplemental Fig. S1). In this case, the isopeptide, which did not carry the extra FR residues, afforded the expected y ion series dominated by y17, y20, and y22 (supplemental Fig. S1A). In contrast, the isopeptide with additional N-terminal FR residues due to missed cleavage afforded strong b4, b12, b21, and b23 ion signals, as well as y17, y20, y22, y31, and y39 ions (Fig. S1B), due to the Arg residue at the C terminus of the sumoylated tryptic peptide, ETAAAKFER.Sumoylation of Ubc9 at the Non-consensus Site—Identification of peptides matching to both SUMO-2 and Ubc9 from band 3 (Fig. 2) implicated a covalent complex of the two proteins. Two putative isopeptides were detected by MALDI-TOF/TOF MS analysis at high mass range, with their monoisotopic [M+H]+ molecular ion signals accurately mass-measured at m/z 4110.9 and 4414.0, respectively, corresponding to a FR residual mass difference. Both isopeptides were subjected to MS/MS sequence mapping and the resulting y and b ions detected (Fig. 4) confirm their identities, localizing the Lys sumoylation site to the C-terminal peptide of Ubc9, _AQAKKFAPS-COOH, similar to analogous position reported for yeast Ubc9 (_AKQYSK-COOH) conjugation with Smt3p (9Bencsath K.P. Podgorski M.S. Pagala V.R. Slaughter C.A. Schulman B.A. J. Biol. Chem. 2002; 277: 47938-47945Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). The putative isopeptide carrying the catalytic Cys93 of Ubc9 with thiol ester bond to SUMO-2 was not detected. This may reflect that such isopeptide is transient and unstable whereby the conjugated SUMO protein will be rapidly transferred to substrate proteins. In the absence of other substrate proteins in the in vitro system, Ubc9 may itself act as an acceptor, utilizing a non-consensus site near its C terminus. It is reasonable to assume that SUMO-1 can likewise sumoylate Ubc9, probably at the same site, to yield the protein band 2 in Fig. 2. Indeed, a candidate SUMO-1/Ubc9 isopeptide was barely detectable by MALDI-MS analysis of its tryptic digestion products, further MS
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