Targeted Identification of SUMOylation Sites in Human Proteins Using Affinity Enrichment and Paralog-specific Reporter Ions
2013; Elsevier BV; Volume: 12; Issue: 9 Linguagem: Inglês
10.1074/mcp.m112.025569
ISSN1535-9484
AutoresFrédéric Lamoliatte, Éric Bonneil, Chantal Durette, Olivier Caron-Lizotte, Dirk Wildemann, Johannes Zerweck, Holger Wenshuk, Pierre Thibault,
Tópico(s)Mass Spectrometry Techniques and Applications
ResumoProtein modification by small ubiquitin-like modifier (SUMO) modulates the activities of numerous proteins involved in different cellular functions such as gene transcription, cell cycle, and DNA repair. Comprehensive identification of SUMOylated sites is a prerequisite to determine how SUMOylation regulates protein function. However, mapping SUMOylated Lys residues by mass spectrometry (MS) is challenging because of the dynamic nature of this modification, the existence of three functionally distinct human SUMO paralogs, and the large SUMO chain remnant that remains attached to tryptic peptides. To overcome these problems, we created HEK293 cell lines that stably express functional SUMO paralogs with an N-terminal His6-tag and an Arg residue near the C terminus that leave a short five amino acid SUMO remnant upon tryptic digestion. We determined the fragmentation patterns of our short SUMO remnant peptides by collisional activation and electron transfer dissociation using synthetic peptide libraries. Activation using higher energy collisional dissociation on the LTQ-Orbitrap Elite identified SUMO paralog-specific fragment ions and neutral losses of the SUMO remnant with high mass accuracy (< 5 ppm). We exploited these features to detect SUMO modified tryptic peptides in complex cell extracts by correlating mass measurements of precursor and fragment ions using a data independent acquisition method. We also generated bioinformatics tools to retrieve MS/MS spectra containing characteristic fragment ions to the identification of SUMOylated peptide by conventional Mascot database searches. In HEK293 cell extracts, this MS approach uncovered low abundance SUMOylated peptides and 37 SUMO3-modified Lys residues in target proteins, most of which were previously unknown. Interestingly, we identified mixed SUMO-ubiquitin chains with ubiquitylated SUMO proteins (K20 and K32) and SUMOylated ubiquitin (K63), suggesting a complex crosstalk between these two modifications. Protein modification by small ubiquitin-like modifier (SUMO) modulates the activities of numerous proteins involved in different cellular functions such as gene transcription, cell cycle, and DNA repair. Comprehensive identification of SUMOylated sites is a prerequisite to determine how SUMOylation regulates protein function. However, mapping SUMOylated Lys residues by mass spectrometry (MS) is challenging because of the dynamic nature of this modification, the existence of three functionally distinct human SUMO paralogs, and the large SUMO chain remnant that remains attached to tryptic peptides. To overcome these problems, we created HEK293 cell lines that stably express functional SUMO paralogs with an N-terminal His6-tag and an Arg residue near the C terminus that leave a short five amino acid SUMO remnant upon tryptic digestion. We determined the fragmentation patterns of our short SUMO remnant peptides by collisional activation and electron transfer dissociation using synthetic peptide libraries. Activation using higher energy collisional dissociation on the LTQ-Orbitrap Elite identified SUMO paralog-specific fragment ions and neutral losses of the SUMO remnant with high mass accuracy (< 5 ppm). We exploited these features to detect SUMO modified tryptic peptides in complex cell extracts by correlating mass measurements of precursor and fragment ions using a data independent acquisition method. We also generated bioinformatics tools to retrieve MS/MS spectra containing characteristic fragment ions to the identification of SUMOylated peptide by conventional Mascot database searches. In HEK293 cell extracts, this MS approach uncovered low abundance SUMOylated peptides and 37 SUMO3-modified Lys residues in target proteins, most of which were previously unknown. Interestingly, we identified mixed SUMO-ubiquitin chains with ubiquitylated SUMO proteins (K20 and K32) and SUMOylated ubiquitin (K63), suggesting a complex crosstalk between these two modifications. SUMOylation is a covalent and reversible post-translational modification that conjugates the small ubiquitin-like modifier (SUMO) 1The abbreviations used are:SUMOSmall Ubiquitin-related ModifierBPCbase peak chromatogramCIDcollision induced dissociationDDAdata dependent acquisitionDIAdata independent acquisitionE2–25KE2 ubiquitin ligase – 25 kiloDaltonETDelectron transfer dissociationFDRfalse discovery rateHCDhigher energy collisional dissociationHEKhuman embryonary kidneyhnRNPHeterogeneous nuclear ribonucleoproteinLC-MS/MSliquid chromatography tandem mass spectrometryMS/MStandem mass spectrometryNEMN-EthylmaleimideNTANickel nitriloacetic acidPIASprotein inhibitor of activated STATRanBP2Ran Binding Protein 2RanGAP1Ran GTPase activating Protein 1RNF4RING finger 4 proteinSAESUMO activating EnzymeSAFB2Scaffold Assembly factor B2SENPSentrin/SUMO-specific proteaseTBSTris buffered salineTIF1β(TRIM28) Transcription intermediary factor 1-beta (TRIM28)Ubc9E2 SUMO conjugating enzymeUBLsUbiquitin-like proteinsXICExtracted ion chromatogram. 1The abbreviations used are:SUMOSmall Ubiquitin-related ModifierBPCbase peak chromatogramCIDcollision induced dissociationDDAdata dependent acquisitionDIAdata independent acquisitionE2–25KE2 ubiquitin ligase – 25 kiloDaltonETDelectron transfer dissociationFDRfalse discovery rateHCDhigher energy collisional dissociationHEKhuman embryonary kidneyhnRNPHeterogeneous nuclear ribonucleoproteinLC-MS/MSliquid chromatography tandem mass spectrometryMS/MStandem mass spectrometryNEMN-EthylmaleimideNTANickel nitriloacetic acidPIASprotein inhibitor of activated STATRanBP2Ran Binding Protein 2RanGAP1Ran GTPase activating Protein 1RNF4RING finger 4 proteinSAESUMO activating EnzymeSAFB2Scaffold Assembly factor B2SENPSentrin/SUMO-specific proteaseTBSTris buffered salineTIF1β(TRIM28) Transcription intermediary factor 1-beta (TRIM28)Ubc9E2 SUMO conjugating enzymeUBLsUbiquitin-like proteinsXICExtracted ion chromatogram. proteins on Lys residues of target proteins (1Hay R.T. SUMO: a history of modification.Mol Cell. 2005; 18: 1-12Abstract Full Text Full Text PDF PubMed Scopus (1325) Google Scholar). Although this modification is common to all species, the number of SUMO genes expressed vary from a single SUMO gene in lower eukaryotes (e.g. yeast, drosophila, nematodes) to eight different paralogs in plants (e.g. Arabidopsis thaliana) (2Lois L.M. Diversity of the SUMOylation machinery in plants.Biochem. Soc Trans. 2010; 38: 60-64Crossref PubMed Scopus (41) Google Scholar). In human, three SUMO genes (SUMO1, SUMO2, and SUMO3) are ubiquitously expressed in all tissues, whereas a forth gene (SUMO4) is uniquely expressed in spleen, lymph nodes, and kidney cells (3Guo D. Li M. Zhang Y. Yang P. Eckenrode S. Hopkins D. Zheng W. Purohit S. Podolsky R.H. Muir A. Wang J. Dong Z. Brusko T. Atkinson M. Pozzilli P. Zeidler A. Raffel L.J. Jacob C.O. Park Y. Serrano-Rios M. Larrad M.T. Zhang Z. Garchon H.J. Bach J.F. Rotter J.I. She J.X. Wang C.Y. A functional variant of SUMO4, a new I kappa B alpha modifier, is associated with type 1 diabetes.Nat. Genet. 2004; 36: 837-841Crossref PubMed Scopus (335) Google Scholar). As for other ubiquitin-like proteins (UBLs) such as NEDD8, ISG15, and FAT10, the structure of SUMO proteins share a characteristic three-dimensional fold with ubiquitin, but differ significantly in their sequences. Small Ubiquitin-related Modifier base peak chromatogram collision induced dissociation data dependent acquisition data independent acquisition E2 ubiquitin ligase – 25 kiloDalton electron transfer dissociation false discovery rate higher energy collisional dissociation human embryonary kidney Heterogeneous nuclear ribonucleoprotein liquid chromatography tandem mass spectrometry tandem mass spectrometry N-Ethylmaleimide Nickel nitriloacetic acid protein inhibitor of activated STAT Ran Binding Protein 2 Ran GTPase activating Protein 1 RING finger 4 protein SUMO activating Enzyme Scaffold Assembly factor B2 Sentrin/SUMO-specific protease Tris buffered saline (TRIM28) Transcription intermediary factor 1-beta (TRIM28) E2 SUMO conjugating enzyme Ubiquitin-like proteins Extracted ion chromatogram. Small Ubiquitin-related Modifier base peak chromatogram collision induced dissociation data dependent acquisition data independent acquisition E2 ubiquitin ligase – 25 kiloDalton electron transfer dissociation false discovery rate higher energy collisional dissociation human embryonary kidney Heterogeneous nuclear ribonucleoprotein liquid chromatography tandem mass spectrometry tandem mass spectrometry N-Ethylmaleimide Nickel nitriloacetic acid protein inhibitor of activated STAT Ran Binding Protein 2 Ran GTPase activating Protein 1 RING finger 4 protein SUMO activating Enzyme Scaffold Assembly factor B2 Sentrin/SUMO-specific protease Tris buffered saline (TRIM28) Transcription intermediary factor 1-beta (TRIM28) E2 SUMO conjugating enzyme Ubiquitin-like proteins Extracted ion chromatogram. Protein SUMOylation is an essential cellular process conserved from yeast to mammals, and is associated with many fundamental pathways in both the nucleus and cytoplasm including DNA replication, genome stability, nuclear transport, gene transcription, mitochondrial fission and fusion events (4Bossis G. Melchior F. SUMO: regulating the regulator.Cell Div. 2006; 1: 13Crossref PubMed Scopus (129) Google Scholar, 5Meulmeester E. Melchior F. Cell biology: SUMO.Nature. 2008; 452: 709-711Crossref PubMed Scopus (133) Google Scholar, 6Seeler J.S. Dejean A. Nuclear and unclear functions of SUMO.Nat. Rev. Mol. Cell Biol. 2003; 4: 690-699Crossref PubMed Scopus (578) Google Scholar). Despite the low occurrence of SUMOylation compared with ubiquitylation, an increasing number of substrates have been characterized based on the presence of a predicted SUMO consensus motif ψKxE/D, where ψ represents a hydrophobic residue (7Rodriguez M.S. Dargemont C. Hay R.T. SUMO-1 conjugation in vivo requires both a consensus modification motif and nuclear targeting.J. Biol. Chem. 2001; 276: 12654-12659Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar). This modification can also take place on Lys residues located on an extended consensus site defined by the phosphorylation-dependent motif (ψKxExxpSP) (8Hietakangas V. Anckar J. Blomster H.A. Fujimoto M. Palvimo J.J. Nakai A. Sistonen L. PDSM, a motif for phosphorylation-dependent SUMO modification.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 45-50Crossref PubMed Scopus (381) Google Scholar) and the negatively charged amino acid-dependent motif (9Yang S.H. Galanis A. Witty J. Sharrocks A.D. An extended consensus motif enhances the specificity of substrate modification by SUMO.EMBO J. 2006; 25: 5083-5093Crossref PubMed Scopus (167) Google Scholar). It is noteworthy that the ψKxE/D motif is also found on many proteins that are not SUMO targets, and that many SUMOylation sites are known to be located in non consensus sites. SUMOylation involves a sequence of enzymatic steps similar to those of ubiquitylation in which SUMO is transferred from the E1 activating enzyme SAE1/SAE2 to the single E2-conjugating enzyme Ubc9 that directly recognize substrates and catalyze the formation of an isopeptide bond between the C-terminal group of SUMO and the ε-NH2 group of Lys residue from the target protein (10Geiss-Friedlander R. Melchior F. Concepts in sumoylation: a decade on.Nat. Rev. Mol. Cell Biol. 2007; 8: 947-956Crossref PubMed Scopus (1359) Google Scholar). Although Ubc9 is sufficient to promote SUMOylation, substrate recognition can be mediated by one of several E3 ligases including nucleoporin RanBP2, polycomb repressor Pc2, and members of the protein inhibitor of STAT (PIAS) ligase family (PIAS1, PIAS3, PIASxα, PIASxβ, PIASy) that facilitate the conjugation process (11Melchior F. Schergaut M. Pichler A. SUMO: ligases, isopeptidases and nuclear pores.Trends Biochem. Sci. 2003; 28: 612-618Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). SUMOylation is reversible and this modification can be cleaved rapidly from its target protein by Sentrin/SUMO-specific proteases (SENPs), a family of conserved proteins that can also process SUMO precursors to expose the di-glycine motif necessary for the conjugation of mature SUMO proteins (12Mukhopadhyay D. Dasso M. Modification in reverse: the SUMO proteases.Trends Biochem. Sci. 2007; 32: 286-295Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar). The identification of SUMOylation sites in protein substrates by mass spectrometry (MS) represents a sizable analytical challenge because of low abundance and dynamic nature of this modification. In contrast to phosphorylation where affinity media such as TiO2 or immobilized metal affinity chromatography are available to enrich phosphopeptides from complex tryptic digests, the purification of UBLs has been typically performed at the protein level using His6- or TAP-tag of the target UBL proteins (13Golebiowski F. Matic I. Tatham M.H. Cole C. Yin Y. Nakamura A. Cox J. Barton G.J. Mann M. Hay R.T. System-wide changes to SUMO modifications in response to heat shock.Sci Signal. 2009; 2: ra24Crossref PubMed Scopus (385) Google Scholar, 14Schimmel J. Larsen K.M. Matic I. van Hagen M. Cox J. Mann M. Andersen J.S. Vertegaal A.C. The ubiquitin-proteasome system is a key component of the SUMO-2/3 cycle.Mol Cell Proteomics. 2008; 7: 2107-2122Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 15Vertegaal A.C. Andersen J.S. Ogg S.C. Hay R.T. Mann M. Lamond A.I. Distinct and overlapping sets of SUMO-1 and SUMO-2 target proteins revealed by quantitative proteomics.Mol Cell Proteomics. 2006; 5: 2298-2310Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). Although these approaches are effective to identify and quantify UBL-modified proteins, the precise identification of SUMOylation sites is still a daunting task in view of the large remnant SUMO sequence appended to the Lys residues of modified tryptic peptides. The identification of SUMOylated peptides is also complicated by their relatively low abundance and their small proportion in complex protein digests (typically < 1% of all identified peptides). The corresponding cross-linked peptides comprise long SUMO remnant chains up to 32 amino acids for SUMO2 and SUMO3 that complicate the interpretation of the MS/MS spectra, and often lead to misidentification of modified peptides using conventional database search engines tailored to the analysis of linear peptides. Different bioinformatics approaches have been proposed to alleviate these difficulties including an automated recognition pattern tool (SUMmOn) (16Pedrioli 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 (101) Google Scholar) and a database containing "linearized branched" peptides (ChopNSpice) (17Hsiao H.H. Meulmeester E. Frank B.T. Melchior F. Urlaub H. "ChopNSpice," a mass spectrometric approach that allows identification of endogenous small ubiquitin-like modifier-conjugated peptides.Mol Cell Proteomics. 2009; 8: 2664-2675Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). To facilitate the enrichment and identification of modification sites of distinct SUMO paralogs from in vivo samples, we recently reported a novel proteomics approach using HEK293 cells stably expressing SUMO (1, 2, or 3) mutant proteins with a His6 tag and an Arg residue at the 6th position from the C terminus (18Galisson F. Mahrouche L. Courcelles M. Bonneil E. Meloche S. Chelbi-Alix M.K. Thibault P. A novel proteomics approach to identify SUMOylated proteins and their modification sites in human cells.Mol Cell Proteomics. 2011; 10 (M110.004796)Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Also, Gln88 of SUMO3 was mutated to an Asn residue to distinguish this paralog from SUMO2. Our SUMO2 mutant is similar to that reported by Matic et al. (19Matic I. Schimmel J. Hendriks I.A. van Santen M.A. van de Rijke F. van Dam H. Gnad F. Mann M. Vertegaal A.C. Site-Specific Identification of SUMO-2 Targets in Cells Reveals an Inverted SUMOylation Motif and a Hydrophobic Cluster SUMOylation Motif.Mol. Cell. 2010; 39: 641-652Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar) except that internal lysines were not replaced by arginines to maintain functional polySUMOylation of protein substrates. Upon tryptic digestion, proteins modified with these SUMO mutants give rise to tryptic peptides with a five amino acid long SUMO remnant that facilitates their identification using conventional database search engines. In the present study, we determined the occurrence and abundance of paralog-specific fragment ions formed by collisional activation (collision induced dissociation, CID; higher energy collision dissociation, HCD) and electron transfer dissociation (ETD) using libraries of synthetic peptides with SUMO1 and SUMO3 remnant chains. We also implemented a data independent acquisition method similar to that of Gillet et al. (20Gillet L.C. Navarro P. Tate S. Röst H. Selevsek N. Reiter L. Bonner R. Aebersold R. Targeted data extraction of the MS/MS spectra generated by data-independent acquisition: a new concept for consistent and accurate proteome analysis.Mol. Cell. Proteomics. 2012; 11 (O111.016717)Abstract Full Text Full Text PDF PubMed Scopus (1795) Google Scholar) that repeatedly cycle through consecutive 100-m/z precursor isolation windows to form paralog-specific fragment ions and selectively identify SUMOylated peptides from HEK293 cells expressing our SUMO3 mutant. The combination of HCD and high resolution fragment ion detection on the LTQ-Orbitrap Elite conferred a unique advantage to map SUMO modification sites in endogenous target proteins, and provided complementary identification to traditional data dependent acquisition (DDA). The peptides (1) Boc-Glu(OtBu)-Qln(Trt)-Thr(tBu)-Gly-Gly-OH and (2) Boc-Asn(Trt)-Gln(Trt)-Thr(tBu)-Gly-Gly-OH were synthesized manually on a 2-chlorotrityl chloride resin using standard procedures of solid phase peptide synthesis. Cleavage of both fragments from the resin was performed using 25% hexafluoroisopropanol in dichloromethane. To a solution of 400 mg of compound 1 or 2 in 10 ml N,N-dimethylformamide were added 1.2 equivalent N-hydroxybenzotriazole and one equivalent ethyl diisopropylcarbodiimide. The mixture was stirred for 30 min at room temperature followed by addition of one equivalent Fmoc-Lys-OH . HCl and one equivalent N-methylmorpholine. After stirring for 2h at room temperature, the solvent was evaporated and the residue was dissolved in 100 ml ethyl acetate. The solution was then extracted four times with 5% K2HSO3, four times with a saturated aqueous solution of sodium chloride and dried over Na2SO4. After evaporation of the solvent, the building blocks (3) Fmoc-Lys[Boc-Glu(OtBu)-Gln(Trt)-Thr(tBu)-Gly-Gly]-OH (44% yield) and (4) Fmoc-Lys[Boc-Asn(Trt)-Gln(Trt)-Thr(tBu)-Gly-Gly]-OH (77% yield) were precipitated with diethyl ether and dried in vacuo over P2O5. All tryptic peptides with SUMO remnant chains were synthesized by automated SPOT synthesis as described by Frank et al. (21Frank F. Spot-synthesis: an easy technique for the positionally addressable, parallel chemical synthesis on a membrane support.Tetrahedron. 1992; 48: 9217-9232Crossref Scopus (925) Google Scholar). Acylation of the peptide chain with 3 and 4 was performed by preactivation of the respective building block with 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate/N,N-diisopropylethylamine. All synthetic peptides were analyzed by LC-MS on a Agilent 1100 series LC/MSD Trap to confirm purity. HEK293 wild type and HEK293 cells stably expressing His6-SUMO mutants (5 × 106 cells/replicate) were cultured in Dulbecco's modified Eagles medium (DMEM) High Glucose (Hyclone SH30081.02) supplemented with 10% fetal bovine serum (FBS) (Fisher), 1% l-glutamine and 1% Penicillin/Streptomycin (Fisher) (18Galisson F. Mahrouche L. Courcelles M. Bonneil E. Meloche S. Chelbi-Alix M.K. Thibault P. A novel proteomics approach to identify SUMOylated proteins and their modification sites in human cells.Mol Cell Proteomics. 2011; 10 (M110.004796)Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Cells were washed with PBS containing 20 mm of N-ethyl maleimide (NEM) and centrifuged at 215 × g for 10 min. NEM is used to stabilize SUMO conjugates by alkylating the sulfhydryl group of the catalytic cysteine on SUMO-specific proteases (SENP). Cells were lysed in hypotonic buffer A (10 mm Tris pH 7.6, 1.5 mm MgCl2, 20 mm NEM, protease inhibitor, Sigma-Aldrich) for 30 min and centrifuged 15 min at 215 × g. Nuclei were washed with buffer A and centrifuged at 215 × g. The nuclei pellet was lysed in denaturing buffer B (6 m Guanidine, 0.1 m NaH2PO4, 10 mm imidazole, 10 mm Tris-HCl pH 8, 20 mm NEM, 10 mm β mercaptoethanol), sonicated and centrifuged at 16000 × g for 15 min. The supernatant was incubated with 200 μl of nickel nitriloacetic acid (NTA) agarose beads (Invitrogen) for 3h at room temperature. Ni-NTA beads were washed once with buffer B and then five times with buffer C (8 m urea, 0.1 m NaH2PO4, 10 mm Tris-HCl pH 6.3, 10 mm β mercaptoethanol, 20 mm imidazole). A portion of the NTA beads were used to determine total protein amounts using a Bradford protein assay. NTA beads were kept frozen until digestion. The enrichment of SUMOylated proteins was verified by immunoblots using rabbit anti-SUMO2/3 and chicken anti-mouse Alexa-Fluor 594-conjugated secondary antibody from Invitrogen, and monoclonal anti-6xHis antibody from Clontech. SUMO-proteins immobilized on NTA beads were solubilized in 4 m urea, reduced in 5 mm tris(2-carboxyethyl)phosphine (TCEP) (Pierce) for 20 min at 37 °C and then alkylated in 5 mm chloroacetamide (Sigma-Aldrich) for 20 min at 37 °C. We used this alkylating agent to differentiate free cysteines (modified by NEM) and residues linked by disulfide-bonds (modified by chloroacetamide). A solution of 5 mm dithiothreitol was added to the protein solution to react with excess chloroacetamide. The solution was diluted to 1 m urea using 50 mm ammonium bicarbonate and digested overnight with modified trypsin (1:50, enzyme:substrate ratio) at 37 °C under high agitation speed. The digest was acidified with trifluoroacetic acid (TFA), desalted using an Oasis HLB cartridge (Waters) and dried using a speed vac prior to MS analyses. LC-MS/MS analyses were performed on a nano-LC 2D pump (Eksigent) coupled to a LTQ-Orbitrap Elite hybrid mass spectrometer via a nanoelectrospray ion source (Thermo Fisher Scientific). Peptides were separated on a Optiguard SCX trap column, 5 μm, 300Å, 0.5 ID × 23 mm (Optimize technologies) and eluted on-line to a 360 μm ID × 4 mm, C18 trap column prior to separation on a 150 μm ID x 10 cm nano-LC column (Jupiter C18, 3 μm, 300 Å, Phenomex). Tryptic digests were loaded on the SCX trap and sequentially eluted using salt plugs of 0, 250, 500, 750, 1000, and 2000 mm ammonium acetate, pH 3.5. Peptides were separated on the analytical column using a linear gradient of 5–40% acetonitrile (0.2% formic acid) in 53 min with a flow rate of 600 nL/min. For the analysis of synthetic peptides, MS/MS spectra were collected in DDA. The conventional MS spectra (survey scan) were acquired in the Orbitrap at a resolution of 60000 for m/z 400 after the accumulation of 106 ions in the linear ion trap. Mass calibration used a lock mass from ambient air [protonated (Si(CH3)2O))6; m/z 445.120029], and provided mass accuracy within 7 ppm for precursor ion mass measurements. The dynamic exclusion of previously acquired precursor ions was enabled (repeat count 1, repeat duration: 15 s; exclusion duration 15 s). MS/MS spectra were acquired in CID, HCD or ETD activation modes using an isolation window of 2 Da. For ETD, up to 12 precursor ions were selected for fragmentation. Precursor cation AGC target was set at 1 × 104, whereas a value of 2 × 105 was used for the fluoranthene anion population and ion/ion reaction duration was fixed at 100 ms. For CID, a normalized collision energy of 35% was used and up to 12 precursor ions were sequentially isolated and accumulated to a target value of 10000 with a maximum injection time of 100 ms. For MS/MS spectra acquired with HCD, a normalized collision energy of 30% was selected. Up to six precursor ions were accumulated to a target value of 50000 with a maximum injection time of 300 ms and fragment ions were transferred to the Orbitrap analyzer operating at a resolution of 30000 at m/z 400. For the analysis of HEK293 cell extracts, MS/MS spectra were acquired in HCD mode with a normalized energy of 30%. We first used the LTQ-Orbitrap Elite in data dependent acquisition with dynamic exclusion of previously acquired precursor ions (repeat count 1, repeat duration: 30 s; exclusion duration 45 s). In data independent acquisition, the MS instrument cycled through the sequential acquisition of a MS scan and a HCD MS/MS scan to transmit in turn intact peptide ions and fragment ions arising from the dissociation of selected precursor ion windows (up to seven ion trap segments of m/z 100–700). Typically, seven segments of m/z 100 each were transmitted in turn to the HCD cell and the collision energy was scaled according to precursor m/z windows (16–25% for precursor ions m/z 300–1000). An injection time of 50 ms for a target value of 106 counts was used for the HCD MS/MS acquisition. This experiment enabled the generation of an inclusion list of potential SUMO peptides for subsequent targeted MS/MS experiments. MS data were acquired using the Xcalibur software (version 2.1). Peak lists were generated using Mascot distiller (version 2.3.2.0, Matrix science) and MS/MS spectra were searched against a concatenated target/decoy UniProtKB/Swiss-Prot Human containing 37275 forward sequences (released Feb 2013) using Mascot (version 2.3.2, Matrix Science) to achieve a false-positive rate of less than 2% (p < 0.02). MS/MS spectra were searched with a mass tolerance of 7 ppm, for precursor ions and 0.5 Da for fragment ions acquired in CID and ETD modes or 0.02 Da for HCD spectra. The number of allowed missed cleavage sites for trypsin was set to 2 and phosphorylation (STY), oxidation (M), deamidation (NQ), carbamidomethylation (C), ethylmaleimidation (C), ubiquitylation (K), acetylation (K, NH2-term), and SUMOylation (K) (EQTGG: SUMO1 or NQTGG: SUMO3) were selected as variable modifications. A software application was developed to search Mascot generic files (mgf) for specific SUMO fragment ions (e.g. SUMO1: m/z 102.0550, 129.0659, 240.0979, 258.1084, 341.1456, 359.1561; SUMO3: m/z 132.0768, 226.0822, 243.1088, 299.1350, 316.1615, 326.1459, 327.1299, 344.1565, 383.1674, 401.1779; and neutral losses of SUMO remnants) to produce a subset mgf file containing only MS/MS spectra of potential SUMOylated peptide candidates. SUMO fragment ions were removed from the corresponding mgf files and searched using Mascot as indicated above. Manual inspection of all MS/MS spectra for modified peptides was performed to validate assignments. For the generation of inclusion lists of potential SUMO peptides, raw LC-MS files that comprised MS and HCD MS/MS data sets were converted into peptide maps using in-house peptide detection and clustering software (22Marcantonio M. Trost M. Courcelles M. Desjardins M. Thibault P. Combined enzymatic and data mining approaches for comprehensive phosphoproteome analyses: application to cell signaling events of interferon-gamma-stimulated macrophages.Mol. Cell. Proteomics. 2008; 7: 645-660Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The HCD MS/MS data sets were searched for specific SUMO fragment ions (e.g. 132.0768, 226.0822, 243.1088 for SUMO3) to identify relevant scans containing SUMO peptide candidates. Multiply charged precursor ions with elution profile overlapping with SUMO fragment ions (± 2 scans) were selected in neighboring MS scans. Only precursor ions with corresponding neutral losses (e.g. 242.1015, 343.1492 for SUMO3, tolerance 10 ppm) identified in the HCD MS/MS scan were retained. Data clustering was then performed to remove redundancy and merge potential precursors from the same peptide. Inclusion lists were created with peptide candidates identified with at least two fragment ions and two neutral losses, and detected in LC-MS/MS runs above an intensity threshold of 5000 counts (supplemental Fig. S1). This script is available at http://www.thibault.iric.ca/ADIA. The identification of SUMOylated peptides by conventional database search engines can be challenging in view of the occurrence of fragment ions from the peptide backbone and the long SUMO remnant chain (up to 32 amino acid long for SUMO2 and SUMO3) that complicates the interpretation of the corresponding MS/MS spectra (16Pedrioli 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 (101) Google Scholar, 17Hsiao H.H. Meulmeester E. Frank B.T. Melchior F. Urlaub H. "ChopNSpice," a mass spectrometric approach that allows identification of endogenous small ubiquitin-like modifier-conjugated peptides.Mol Cell Proteomics. 2009; 8: 2664-2675Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). To facilitate the identification of SUMOylated peptides from digests of cell extracts, we previously generated HEK293 cell lines stably expressing His6-SUMO mutants that comprised an arginine residue strategically located near the C terminus thus leaving a short five amino acid long SUMO remnant
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