In Vivo Identification of Human Small Ubiquitin-like Modifier Polymerization Sites by High Accuracy Mass Spectrometry and an in Vitro to in Vivo Strategy
2007; Elsevier BV; Volume: 7; Issue: 1 Linguagem: Inglês
10.1074/mcp.m700173-mcp200
ISSN1535-9484
AutoresIvan Matić, Martijn van Hagen, Joost Schimmel, Boris Maček, Stephen C. Ogg, Michael H. Tatham, Ronald T. Hay, Angus I. Lamond, Matthias Mann, Alfred C.O. Vertegaal,
Tópico(s)Cancer, Hypoxia, and Metabolism
ResumoThe length and precise linkage of polyubiquitin chains is important for their biological activity. Although other ubiquitin-like proteins have the potential to form polymeric chains their identification in vivo is challenging and their functional role is unclear. Vertebrates express three small ubiquitin-like modifiers, SUMO-1, SUMO-2, and SUMO-3. Mature SUMO-2 and SUMO-3 are nearly identical and contain an internal consensus site for sumoylation that is missing in SUMO-1. Combining state-of-the-art mass spectrometry with an "in vitro to in vivo" strategy for post-translational modifications, we provide direct evidence that SUMO-1, SUMO-2, and SUMO-3 form mixed chains in cells via the internal consensus sites for sumoylation in SUMO-2 and SUMO-3. In vitro, the chain length of SUMO polymers could be influenced by changing the relative amounts of SUMO-1 and SUMO-2. The developed methodology is generic and can be adapted for the identification of other sumoylation sites in complex samples. The length and precise linkage of polyubiquitin chains is important for their biological activity. Although other ubiquitin-like proteins have the potential to form polymeric chains their identification in vivo is challenging and their functional role is unclear. Vertebrates express three small ubiquitin-like modifiers, SUMO-1, SUMO-2, and SUMO-3. Mature SUMO-2 and SUMO-3 are nearly identical and contain an internal consensus site for sumoylation that is missing in SUMO-1. Combining state-of-the-art mass spectrometry with an "in vitro to in vivo" strategy for post-translational modifications, we provide direct evidence that SUMO-1, SUMO-2, and SUMO-3 form mixed chains in cells via the internal consensus sites for sumoylation in SUMO-2 and SUMO-3. In vitro, the chain length of SUMO polymers could be influenced by changing the relative amounts of SUMO-1 and SUMO-2. The developed methodology is generic and can be adapted for the identification of other sumoylation sites in complex samples. The ubiquitin family (1Welchman R.L. Gordon C. Mayer R.J. Ubiquitin and ubiquitin-like proteins as multifunctional signals.Nat. Rev. Mol. Cell Biol. 2005; 6: 599-609Crossref PubMed Scopus (673) Google Scholar, 2Pickart C.M. Eddins M.J. Ubiquitin: structures, functions, mechanisms.Biochim. Biophys. Acta. 2004; 1695: 55-72Crossref PubMed Scopus (1039) Google Scholar) includes small ubiquitin-like modifiers (SUMOs) 1The abbreviations used are: SUMO, small ubiquitin-like modifier; E1, SUMO-activating enzyme; E2, SUMO protein carrier protein; E3, SUMO ligase; HIF, hypoxia-inducible factor; LTQ, linear quadrupole ion trap; LDS, lithium dodecyl sulfate; RanBP2, Ran-binding protein 2; SAE, SUMO-activating enzyme; Ubc9, ubiquitin-conjugating enzyme 9; aa, amino acids; HRP, horseradish peroxidase; GPMAW, General Protein/Mass Analysis for Windows. that are similar in structure to ubiquitin (3Bayer P. Arndt A. Metzger S. Mahajan R. Melchior F. Jaenicke R. Becker J. Structure determination of the small ubiquitin-related modifier SUMO-1.J. Mol. Biol. 1998; 280: 275-286Crossref PubMed Scopus (328) Google Scholar). In contrast to the well known role of ubiquitin in protein degradation by the proteasome, SUMO conjugation does not directly target proteins for destruction (4Gill G. SUMO and ubiquitin in the nucleus: different functions, similar mechanisms?.Genes Dev. 2004; 18: 2046-2059Crossref PubMed Scopus (625) Google Scholar, 5Hay R.T. SUMO: a history of modification.Mol. Cell. 2005; 18: 1-12Abstract Full Text Full Text PDF PubMed Scopus (1338) Google Scholar, 6Johnson E.S. Protein modification by SUMO.Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1392) Google Scholar). In general, sumoylation regulates the function of target proteins by affecting protein-protein interactions, which can result in altered subcellular localization and activity. Sumoylation is essential for the viability of eukaryotic cells (7Hayashi T. Seki M. Maeda D. Wang W. Kawabe Y. Seki T. Saitoh H. Fukagawa T. Yagi H. Enomoto T. Ubc9 is essential for viability of higher eukaryotic cells.Exp. Cell Res. 2002; 280: 212-221Crossref PubMed Scopus (96) Google Scholar, 8Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. The ubiquitin-like protein Smt3p is activated for conjugation to other proteins by an Aos1p/Uba2p heterodimer.EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (445) Google Scholar, 9Jones D. Crowe E. Stevens T.A. Candido E.P. Functional and phylogenetic analysis of the ubiquitylation system in Caenorhabditis elegans: ubiquitin-conjugating enzymes, ubiquitin-activating enzymes, and ubiquitin-like proteins.Genome Biol. 2002; 3 (RESEARCH0002)Google Scholar, 10Kamath R.S. Fraser A.G. Dong Y. Poulin G. Durbin R. Gotta M. Kanapin A. Le Bot N. Moreno S. Sohrmann M. Welchman D.P. Zipperlen P. Ahringer J. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi.Nature. 2003; 421: 231-237Crossref PubMed Scopus (2742) Google Scholar, 11Li S.J. Hochstrasser M. A new protease required for cell-cycle progression in yeast.Nature. 1999; 398: 246-251Crossref PubMed Scopus (608) Google Scholar, 12Nacerddine K. Lehembre F. Bhaumik M. Artus J. Cohen-Tannoudji M. Babinet C. Pandolfi P.P. Dejean A. The SUMO pathway is essential for nuclear integrity and chromosome segregation in mice.Dev. Cell. 2005; 9: 769-779Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar). A significant number of target proteins have been identified for Smt3, the yeast SUMO family member, and for mammalian SUMOs (13Xu P. Peng J. Dissecting the ubiquitin pathway by mass spectrometry.Biochim. Biophys. Acta. 2006; 1764: 1940-1947Crossref PubMed Scopus (73) Google Scholar). These proteomics studies have highlighted the broad cellular impact of SUMOs on processes including transcription, replication, RNA processing, translation, signaling, and transport. Conjugation of SUMOs to target proteins, analogous to the ubiquitin system, involves E1, E2, and E3 enzymes (1Welchman R.L. Gordon C. Mayer R.J. Ubiquitin and ubiquitin-like proteins as multifunctional signals.Nat. Rev. Mol. Cell Biol. 2005; 6: 599-609Crossref PubMed Scopus (673) Google Scholar, 2Pickart C.M. Eddins M.J. Ubiquitin: structures, functions, mechanisms.Biochim. Biophys. Acta. 2004; 1695: 55-72Crossref PubMed Scopus (1039) Google Scholar, 4Gill G. SUMO and ubiquitin in the nucleus: different functions, similar mechanisms?.Genes Dev. 2004; 18: 2046-2059Crossref PubMed Scopus (625) Google Scholar, 5Hay R.T. SUMO: a history of modification.Mol. Cell. 2005; 18: 1-12Abstract Full Text Full Text PDF PubMed Scopus (1338) Google Scholar, 6Johnson E.S. Protein modification by SUMO.Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1392) Google Scholar). The E1 enzyme is a dimer that consists of SAE1 and SAE2, and in contrast to the large set of E2 enzymes involved in ubiquitination, a single E2 enzyme, Ubc9, is responsible for sumoylation. In addition, E3 enzymes, including protein inhibitor of activated signal transducer and activator of transcription family members and RanBP2, can enhance the sumoylation of target proteins but are not strictly required in vitro (14Hochstrasser M. SP-RING for SUMO: new functions bloom for a ubiquitin-like protein.Cell. 2001; 107: 5-8Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 15Johnson E.S. Gupta A.A. An E3-like factor that promotes SUMO conjugation to the yeast septins.Cell. 2001; 106: 735-744Abstract Full Text Full Text PDF PubMed Scopus (529) Google Scholar, 16Pichler A. Gast A. Seeler J.S. Dejean A. Melchior F. The nucleoporin RanBP2 has SUMO1 E3 ligase activity.Cell. 2002; 108: 109-120Abstract Full Text Full Text PDF PubMed Scopus (645) Google Scholar, 17Sachdev S. Bruhn L. Sieber H. Pichler A. Melchior F. Grosschedl R. PIASy, a nuclear matrix-associated SUMO E3 ligase, represses LEF1 activity by sequestration into nuclear bodies.Genes Dev. 2001; 15: 3088-3103Crossref PubMed Scopus (466) Google Scholar). Sumoylation is reversible; SUMOs can be removed from target proteins by specific SUMO proteases (4Gill G. SUMO and ubiquitin in the nucleus: different functions, similar mechanisms?.Genes Dev. 2004; 18: 2046-2059Crossref PubMed Scopus (625) Google Scholar, 5Hay R.T. SUMO: a history of modification.Mol. Cell. 2005; 18: 1-12Abstract Full Text Full Text PDF PubMed Scopus (1338) Google Scholar, 6Johnson E.S. Protein modification by SUMO.Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1392) Google Scholar, 11Li S.J. Hochstrasser M. A new protease required for cell-cycle progression in yeast.Nature. 1999; 398: 246-251Crossref PubMed Scopus (608) Google Scholar). These proteases are also responsible for the maturation of SUMO precursors, a process that exposes the carboxyl-terminal diglycine motif that is characteristic for ubiquitin-like proteins and required for conjugation to target proteins. Ubiquitin is able to form chains on target proteins via all seven internal lysines (18Pickart C.M. Fushman D. Polyubiquitin chains: polymeric protein signals.Curr. Opin. Chem. Biol. 2004; 8: 610-616Crossref PubMed Scopus (840) Google Scholar, 19Peng 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: 921-926Crossref PubMed Scopus (1319) Google Scholar). Ubiquitin chains were initially discovered by studying the role of ubiquitin in targeting protein substrates for proteolysis. These chains are Lys-48-linked polymers that mark target proteins for proteasome-mediated destruction (20Chau V. Tobias J.W. Bachmair A. Marriott D. Ecker D.J. Gonda D.K. Varshavsky A. A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein.Science. 1989; 243: 1576-1583Crossref PubMed Scopus (1122) Google Scholar). Structurally different ubiquitin chains can also play other roles in cells that are unrelated to protein degradation (18Pickart C.M. Fushman D. Polyubiquitin chains: polymeric protein signals.Curr. Opin. Chem. Biol. 2004; 8: 610-616Crossref PubMed Scopus (840) Google Scholar, 21Sun L. Chen Z.J. The novel functions of ubiquitination in signaling.Curr. Opin. Cell Biol. 2004; 16: 119-126Crossref PubMed Scopus (375) Google Scholar). For example, Lys-63-linked chains are involved in translation, protein kinase activation, vesicle trafficking, and DNA repair. An interesting NMR study has revealed that the conformation of a Lys-63-linked ubiquitin dimer is distinct from a Lys-48-linked dimer (22Varadan R. Assfalg M. Haririnia A. Raasi S. Pickart C. Fushman D. Solution conformation of Lys63-linked di-ubiquitin chain provides clues to functional diversity of polyubiquitin signaling.J. Biol. Chem. 2004; 279: 7055-7063Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). Yeast cells that express a K63R mutant of ubiquitin are compromised in DNA repair, but proteolysis is not affected in these cells (23Spence J. Sadis S. Haas A.L. Finley D. A ubiquitin mutant with specific defects in DNA repair and multiubiquitination.Mol. Cell Biol. 1995; 15: 1265-1273Crossref PubMed Google Scholar). In contrast to the extensive amount of data on ubiquitin chain formation (19Peng 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: 921-926Crossref PubMed Scopus (1319) Google Scholar, 24Hoeller D. Crosetto N. Blagoev B. Raiborg C. Tikkanen R. Wagner S. Kowanetz K. Breitling R. Mann M. Stenmark H. Dikic I. Regulation of ubiquitin-binding proteins by monoubiquitination.Nat. Cell Biol. 2006; 8: 163-169Crossref PubMed Scopus (267) Google Scholar, 25Kirkpatrick D.S. Weldon S.F. Tsaprailis G. Liebler D.C. Gandolfi A.J. Proteomic identification of ubiquitinated proteins from human cells expressing His-tagged ubiquitin.Proteomics. 2005; 5: 2104-2111Crossref PubMed Scopus (81) Google Scholar), very little is known about multimerization of ubiquitin-like proteins. The single SUMO family member in Saccharomyces cerevisiae, Smt3, has been shown to form chains, but these chains are not required for viability (26Bylebyl G.R. Belichenko I. Johnson E.S. The SUMO isopeptidase Ulp2 prevents accumulation of SUMO chains in yeast.J. Biol. Chem. 2003; 278: 44113-44120Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). A yeast strain in which wild-type Smt3 was replaced by a lysine-deficient Smt3 mutant was viable; it had no obvious growth defects or stress sensitivities. The amount of Smt3 chains in yeast is limited due to the activity of the Smt3 protease Ulp2 (26Bylebyl G.R. Belichenko I. Johnson E.S. The SUMO isopeptidase Ulp2 prevents accumulation of SUMO chains in yeast.J. Biol. Chem. 2003; 278: 44113-44120Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Interestingly Smt3 chains accumulate during meiosis (27Cheng C.H. Lo Y.H. Liang S.S. Ti S.C. Lin F.M. Yeh C.H. Huang H.Y. Wang T.F. SUMO modifications control assembly of synaptonemal complex and polycomplex in meiosis of Saccharomyces cerevisiae.Genes Dev. 2006; 20: 2067-2081Crossref PubMed Scopus (209) Google Scholar). Here we investigated the polymerization of the three mammalian SUMOs by mass spectrometry. Trypsin digestion of ubiquitinated proteins produces diglycine-modified lysines, which are easily detected in MS and MS/MS spectra because of their predictable mass shift. In contrast, it is technically challenging to map attachment sites for human SUMO family members due to the fact that the long SUMO tryptic peptides attached to modified lysines substantially increase the mass of the peptide and also fragment during MS/MS. The resulting fragmentation patterns are very complex and not readily interpretable with currently available software for analyzing MS/MS spectra. Recently an automated pattern recognition tool (29Pedrioli P.G. Raught B. Zhang X.D. Rogers R. Aitchison J. Matunis M. Aebersold R. Automated identification of SUMOylation sites using mass spectrometry and SUMmOn pattern recognition software.Nat. Methods. 2006; 3: 533-539Crossref PubMed Scopus (102) Google Scholar) has been developed to overcome this limitation, but further work is needed to test its utility in vivo. Mutational strategies where trypsin cleavage sites are introduced close to the SUMO carboxyl-terminal diglycine (28Knuesel M. Cheung H.T. Hamady M. Barthel K.K. Liu X. A method of mapping protein sumoylation sites by mass spectrometry using a modified small ubiquitin-like modifier 1 (SUMO-1) and a computational program.Mol. Cell. Proteomics. 2005; 4: 1626-1636Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 30Wohlschlegel J.A. Johnson E.S. Reed S.I. Yates III, J.R. Improved identification of SUMO attachment sites using C-terminal SUMO mutants and tailored protease digestion strategies.J. Proteome Res. 2006; 5: 761-770Crossref PubMed Scopus (47) Google Scholar) simplify the mass spectrometric analysis but suffer from the use of non-physiological modifiers. Here we developed an alternative mass spectrometric strategy based on high resolution MS and the transfer of in vitro MS data to the in vivo data generated from very small sample amounts and high sample complexity. We used this strategy to identify conjugation sites for human SUMO family members and to unambiguously detect SUMO branched peptides. This approach allowed us to map the internal lysines that are used for SUMO chain formation and to demonstrate the ability of SUMOs to form chains in vivo. SUMO-1 and SUMO-2 proteins were produced in Escherichia coli and purified as described previously (31Tatham M.H. Jaffray E. Vaughan O.A. Desterro J.M. Botting C.H. Naismith J.H. Hay R.T. Polymeric chains of SUMO-2 and SUMO-3 are conjugated to protein substrates by SAE1/SAE2 and Ubc9.J. Biol. Chem. 2001; 276: 35368-35374Abstract Full Text Full Text PDF PubMed Scopus (645) Google Scholar). GST-SUMO-1, GST-SAE2-SAE1, GST-Ubc9, and control GST were produced in E. coli and purified as described previously (31Tatham M.H. Jaffray E. Vaughan O.A. Desterro J.M. Botting C.H. Naismith J.H. Hay R.T. Polymeric chains of SUMO-2 and SUMO-3 are conjugated to protein substrates by SAE1/SAE2 and Ubc9.J. Biol. Chem. 2001; 276: 35368-35374Abstract Full Text Full Text PDF PubMed Scopus (645) Google Scholar, 32Mohrmann L. Kal A.J. Verrijzer C.P. Characterization of the extended Myb-like DNA-binding domain of trithorax group protein Zeste.J. Biol. Chem. 2002; 277: 47385-47392Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). The GST tag was removed from the E2 by thrombin cleavage to increase the enzymatic activity. T7-HIF-1α-His6 (aa 373–605) was produced in E. coli and purified as described previously (33Groot A.J. Verheesen P. Westerlaken E.J. Gort E.H. van der Groep P. Bovenschen N. van der Wall E. van Diest P.J. Shvarts A. Identification by phage display of single-domain antibody fragments specific for the ODD domain in hypoxia-inducible factor 1α.Lab. Investig. 2006; 86: 345-356Crossref PubMed Scopus (32) Google Scholar). Peptide antibody AV-SM23-0100 against SUMO-2/3 was generated in a rabbit using the peptide MEDEDTIDVFQQQTG (Eurogentec) (34Vertegaal A.C. Ogg S.C. Jaffray E. Rodriguez M.S. Hay R.T. Andersen J.S. Mann M. Lamond A.I. A proteomic study of SUMO-2 target proteins.J. Biol. Chem. 2004; 279: 33791-33798Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Monoclonal antibody 21C7 against SUMO-1 was obtained from Zymed Laboratories Inc., and monoclonal antibody 610958 against hypoxia-inducible factor-1α (HIF-1α) was obtained from BD Biosciences. Anti-T7 antibody coupled to HRP was obtained from Novagen (1:5000). Secondary antibodies used were anti-rabbit HRP and anti-mouse HRP (1:5000, Pierce) and Texas Red-conjugated anti-rabbit and fluorescein isothiocyanate-conjugated anti-mouse (1:350, Jackson ImmunoResearch Laboratories). Protein samples were size-fractionated on Novex 4–12% 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol gradient gels using 4-morpholinepropanesulfonic acid buffer (Invitrogen). Total protein was visualized by silver staining. For immunoblotting experiments, size-fractionated proteins were subsequently transferred onto Hybond-C extra membranes (Amersham Biosciences) using a submarine system (Invitrogen). The membranes were incubated with specific antibodies as indicated. Bound antibodies were detected via chemiluminescence with ECL Plus (Amersham Biosciences). HeLa cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% FCS and 100 units/ml penicillin and streptomycin (Invitrogen). HIF-1α was stabilized by 0.9 mm CoCl2 (Sigma) treatment for 3 h. HeLa cells stably expressing His6-SUMO-2 were described previously (34Vertegaal A.C. Ogg S.C. Jaffray E. Rodriguez M.S. Hay R.T. Andersen J.S. Mann M. Lamond A.I. A proteomic study of SUMO-2 target proteins.J. Biol. Chem. 2004; 279: 33791-33798Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). GST-SUMO-1 conjugates were obtained by incubating 20 μg of GST-SUMO-1 or control GST with 100 μl of HeLa nuclear extract (CILBiotech) in a buffer containing 1.5 mm ATP, 5 mm creatine phosphate (Sigma), 5 mm DTT, and 2 mm MgCl2 for 2.5 h at 30 °C. GST-SUMO-1 conjugates were bound to 30 μl of glutathione beads (GE Healthcare) for 1 h at 4 °C. Beads were successively washed with conjugation buffer, PBS, PBS containing 0.1% Triton X-100, and PBS only at 4 °C. Bound proteins were eluted successively in 8 m urea, pH 7, and NuPage LDS protein sample buffer (Invitrogen). His6-SUMO-2 conjugates were purified essentially as described previously (34Vertegaal A.C. Ogg S.C. Jaffray E. Rodriguez M.S. Hay R.T. Andersen J.S. Mann M. Lamond A.I. A proteomic study of SUMO-2 target proteins.J. Biol. Chem. 2004; 279: 33791-33798Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Endogenous SUMO-2/3 conjugates were purified from HeLa cells lysed in 2% SDS, 50 mm Tris-HCl, pH 7.5, and 10 mm iodoacetamide supplemented with protease inhibitor mixture 1873580 (Roche Diagnostics GmbH) (35Jaffray E.G. Hay R.T. Detection of modification by ubiquitin-like proteins.Methods. 2006; 38: 35-38Crossref PubMed Scopus (30) Google Scholar). Lysates were sonicated and diluted 20-fold in 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.5 mm β-mercaptoethanol, and 0.5% Nonidet P-40 supplemented with protease inhibitor mixture. Immunoprecipitations were performed with antibody AV-SM23-0100 or preimmune serum covalently cross-linked to protein G-Sepharose beads (GE Healthcare) for 3 h at room temperature. After extensive washing, bound proteins were eluted in NuPage LDS protein sample buffer (Invitrogen). SUMO polymer formation described in Fig. 2A was carried out in 10-μl volumes containing 120 ng of SAE1/2, 2 mm ATP, 0.6 units·ml−1 inorganic pyrophosphatase, 10 mm creatine phosphate, 3.5 units·ml−1 creatine kinase (Sigma), 5 mm MgCl2, 50 mm Tris-HCl, pH 7.5, 800 ng of Ubc9, protease inhibitor mixture, and the amounts of Ubc9, SUMO-1, and/or SUMO-2 indicated in the figure. Experiments described in Fig. 2B were carried out in 5-μl volumes and contained the ATP regeneration mixture, 60 ng of SAE1/2, 400 ng of Ubc9, and the indicated amounts of SUMO-1 and/or SUMO-2. For mass spectrometric analysis, a similar experiment without protease inhibitors was carried out using 2 μg of SUMO-1, 2 μg of SUMO-2, 480 ng of SAE1/2, and 4 μg of Ubc9 in a total volume of 40 μl. Assays were incubated for 3 h at 37 °C before either endopeptidase Lys-C and trypsin digestion and mass spectrometric analysis or addition of SDS sample buffer for immunoblotting analysis. Aliquots representing 6% of the reaction mixtures were loaded on the gel. 5 μg of recombinant T7-HIF-1α-His6 (aa 373–605) was sumoylated in vitro and subsequently purified in 8 m urea on Talon beads for mass spectrometric analysis. Mass spectrometric analysis was performed by nanoscale LC-MS/MS using a linear ion trap-Fourier transform-ion cyclotron resonance mass spectrometer (LTQ-FT-ICR, Thermo Fisher Scientific, Bremen, Germany) or an LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific) equipped with a nanoelectrospray ion source (Proxeon Biosystems, Odense, Denmark) and coupled to an Agilent 1100 nano-HPLC system (Agilent Technologies) fitted with an in-house made 75-μm reverse phase C18 column as described previously (36Olsen J.V. Ong S.E. Mann M. Trypsin cleaves exclusively C-terminal to arginine and lysine residues.Mol. Cell. Proteomics. 2004; 3: 608-614Abstract Full Text Full Text PDF PubMed Scopus (881) Google Scholar, 37Olsen J.V. de Godoy L.M. Li G. Macek B. Mortensen P. Pesch R. Makarov A. Lange O. Horning S. Mann M. Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap.Mol. Cell. Proteomics. 2005; 4: 2010-2021Abstract Full Text Full Text PDF PubMed Scopus (1246) Google Scholar). In-solution digestion was performed essentially as described previously (38Foster L.J. de Hoog C.L. Mann M. Unbiased quantitative proteomics of lipid rafts reveals high specificity for signaling factors.Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5813-5818Crossref PubMed Scopus (730) Google Scholar). The 50-kDa band from a silver-stained gel containing GST-SUMO-1 conjugates (Fig. 4A) was excised, cut into 1-mm3 cubes, and subjected to in-gel digestion according to Olsen et al. (37Olsen J.V. de Godoy L.M. Li G. Macek B. Mortensen P. Pesch R. Makarov A. Lange O. Horning S. Mann M. Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap.Mol. Cell. Proteomics. 2005; 4: 2010-2021Abstract Full Text Full Text PDF PubMed Scopus (1246) Google Scholar). The resulting peptides were desalted on RP-C18 stop and go extraction tips (39Rappsilber J. Ishihama Y. Mann M. Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics.Anal. Chem. 2003; 75: 663-670Crossref PubMed Scopus (1833) Google Scholar). Peptides were eluted with a 140-min linear gradient of 98% solvent A (0.5% acetic acid in H2O) to 50% solvent B (80% acetonitrile and 0.5% acetic acid in H2O). Data were acquired in the data-dependent mode: in the case of the LTQ-FT-ICR instrument, full scan spectra (m/z 300–1800, R = 50,000, and ion accumulation to a target value of 3,000,000) were acquired in the ICR cell. The three most intense ions were sequentially isolated for accurate mass measurements by selected ion monitoring scans with 10-Da mass range, R = 50,000, and a target accumulation value of 50,000 and fragmented in the linear ion trap by collisionally induced dissociation followed by MS3 analysis of the most intense product ion in the MS/MS scan. In the case of the LTQ-Orbitrap, the precursor ion spectra were acquired in the orbitrap analyzer (m/z 300–1600, R = 60,000, and ion accumulation to a target value of 1,000,000), and the five most intense ions were fragmented and recorded in the ion trap. In a separate experiment, peptides derived from the digestion of in vitro produced SUMO polymers were fragmented in the linear ion trap, and the fragment ions were recorded in the orbitrap (R = 15,000). The lock mass option enabled accurate mass measurement in both MS and orbitrap MS/MS mode as described previously (37Olsen J.V. de Godoy L.M. Li G. Macek B. Mortensen P. Pesch R. Makarov A. Lange O. Horning S. Mann M. Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap.Mol. Cell. Proteomics. 2005; 4: 2010-2021Abstract Full Text Full Text PDF PubMed Scopus (1246) Google Scholar). Target ions already selected for MS/MS were dynamically excluded for 30 s. The detection and fragmentation of the SUMO-1/SUMO-2 parent ion, derived from the digestion of the His6-SUMO-2 and GST-SUMO-1 conjugates, were obtained with the selected ion monitoring mode with 10-Da mass range in which the dynamic exclusion option was not active and only ions with charge state equal to or larger than 4+ or unassigned were fragmented and recorded in the LTQ. All spectra were acquired in the profile mode. The monoisotopic m/z values for the SUMO-1/SUMO-2, SUMO-1/SUMO-3, SUMO-2/SUMO-2, and SUMO-2/SUMO-3 branched precursor peptides were calculated with GPMAW software (Lighthouse Data, Hanstholm, Denmark) and used to search for the corresponding ions with Xcalibur software (Thermo Fisher Scientific). Assignment was confirmed by manually interpreting all MS/MS spectra. The corresponding "virtual" peptides were fragmented in silico with GPMAW, and the resulting m/z values were used to manually assign fragment ions to the peaks in the experimental fragmentation spectra. All reported MS/MS spectra were manually validated. Only branched peptides having an extensive coverage of y ions were considered. The peptides modified by SUMO-2, containing two prolines, were required to show pronounced cleavage amino-terminal to the proline residue. Parent ion charge and retention time were derived from a pilot LC-MS run of simple peptide mixtures from an in vitro sumoylation reaction and used together with precursor m/z values and fragmentation spectra to search for the branched peptides in a more complex mixture. The LC-MS runs were visualized by using the Viewer tool of our in-house quantitative proteomics processing pipeline (40Cox J. Mann M. Is proteomics the new genomics?.Cell. 2007; 130: 395-398Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar). The resulting two-dimensional LC-MS plots show the peptide m/z values (x axis) along the retention time axis. All the precursor isotope peaks were incorporated into the plots, and their signal intensities are color-coded with white representing the lowest intensities and green representing the highest intensities. The "full range" mode visualizes the peaks present in all MS spectra acquired during a 140-min chromatographic gradient. The enlarged view of specific SUMO branched peptides was obtained by selecting the corresponding m/z and time range and was used for a visual comparison of their abundances between different samples. HeLa cells were grown on glass coverslips and fixed for 10 min in 3.7% paraformaldehyde in 37 °C PHEM buffer (60 mm PIPES, 25 mm HEPES, 10 mm EGTA, and 2 mm MgCl2, pH 6.9) (41Trinkle-Mulcahy L. Andrews P.D. Wickramasinghe S. Sleeman J. Prescott A. Lam Y.W. Lyon C. Swedlow J.R. Lamond A.I. Time-lapse imaging reveals dynamic relocalization of PP1γ throughout the mammalian cell cycle.Mol. Biol. Cell. 2003; 14: 107-117Crossref PubMed Scopus (124) Google Scholar). Subsequent manipulations were carried out at room temperature. Permeabilization was carried out for 20 min in PBS containing 0.5% Triton X-100. Cells were incubated with primary antibodies AV-SM23-0100 against SUMO-2/3 (1:2000) and 21C7 against SUMO-1 (1:50), washed, and incubated with secondary antibodies. DNA was stained with 0.3 μg/ml 4`,6-diamidino-2-phenylindole (Sigma). After washing, cells were mounted in Vectashield (Vector Laboratories). Three-dimensional images and sections were recorded on a Zeiss Axiovert S100 2TV DeltaVision Restoration microscope (Applied Precision) using a Zeiss Plan-Achromat 100 × 1.40-numerical aperture objective and a CCD-1300-Y/HS camera (Roper Scientific). Images were captured and processed by constrained iterative deconvolution using SoftWorx (Applied Precision). Images presented here are maximal intensity projections of Z stacks. Human SUMO-2 and SUMO-3 contain an internal consensus site for sumoylation that is absent from SUMO-1 (Fig. 1A). This allows SUMO-2 and SUMO-3 to polymerize in vitro (31Tatham M.H. Jaffray E. Vaughan O.A. Desterro J.M. Botting C.H. Naismith J.H. Hay R.T. Polymeric chains of SUMO-2 and SUMO-3 are conjugated to protein substrates by SAE1
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