Automated identification of SUMOylation sites using mass spectrometry and SUMmOn pattern recognition software

Pedrioli, Patrick G. A.; Raught, Brian; Zhang, Xiang Dong; Rogers, Richard S.; Aitchison, John D.; Matunis, Michael J.; Aebersold, Ruedi

doi:10.1038/nmeth891

Cited by 115 publications

(112 citation statements)

References 30 publications

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“…Potential SUMO peptide candidates were also obtained using ChopNSpice by generating a modified IPI human database containing the five-amino acid SUMO-specific tag on each lysine residue (35). Database searches using SUMmOn were performed using custom software available from Dr. Brian Raught (University Health Network, Toronto, Canada) (34). Manual inspection of all MS/MS spectra for modified peptides was performed to validate assignments (supplemental Fig.…”

Section: Methodsmentioning

confidence: 99%

“…This limitation was also described in an earlier report by Wohlschlegel et al (33) who indicated that yeast Smt3 mutant with an arginine at the third residue from the C terminus yielded tryptic peptides identical to those of a ubiquitin remnant and facilitated their identification using the database engine SEQUEST. To overcome some of these limitations, different database searching strategies including an automated recognition pattern tool (SUMmOn) (34) and combined search engines (ChopNSpice) (35) were developed to identify potential acceptor sites from MS/MS spectra of these large precursor ions.…”

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confidence: 99%

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A Novel Proteomics Approach to Identify SUMOylated Proteins and Their Modification Sites in Human Cells

Galisson

Mahrouche

Courcelles

et al. 2011

Molecular & Cellular Proteomics

136

139

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The small ubiquitin-related modifier (SUMO) is a small group of proteins that are reversibly attached to protein substrates to modify their functions. The large scale identification of protein SUMOylation and their modification sites in mammalian cells represents a significant challenge because of the relatively small number of in vivo substrates and the dynamic nature of this modification. We report here a novel proteomics approach to selectively enrich and identify SUMO conjugates from human cells. We stably expressed different SUMO paralogs in HEK293 cells, each containing a His 6 tag and a strategically located tryptic cleavage site at the C terminus to facilitate the recovery and identification of SUMOylated peptides by affinity enrichment and mass spectrometry. Tryptic peptides with short SUMO remnants offer significant advantages in large scale SUMOylome experiments including the generation of paralog-specific fragment ions following CID and ETD activation, and the identification of modified peptides using conventional database search engines such as Mascot. We The small ubiquitin-like modifier (SUMO) 1 proteins are structurally similar to ubiquitin, although they share less than 20% sequence identity (1). Like ubiquitylation, protein SUMOylation is regulated by a cascade of reactions involving SUMO-activating enzymes (SAE1/SAE2), -conjugating enzymes (Ubc9), and one of several SUMO E3 ligases (e.g. PIAS1, PIAS3, PIASx␣, PIASx␤, PIASy, RanBP2, and Pc2) that covalently attach SUMO to specific protein substrates (2, 3). SUMO proteins are expressed as an immature proform that comprises an invariant Gly-Gly motif followed by a Cterminal stretch of variable length (2-11 amino acids). Removal of this C-terminal extension by sentrin-specific proteases (SENPs) to expose the diglycine motif is necessary for the conjugation of SUMO to protein targets. These SUMO proteases are able to cleave both a peptide bond during the formation of mature SUMO and an isopeptide bond to deconjugate modified protein substrates (4). This covalent modification arises from the formation of an isopeptide bond between the -amino group of a lysine within the protein substrate and the C terminus carboxyl group of the SUMO glycine residue. SUMO conjugation frequently occurs at the lysine residue within the consensus motif KXE (where is an aliphatic residue and X is any amino acid) that is recognized by Ubc9 (5, 6). Recent studies have also identified a phosphorylation-dependent motif (⌿KXEXXpSP where pS is phosphoserine) (7) and a negatively charged amino acid-dependent motif (8) that harbor negative charges next to the basic SUMO consensus site to enhance protein SUMOylation. However, several other SUMOylated proteins including proliferating cell nuclear antigen, E2-25K, Daxx (death domainassociated protein), and USP25 are modified at non-consensus sites (9 -11). Whether these types of sites are rare 1 The abbreviations used are: SUMO, small ubiquitin-related modifier; As 2 O 3 , arsenic trioxide; E2-25K, E2 ubiquitin ligase, 25 kilodalto...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

A Novel Proteomics Approach to Identify SUMOylated Proteins and Their Modification Sites in Human Cells

Galisson

Mahrouche

Courcelles

et al. 2011

Molecular & Cellular Proteomics

136

139

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“…The resolution and mass accuracy of this technique is ideal for analysis of large isopeptides containing the human SUMO signature tags, though it still likely limited to samples of low complexity. What should further help these analyses are the development of improved software packages, such as SUMnOn [61]. This program can be tailored for identification of fragment ion patterns generated by complex post-translational modifications, such as sumoylation.…”

Section: Identification Of Target Lysine In Sumo Substratesmentioning

confidence: 99%

Ubiquitin proteolytic system: focus on SUMO

Wilson¹,

Heaton²

2008

Expert Review of Proteomics

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The Small Ubiquitin-like Modifier proteins (Smt3 in yeast and SUMOs 1-4 in vertebrates) are members of the ubiquitin super family. Like ubiquitin, the SUMOs are protein modifiers that are covalently attached to the epsilon amino group of lysine residues in the substrates. The application of proteomics to the SUMO field has greatly expanded both the number of known targets and the number of identified target lysines. As new refinements of proteomic techniques are developed and applied to sumoylation, an explosion of novel data is likely in the next five years. This ability to examine sumoylated proteins globally, rather than individually, will lead to new insights into both the functions of the individual SUMO types and to how dynamic changes in overall sumoylation occur in response to alterations in cellular environment. In addition, there is a growing appreciation for the existence of crosstalk mechanisms between the sumoylation and ubiquitinylation processes. Rather than being strictly parallel, these two systems have many points of intersection, and It is likely that coordination of these two systems is a critical contributor to the regulation of many fundamental cellular events. KeywordsMass spectrometry; Proteomics; Tandem Affinity Purification; Cross-talk; Signature Tags; STUbL; Ubl; SUMOeome SUMOs and the ubiquitin super familyThe small protein modifier, ubiquitin, is the prototype of the ubiquitin superfamily [1]. The biochemistry of ubiquitinylation is well understood as is the role of polyubiquitinylation in targeting proteins for proteasomal degradation. Over the last decade, a number of ubiquitinlike proteins (Ulps) have been discovered, including the SUMO family (Smt3 in yeast and SUMOs 1-4 in vertebrates), whose biological functions are less well-defined. The three most well-characterized vertebrate SUMOs fall into two subfamilies, SUMO1 and SUMO2/3. SUMO2 and SUMO3 share 95% sequence identity compared to only ~ 50% identity between SUMO2/3 and SUMO1, though the common and distinct functional roles of the various SUMOs has not been clearly delineated. While SUMO1 shares only about 18% with ubiquitin, all the Ulps have very similar tertiary structures with a common ubiquitin fold and a C-terminal diglycine in the mature forms. Like ubiquitin, SUMOs are post-translationally attached to target protein lysine residues using an enzymatic pathway that is biochemically analogous to, but functionally distinct from the classical ubiquitinylation system [2]. The overall process has four steps ( Fig. 1): 1) processing of the precursor SUMO by SUMO proteases (SENPS) which remove C-terminal residues to expose the diglycine motif, 2)

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“…This is due in part to the low stoichiometry of protein SUMOylation and the relatively long SUMO remnant appended on lysine residues following tryptic digestion (for example, up to 32 amino acids for SUMO2/3). The branched structure of the corresponding peptides not only complicates the interpretation of the MS/MS spectra, but often require specialized pattern recognition tools or database search engines such as SUMmOn 27 or ChopNSpice 28 to facilitate their identification. Here we developed a novel proteomics approach for antibodybased capture and MS analyses of SUMO-modified peptides.…”

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confidence: 99%

Large-scale analysis of lysine SUMOylation by SUMO remnant immunoaffinity profiling

Lamoliatte¹,

et al. 2014

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Small ubiquitin-related modifiers (SUMO) are evolutionarily conserved ubiquitin-like proteins that regulate several cellular processes including cell cycle progression, intracellular trafficking, protein degradation and apoptosis. Despite the importance of protein SUMOylation in different biological pathways, the global identification of acceptor sites in complex cell extracts remains a challenge. Here we generate a monoclonal antibody that enriches for peptides containing SUMO remnant chains following tryptic digestion. We identify 954 SUMO3-modified lysine residues on 538 proteins and profile by quantitative proteomics the dynamic changes of protein SUMOylation following proteasome inhibition. More than 86% of these SUMOylation sites have not been reported previously, including 5 sites on the tumour suppressor parafibromin (CDC73). The modification of CDC73 at K136 affects its nuclear retention within PML nuclear bodies on proteasome inhibition. In contrast, a CDC73 K136R mutant translocates to the cytoplasm under the same conditions, further demonstrating the effectiveness of our method to characterize the dynamics of lysine SUMOylation.

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Automated identification of SUMOylation sites using mass spectrometry and SUMmOn pattern recognition software

Cited by 115 publications

References 30 publications

A Novel Proteomics Approach to Identify SUMOylated Proteins and Their Modification Sites in Human Cells

A Novel Proteomics Approach to Identify SUMOylated Proteins and Their Modification Sites in Human Cells

Ubiquitin proteolytic system: focus on SUMO

Large-scale analysis of lysine SUMOylation by SUMO remnant immunoaffinity profiling

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