“ChopNSpice,” a Mass Spectrometric Approach That Allows Identification of Endogenous Small Ubiquitin-like Modifier-conjugated Peptides

Conjugation of small ubiquitin-like modifier (SUMO) to substrates is involved in a large number of cellular processes. Typically, SUMO is conjugated to lysine residues within a SUMO consensus site; however, an increasing number of proteins are sumoylated on non-consensus sites. To appreciate the functional consequences of sumoylation, the identification of SUMO attachment sites is of critical importance. Discovery of SUMO acceptor sites is usually performed by a laborious mutagenesis approach or using MS. In MS, identification of SUMO acceptor sites in higher eukaryotes is hampered by the large tryptic fragments of SUMO1 and SUMO2/3. MS search engines in combination with known databases lack the possibility to search MSMS spectra for larger modifications, such as sumoylation. Therefore, we developed a simple and straightforward database search tool (“ChopNSpice”) that successfully allows identification of SUMO acceptor sites from proteins sumoylated in vivo and in vitro. By applying this approach we identified SUMO acceptor sites in, among others, endogenous SUMO1, SUMO2, RanBP2, and Ubc9. Conjugation of small ubiquitin-like modifier (SUMO) to substrates is involved in a large number of cellular processes. Typically, SUMO is conjugated to lysine residues within a SUMO consensus site; however, an increasing number of proteins are sumoylated on non-consensus sites. To appreciate the functional consequences of sumoylation, the identification of SUMO attachment sites is of critical importance. Discovery of SUMO acceptor sites is usually performed by a laborious mutagenesis approach or using MS. In MS, identification of SUMO acceptor sites in higher eukaryotes is hampered by the large tryptic fragments of SUMO1 and SUMO2/3. MS search engines in combination with known databases lack the possibility to search MSMS spectra for larger modifications, such as sumoylation. Therefore, we developed a simple and straightforward database search tool (“ChopNSpice”) that successfully allows identification of SUMO acceptor sites from proteins sumoylated in vivo and in vitro. By applying this approach we identified SUMO acceptor sites in, among others, endogenous SUMO1, SUMO2, RanBP2, and Ubc9. Post-translational modification with ubiquitin and ubiquitin-like modifiers (Ubls) 1The abbreviations used are:Ublubiquitin-like modifierSUMOsmall ubiquitin-like modifierPIASprotein inhibitors of activated STAT (signal transducers and activators of transcription)NEMN-ethylmaleimideLTQlinear trap quadrupole. such as SUMO plays an important role in most, if not all, cellular processes (1Kerscher O. Felberbaum R. Hochstrasser M. Modification of proteins by ubiquitin and ubiquitin-like proteins.Annu. Rev. Cell Dev. Biol. 2006; 22: 159-180Crossref PubMed Scopus (1232) Google Scholar, 2Hay R.T. SUMO: a history of modification.Mol. Cell. 2005; 18: 1-12Abstract Full Text Full Text PDF PubMed Scopus (1338) Google Scholar, 3Meulmeester E. Melchior F. Cell biology: SUMO.Nature. 2008; 452: 709-711Crossref PubMed Scopus (133) Google Scholar, 4Geiss-Friedlander R. Melchior F. Concepts in sumoylation: a decade on.Nat. Rev. Mol. Cell Biol. 2007; 8: 947-956Crossref PubMed Scopus (1372) Google Scholar, 5Hershko A. Ciechanover A. The ubiquitin system.Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6959) Google Scholar, 6Johnson E.S. Protein modification by SUMO.Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1392) Google Scholar). Conjugation of Ubls to their targets involves an isopeptide bond between the carboxyl group of the modifier and the ε-amino group of a lysine residue within the targets. Attachment of Ubls to specific targets involves an enzymatic cascade. First the Ubls are processed to expose their C-terminal diglycine motif. The mature Ubl is then transferred to its target via a cascade of E1 (activating), E2 (conjugating), and E3 (ligase) enzymes. The conjugation system for SUMO consists of a heterodimeric activating enzyme, Aos1/Uba2; a conjugating enzyme, Ubc9; and E3 ligases, such as RanBP2 or members of the PIAS family. The conjugation status undergoes perpetual change and is governed by a small family of SUMO proteases that hydrolyze the isopeptide bond between SUMO and its target (7Hay R.T. SUMO-specific proteases: a twist in the tail.Trends Cell Biol. 2007; 17: 370-376Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 8Mukhopadhyay D. Dasso M. Modification in reverse: the SUMO proteases.Trends Biochem. Sci. 2007; 32: 286-295Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar). Although in lower eukaryotes only one SUMO is present, vertebrates express at least three different SUMO paralogs: SUMO1, SUMO2, and SUMO3. Mature SUMO2 and SUMO3 (referred to as SUMO2/3) are 97% identical but differ substantially from SUMO1 (∼50% identity). ubiquitin-like modifier small ubiquitin-like modifier protein inhibitors of activated STAT (signal transducers and activators of transcription) N-ethylmaleimide linear trap quadrupole. Although the list of known SUMO substrates is growing rapidly, our understanding of the functional consequences for many of these targets is lagging behind. At a molecular level, the functional consequences of SUMO conjugation can be explained by a gain or loss of interaction with other macromolecules (3Meulmeester E. Melchior F. Cell biology: SUMO.Nature. 2008; 452: 709-711Crossref PubMed Scopus (133) Google Scholar, 4Geiss-Friedlander R. Melchior F. Concepts in sumoylation: a decade on.Nat. Rev. Mol. Cell Biol. 2007; 8: 947-956Crossref PubMed Scopus (1372) Google Scholar). SUMO-dependent intramolecular conformational changes have also been described (9Steinacher R. Schär P. Functionality of human thymine DNA glycosylase requires SUMO-regulated changes in protein conformation.Curr. Biol. 2005; 15: 616-623Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 10Baba D. Maita N. Jee J.G. Uchimura Y. Saitoh H. Sugasawa K. Hanaoka F. Tochio H. Hiroaki H. Shirakawa M. Crystal structure of thymine DNA glycosylase conjugated to SUMO-1.Nature. 2005; 435: 979-982Crossref PubMed Scopus (186) Google Scholar). Thus, to appreciate the role that SUMO plays in the regulation of specific substrates, identification of the acceptor site(s) for SUMO conjugation is of key importance. So far, identification of SUMO acceptor sites has relied largely on mutation of the SUMO consensus site, which consists of a short motif with the sequence ψKXE (ψ represents a bulky hydrophobic residue, and X represents any amino acid). This motif is recognized by Ubc9 if presented in an extended conformation (11Sampson D.A. Wang M. Matunis M.J. The small ubiquitin-like modifier-1 (SUMO-1) consensus sequence mediates Ubc9 binding and is essential for SUMO-1 modification.J. Biol. Chem. 2001; 276: 21664-21669Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar, 12Lin D. Tatham M.H. Yu B. Kim S. Hay R.T. Chen Y. Identification of a substrate recognition site on Ubc9.J. Biol. Chem. 2002; 277: 21740-21748Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 13Bernier-Villamor V. Sampson D.A. Matunis M.J. Lima C.D. Structural basis for E2-mediated SUMO conjugation revealed by a complex between ubiquitin-conjugating enzyme Ubc9 and RanGAP1.Cell. 2002; 108: 345-356Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar). However, an increasing number of proteins, such as PCNA, E2-25K, Daxx, and USP25, turned out to be sumoylated on lysine residues that do not conform to the SUMO consensus site (14Hoege C. Pfander B. Moldovan G.L. Pyrowolakis G. Jentsch S. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO.Nature. 2002; 419: 135-141Crossref PubMed Scopus (1751) Google Scholar, 15Pichler A. Knipscheer P. Oberhofer E. van Dijk W.J. Körner R. Olsen J.V. Jentsch S. Melchior F. Sixma T.K. SUMO modification of the ubiquitin-conjugating enzyme E2–25K.Nat. Mol. Biol. 2005; PubMed Scopus Google Scholar, Chen M.J. of motif in SUMO and of sumoylated Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, E. M. H. Melchior F. and consequences for of Cell. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). this of proteins, as as for proteins that a large number of SUMO consensus the identification of acceptor is a that involves mutagenesis of lysine residue within the substrate in MS is one of the to protein and their in an and have using MS can be to endogenous substrates for SUMO conjugation C. D. for protein in Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, Hay R.T. M. and of SUMO-1 and target proteins revealed by Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, A. H. A. Hochstrasser M. the by in Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). However, the identification of SUMO acceptor using MS has a C. D. for protein in Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, van M. B. Tatham M.H. Hay R.T. M. In vivo identification of human small ubiquitin-like modifier sites by and an in to in vivo Cell. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. of protein sites by using a small ubiquitin-like modifier (SUMO-1) and a Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, B. R. Matunis M. R. identification of sites using and recognition 2006; PubMed Scopus Google Scholar). So far, using identification of acceptor for endogenous substrates has only been in C. D. for protein in Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). The identification of substrates in higher eukaryotes has been hampered by the large conjugated SUMO that tryptic with human SUMO1 and with human with for in S. large in to the of the conjugated can their and in MS. To of these different have been mutation of the tryptic of a tryptic M. M. of protein sites by using a small ubiquitin-like modifier (SUMO-1) and a Cell. 2005; Full Text Full Text PDF PubMed Scopus Google of an recognition tool B. R. Matunis M. R. identification of sites using and recognition 2006; PubMed Scopus Google and identification of targets using an in to in vivo approach van M. B. Tatham M.H. Hay R.T. M. In vivo identification of human small ubiquitin-like modifier sites by and an in to in vivo Cell. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). Although these have been successfully for the identification of SUMO in and in identification of SUMO in vivo has not been in higher to such identification of SUMO is the of that can for one lysine in a protein of tryptic Thus, the identification of SUMO acceptor sites requires the of the the conjugated SUMO to be with and requires sequence of the search engines lack the possibility to search MSMS spectra for larger modifications, that sumoylation, we developed a and straightforward database search tool (“ChopNSpice”) in combination with search engines as protein identification by sequence databases using PubMed Scopus Google or K. 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M. H. Melchior F. and consequences for of Cell. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar, A. A. A. Melchior F. The RanBP2 has SUMO1 E3 2002; 108: Full Text Full Text PDF PubMed Scopus Google Scholar). for by Protein for SUMO1, SUMO2, and has been described E. M. H. Melchior F. and consequences for of Cell. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar, A. A. A. Melchior F. The RanBP2 has SUMO1 E3 2002; 108: Full Text Full Text PDF PubMed Scopus Google Scholar, R. C. Melchior F. small involved in to complex protein Full Text Full Text PDF PubMed Scopus Google Scholar, A. Melchior F. in using Mol. Biol. 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