Supplementary Figure 1. Immunoprecipitation of synthetic SUMOm-remnant peptides using UMO monoclonal antibody. (a) LC-MS analyses of tryptic

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1 Supplementary Figure 1. Immunoprecipitation of synthetic SUMOm-remnant peptides using UMO monoclonal antibody. (a) LC-MS analyses of tryptic digest from HEK293 cells spiked with 6 SUMOmremnant peptides prior to and after immunoaffinity enrichment. All the spiked peptides have the same amino acid sequence flanking the SUMOylated lysine. Asterisks indicate polyoxoethylene glycol contaminants arising from antibody/plasticware. (b) Recovery yields of paralog-specific SUMOmremnant peptides following immunoaffinity purification.

2 Supplementary Figure 2. SUMO-remnant immunoaffinity purification with UMO monoclonal antibody provides high specificity for a wide range of peptide sequences. (a) LC-MS analyses of tryptic digest from HEK293 cells spiked with 66 SUMO3-remnant peptides prior to and after immunoaffinity enrichment. Asterisks indicate polyoxoethylene glycol contaminants arising from antibody/plasticware. Insets show the number of identified tryptic peptides with and without SUMO3 remnant chain. (b) Recovery yields of SUMO3m-remnant synthetic peptides spiked in HEK293 tryptic digest. Peptide identification number correspond to: (1) IESESK*GIKGGK, (2) TVTITK*EDESTEK, (3) ATGDETGAK*VER, (4) YK*EETIEK, (5) EMSGSTSELLIK*ENK, (6) VK*EDPDGEHAR, (7) SSVK*VEAEASR, (8) NDQNNSDTK*ISETETLK, (9) K*VEEDEAGGR, (10) NSIDASEEK*PVMR, (11) TK*PDPEVEEQEK, (12) VGEPEVK*EEK, (13) K*DDEVQVVR, (14) IGAIK*QESEEPPTK, (15) NISIK*QEPK, (16) SAEEVEEIK*AEK, (17) VLLGETGK*EK, (18) VNLDSEQAVK*EEK, (19) STPK*EETVNDPEEAGHR, (20) VQIK*QETIESR, (21) VK*LDSVR, (22) IK*EDDAPR, (23) TFSESLK*SEK, (24) IK*DEPDNAQEYSHGQQQK, (25) IK*SGEVAEGEAR, (26) IK*IEPGIEPQR, (27) VK*FEQNGSSK, (28) K*NAALVTR, (29) K*EETVEDEIDVR, (30) NQTILK*K, (31) MNKSEDDESGAGELTR, (32) TK*AEEPSDLIGPEAPK, (33) QFNK*LTEDK, (34) IK*VEPASEKDPESLR, (35) TSDADIK*SSETGAFR, (36)

3 EEDAEK*AVIDLNNR, (37) APTASQERPK*EELGAGR, (38) STYADEELVIK*AEGLAR, (39) VK*EEHLDVASPDK, (40) FK*QEPEDELPEAPPK, (41) ATMHLK*QEVTPR, (42) LK*EDVLEQR, (43) DGK*YSQVLANGLDNK, (44) AEAMNIK*IEPEETTEAR, (45) WTVVK*TEEGR, (46) ADSLLAVVK*R, (47) EK*LEMEMEAAR, (48) SGLK*HELVTR, (49) ELSEVK*NVLEK, (50) SVHSQDPSGDSK*LYR, (51) VSISEGDDK*IEYR, (52) EK*LTADPDSEIATTSLR, (53) EGVK*TENDHINLK, (54) IFDEEPANGVK*IER, (55) YQK*STELLIR, (56) LK*TEEGEIDYSAEEGENR, (57) TDLDDDITALK*QR, (58) QQEGFK*GTFPDAR, (59) LTEDK*ADVQSIIGLQR, (60) NLLHDNELSDLK*EDKPR, (61) FQIIK*QEPMELESYTR, (62) HHSLALTSFK*R, (63) IEEIK*DFLLTAR, (64) MK*FNPFVTSDR, (65) NLYK*NVILENYR, (66) YEEALSQLEESVK*EER. (*designates lysine residue modified with SUMO3m remnant (NQTGG))

4 Supplementary Figure 3. Schematic workflow illustrating the selection and identification of SUMOylated peptides. (a) The algorithm first searches for fragment ions characteristic to SUMO3m remnant chains (diagnostic ions) with intensities > 5000 counts detected in the HCD MS/MS spectra (mass tolerance ± 8 ppm). If at least two diagnostic ions are present in the scan, the corresponding spectra are selected for further consideration. For each MS/MS of potential SUMO3m remnant peptides the algorithm calculates the position of neutral loss fragments from the precursor ion, including internal fragment ions arising from consecutive cleavages of the SUMO3m remnant chain. Diagnostic ions, neutral loss and internal fragment ions are removed from the MS/MS spectrum. The internal fragment ions are replaced by a single fragment ion corresponding to the pentapeptide SUMO3m remnant chain to facilitate the assignment of a linearized peptide without side chain cleavages. The algorithm repeats this operation until all peaks and all scan are processed. The corresponding MS/MS spectra are then searched with Mascot database engine with conventional search parameters. (b) Example of a MS/MS spectrum after each processing step for a tryptic peptide from 40S ribosomal protein S3a modified at K249. The effect of peak removal is reflected by the increasing Mascot scores during the data processing stages.

5 Supplementary Figure 4. Comparison of Mascot scores from identified of SUMOylated peptides with and without MS/MS editing. (a) Venn diagram showing the number of identified SUMOylated peptides from LC-MS/MS analysis of tryptic digest from HEK293 SUMO3m cells without MS/MS editing (HCD), with removal of diagnostic fragment ions and neutral losses (MS/MS edition 1) 1, and conversion of internal fragment ions (MS/MS edition 2, this study). (b) Improvement of Mascot scores using MS/MS edition 1 and 2. (c) Distribution of Mascot scores with and without MS/MS edition 2.

6 Supplementary Figure 5. Dynamic changes in protein SUMOylation of HEK293 SUMO3 mutant cells upon proteasome inhibition. Immunoblots of nuclear extracts (left panel) and IMAC-purified nuclear extracts (right panel) for the first 24 h following treatment of HEK293 SUMO3m cells with 10 M of MG132 for different time periods. Immunoblots revealed with anti SUMO2/3 antibody. Histone H3 loading control shown for nuclear fraction.

7 Supplementary Figure 6. Distribution of SUMOylated peptide intensities from replicate injections. (a) Venn diagrams showing the distribution of identified SUMOylated peptides from each replicate and conditions. (b) Scatter plots of intensities for peptides detected in three different replicates of control (DMSO) and MG132 treated HEK293 SUMO3m cells. The correlation coefficient (R 2 ) between average and individual replicates is provided as inset.

8 Supplementary Figure 7. Distribution of all 20 amino acids for their propensity to be found proximal to SUMOylated lysines. The heat map represents the frequency of each residue at any of the 10 positions upstream and downstream of the modified lysines compared to that observed for lysines of the human proteome. Normalized frequency of each of the 20 amino acids within a ten amino acid span on either side of the SUMOylated lysines was carried out against the frequency calculated for each amino acid using all lysines in all human proteins in the Swiss-Prot database (released Feb. 2013). Residues adjacent to the modified lysine in SUMO remnant peptides, Ile and Val (position -1), and Glu, Asp and Lys (position -2) are enriched (student s t-test, p < ) while Cys and Trp (positions -2, -1, +2) are depleted (student s t-test, p < ).

9 Supplementary Figure 8. Confirmation of SUMOylation sites on CDC73 using an Ubc9 in vitro assay. MS/MS spectra (HCD mode) from in vitro SUMOylation assay confirming the SUMOylation of residues K136, K161, K198 and K283 residues by Ubc9.

10 Supplementary Figure 9. CDC73 colocalizes with SUMO3 and PML within the NBs upon MG132 treatment. HEK293 cells were co-transfected with pcdna3-myc-cdc73 and His-SUMO3. Cells were either treated with DMSO or with 10 um MG132 for 6 hours. Double immunofluorescence analyses were performed using rabbit anti-myc (red) and mouse anti-his (green) antibodies (upper panel) or rabbit anti-myc (red) and mouse anti-pml (green) antibodies (lower panel). The cells were mounted onto glass slides by using Immu-Mount (Shandon) containing 4,6-diamidino-2-phenylindole (DAPI) to stain nuclei. Immunofluorescence images were acquired on a Nikon Eclipse TE2000-E inverted confocal laser scanning microscope using an Apochromat 100X oil immersion objective. Scale bar 10 µm.

11 Supplementary Figure 10. SUMOylation of CDC73 affects its nucleocytoplasmic localization. (a) Cells cotransfected with His-SUMO3 and Myc-CDC73 WT or Myc-CDC73 mutants (K136R, K161R, K198R or K263R) were treated or not with MG132 and double immunofluorescence was performed with anti-myc (green) and anti-his antibodies (red). Scale bar 10 µm. (b) Intracellular distribution of CDC73 fluorescent signal in control and MG132-treated cells expressing CDC73 WT or CDC73 mutants. The graph shows the ratio of the nuclear signal compared to the total cell signal (*p=0.01, **p=0.0001, Student t-test, n=10 cells/condition).

12 Supplementary Figure 11. Full images of Western blots.

13 Supplementary Figure 11 continued. Full images of Western blots.

14 Supplementary Reference: 1. Lamoliatte, F. et al. Targeted identification of SUMOylation sites in human proteins using affinity enrichment and paralog-specific reporter ions. Mol Cell Proteomics 12, (2013).