Nature Protocols: doi: /nprot Supplementary Figure 1

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1 Supplementary Figure 1 HPLC chromatogram, ESI and MALDI-TOF spectra for purified SEA off peptide segment 1. Figure adapted from ref.16 with permission.

2 Supplementary Figure 2 HPLC chromatogram, ESI and MALDI-TOF spectra for SEA off peptide segment 3. Figure adapted from ref.16 with permission.

3 Supplementary Figure 3 HPLC analysis of the exchange reaction of the bis(2-sulfanylethyl)amino group by 3-mercaptopropionic acid (MPA). Synthesis of MPA thioester peptide 2. Figure adapted from ref.16 with permission.

4 Supplementary Figure 4 HPLC chromatogram, ESI and MALDI-TOF spectra for purified MPA thioester peptide 2. Figure adapted from ref.16 with permission.

5 Supplementary Figure 5 HPLC chromatogram for purified SUMO-1-SEA off protein 4. Figure adapted from ref.16 with permission.

6 Supplementary Figure 6 MALDI-TOF analysis of SUMO-1-SEA off protein 4. Figure adapted from ref.16 with permission.

7 Supplementary Figure 7 MALDI-TOF analysis of the purified SUMO-1-peptide conjugate 6. Figure adapted from ref.16 with permission.

8 Supplementary Figure 8 MALDI-TOF and in source fragmentation analysis of the purified SUMO-1-peptide conjugate 6 (part 1). Figure adapted from ref.16 with permission.

9 Supplementary Figure 9 MALDI-TOF and in source fragmentation analysis of the purified SUMO-1-peptide conjugate 6 (part 2). Figure adapted from ref.16 with permission.

10 Supplementary Figure 10 Desumoylation of SUMO-1 peptide conjugate 6 by Ulp1 protease. Micro HPLC analysis of the desumoylation of SUMO-1 peptide conjugate 6 by Ulp1 protease.1 Trace (a) Synthetic peptide 5; Trace (b) Synthetic SUMO-1 peptide conjugate 6 in the cleavage buffer. Trace (c) Cleavage mixture few seconds after addition of the enzyme Ulp1; SUMO-1 peptide conjugate 6 (0.75 mg/ml final concentration) and Ulp1 (2 units) were reacted in 41.5 mm Tris.HCl buffer, ph 8.0, 0.17% Igepal (NP-40) and 0.83 mm DTT at room temperature (20 C). The peaks were collected and analyzed by MALTI-TOF MS using sinapinic acid as matrix. Experimental conditions for the microlc analysis: A: deionized H 2O containing 0.1% (vol/vol) TFA; B: ACN 60% containing 0.1% (vol/vol) TFA. Flow rate 50 µl/min, gradient 0-100% of B in 30 min, Waters BEH300 C18 column, 5 µm, mm, UV detection at 215 nm. Figure adapted from ref.16 with permission.

11 Supplementary Note 1 HPLC chromatogram, ESI and MALDI-TOF spectra for peptide 5 Figure adapted from ref.16 with permission.

12 Supplementary Note 2 ESI-MS chromatogram for SUMO-1-SEA off protein 4 Figure adapted from ref.16 with permission.

13 Supplementary Note 3 ESI-MS chromatogram for SUMO-1-SEA off protein 4-MPAA mixed disulfide Figure adapted from ref.16 with permission.

14 Supplementary Note 4 MALDI-TOF analysis of the purified SUMO-1-peptide conjugate 6 Figure adapted from ref.16 with permission.

15 Supplementary Note 5 LC-MS analysis of bis(2-tritylsulfanylethyl)aminee Figure adapted from ref.17 with permission.

16 Supplementary Note 6 1 H NMR spectrum of bis(2-tritylsulfanylethyl)amine Figure adapted from ref.17 with permission.

17 Supplementary Note 7 13 C NMR spectrum of bis(2-tritylsulfanylethyl)amine Figure adapted from ref.17 with permission.

18 Supplementary Note 8 Principle of the acetaldehyde/chloranil test

19 Supplementary Method 1 Setup of the automated column peptide synthesizer (Intavis, 6 column module, Germany) The synthesis columns (5 ml, diameter 160 mm) are closed with a septum at the top through which the needle delivers the solvents and reagents. The bottoms of the columns are connected to the waste bottles with a plastic tubing. The excess of solvents and reagents are eliminated by gravimetry during the waiting s (see for example Step 1 in Table S1). The columns can be drained by injection of air in the column by the needle, thereby forcing the elimination of the solvents or reagents through the bottom (see for example Step 9 in Table S1). The following reagents are used in the automated Fmoc SPPS protocol: DMF/thioanisole: 1% thioanisole in DMF for peptide synthesis (vol/vol) Fmoc amino acid solution : 0.6 M in DMF for peptide synthesis HATU solution: HATU 0.6 M in DMF for peptide synthesis DIPEA solution : DIPEA 33% in NMP (vol/vol) Piperidine solution : piperidine 20% in DMF for peptide synthesis (vol/vol) Capping solution: acetic anhydride10%/dipea 5%/ DMF for peptide synthesis 85% (vol/vol/vol) Table S1. Fmoc SPPS protocol for the synthesis of 50 Aa SEA off peptide segments using 30 µmol of SEA Trt ChemMatrix resin. Step 1 Name of chemical Action Volume Solvent or reagent Time Add solvent to the reactor and wait 2500 µl DMF/thioanisole 3 min Resin swelling 2 Add solvent to the reactor and wait 2000 µl DCM 4 min 3 Add solvent to the reactor and wait 2000 µl DMF/thioanisole 3 min 4 Add solvent to the reactor and wait 2500 µl DMF/thioanisole 4 min 5 Add reagent to the reactor Piperidine Fmoc removal and wait 3000 µl solution 3 min 6 Add reagent to the reactor Piperidine and wait 3000 µl solution 17 min 7 Add solvent to the reactor and wait Washing 4000 µl DMF/thioanisole 2 min

20 8 Add solvent to the reactor and wait 2000 µl DMF/thioanisole 3 1 min 9 Drain 10 Activation, 1: The First coupling solutions are aspirated by the needle with air in between 1245 µl HATU solution 11 Aspirate in the needle line 375 µl DIPEA solution 12 Aspirate in the needle line 30 µl DMF/thioanisole 13 Aspirate in the needle line Fmoc amino acid 1260 µl solution 14 Activation, 2: the solutions are transferred to a tube to start the activation Few seconds s 15 Activation, 3: the content of the tube is aspirated and transferred to the synthesis column for the coupling 16 wait 17 Add solvent to the reactor 42 seconds after the activation, 2 30 min and wait 250 µl DCM 1 min 18 Activation, 1: The Second coupling solutions are aspirated by the needle with air in between 1245 µl HATU solution 19 Aspirate in the needle line 375 µl DIPEA slution 20 Aspirate in the needle line 30 µl DMF/thioanisole 21 Aspirate in the needle line Fmoc amino acid 1260 µl solution 22 Activation, 2: the four solutions are transferred to a tube to start the activation Few seconds s 23 Activation, 3: the content of the tube is aspirated and transferred to the reaction column for the coupling 24 wait seconds after the Activation, 2 30 min Add solvent to the reactor and wait 4000 µl DMF/thioanisole 3 min Washing 26 Add solvent to the reactor and wait 2000 µl DMF/thioanisole 3 3 min

21 27 Drain 28 Add reagent to the reactor Capping and wait 3000 µl Capping solution 6 min 29 Add solvent to the reactor and wait Washing 4000 µl DMF/thioanisole 1 min 30 Add solvent to the reactor and wait 2000 µl DMF/thioanisole 5 1min 31 Go to 5 for the next amino acid 32 Last Fmoc Add reagent to the reactor Piperidine removal and wait 3000 µl solution 3 min 33 Add reagent to the reactor Piperidine and wait 3000 µl solution 17 min 34 Add solvent to the reactor Washing and wait 4000 µl DMF/thioanisole 2 min Add solvent to the reactor and wait 2000 µl DMF/thioanisole 3 1 min Drain 35 Add solvent to the reactor Last washing and wait 36 Drain 3000 µl DCM 5 5min

22 Supplementary Method 2 Setup of the standard batch automated peptide synthesizer (one reactor) CEM Liberty peptide synthesizer operated without microwave heating. The following reagents are used in the automated Fmoc SPPS protocol: Fmoc amino acid solution : 0.2 M in DMF for peptide synthesis HBTU solution: HBTU 0.5 M in DMF for peptide synthesis DIPEA solution : DIPEA 37% in NMP (vol/vol) Piperidine solution : piperidine 20% in DMF for peptide synthesis (vol/vol) Capping solution: acetic anhydride 10%/DIPEA 5%/ DMF for peptide synthesis 85% (vol/vol/vol) Table S2. Fmoc SPPS protocol for the synthesis of peptide 5 using 0.1 mmol of Rink TG Novasyn resin (0.25 mmol/g) Step 1 Name of chemical Action Volume Resin swelling Solvent or reagent Time Add solvent to the reactor, mix and drain the vessel after the indicated time 7000 µl DMF 1 min 2 Add reagent to the reactor, mix Fmoc removal and drain the vessel after the indicated time 7000 µl 3 Add reagent to the reactor, mix and drain the vessel after the indicated time 7000 µl 4 Add solvent to the reactor, mix and drain the vessel after the indicated Washing time 5 Repeat Step 4 three times 6 First coupling Add reagent to the reactor 7 Add reagent to the reactor 8 Add reagent to the reactor, mix and drain the vessel after the indicated time Piperidine solution Piperidine solution 5 min 10 min 7000 µl DMF 1 min 7000 µl DMF 1 min 2500 µl Fmoc amino acid solution 1000 µl HBTU solution 500 µl DIPEA solution 60 min

23 9 Washing Repeat Step 4 twice 10 Second Repeat Steps 6 8 coupling 11 Washing Repeat Step 4 twice 12 Add reagent to the reactor, mix Capping and drain after the indicated time 7000 µl Capping solution 15.5 min 13 Washing Repeat Step 4 three times 14 Go to 2 for the next amino acid 15 Add solvent to the reactor, mix and Washing drain after the indicated time 7000 µl DMF 1 min 16 Final Fmoc removal Repeat Steps 2 and 3 17 Washing Repeat Step 4 three times 18 Add reagent to the reactor, mix N terminal and drain after the indicated time acetylation 7000 µl Capping solution 15.5 min 19 Final washing Repeat Step 4 three times