A central cysteine residue is essential for the thermal stability and function of SUMO-1 protein and SUMO-1 peptide protein conjugates

Transcription

1 Supporting information for A central cysteine residue is essential for the thermal stability and function of SUM-1 protein and SUM-1 peptide protein conjugates ervé Drobecq, a Emmanuelle Boll, a Magalie Sénéchal, a Rémi Desmet, a Jean-Michel Saliou, b Jean-Jacques Lacapère, c Alexandra Mougel, a Jérôme Vicogne, a * leg Melnyk a * a Univ. Lille, CRS, Institut Pasteur de Lille, UMR M3T - Mechanisms of Tumorigenesis and Target Therapies, F-59 Lille, France b Univ. Lille, CRS, Inserm, CU Lille, Institut Pasteur de Lille, U119 - UMR 824, F-59 Lille, France. c Sorbonne Universités, UPMC Univ Paris 6, École ormale Supérieure, PSL Research University, CRS UMR 723 LBM, F-755, Paris, France. Corresponding authors: Dr leg Melnyk, oleg.melnyk@ibl.cnrs.fr Web site: Phone: +33 () and Dr Jérôme Vicogne, jerome.vicogne@ibl.cnrs.fr Phone: +33 () Cancer Chemistry & Biology team Cent at de la Recherche Scientifique (CRS) Institut de Biologie de Lille 1 rue du Pr Calmette, CS 5447, 5921 Lille cedex, France. S1

2 Table of content 1. General Methods Reagents and solvents... 4 Analyses Peptide synthesis... 6 Synthesis of peptides 1 and Synthesis of thioester 1 by thiol-sea exchange Synthesis of p53 peptide 4a Synthesis of p53 peptide 4b Synthesis of p53 SUM-1 peptide protein conjugates ne-pot synthesis of p53 SUM-1 peptide conjugate 5a Characterization of p53 SUM-1 conjugate 5a... 2 MALDI-TF in source fragmentation of p53 SUM-1 conjugate 5a. Proof of structure ne-pot synthesis of p53 SUM-1 peptide conjugate 5b Characterization of p53 SUM-1 conjugate 5b MALDI-TF in source fragmentation of p53 SUM-1 conjugate 5b. Proof of structure... 3 Synthesis of conjugate 6a by selective desulfurization of conjugate 5a Desulfurization of conjugate 5a in native conditions Characterization of conjugate 6a MALDI-TF in source fragmentation of p53 SUM-1 conjugate 6a. Proof of structure Desulfurization of conjugate 5a in denaturing conditions. Preparation of 7a Alkylation of 5a, 6a and 7a with iodoacetamide, enzymatic cleavage and analysis of the peptide fragments by MALDI-TF MS or LC-MS Synthesis of conjugate 6b by selective desulfurization of conjugate 5b Desulfurization of conjugate 5b in native conditions Characterization of conjugate 6b MALDI-TF in source fragmentation of p53 SUM-1 conjugate 6b. Proof of structure Alkylation of conjugates 5b and 6b with iodoacetamide, enzymatic cleavage and analysis of the peptide fragments by MALDI-TF MS or LC-MS Preparation of SUM-1 proteins 8 and MALDI-TF characterization of commercial hsum-1 (mixed disulfide with β-mercaptoethanol) 5 Preparation of SUM-1 protein Preparation of SUM-1 C52A protein Structure and functionality of p53 SUM-1 conjugates S2

3 Thermal shift assay (TSA) Circular dichroism (Fig. 4) Cleavage of the p53 SUM-1 conjugates 6b and 7b by Ulp1 enzyme Monitoring by MALDI-TF mass spectrometry Monitoring by SDS-PAGE and Coomassie staining (Fig. 5A)... 6 In vitro SUMylation Assay (Fig. 5B)... 6 References... 6 S3

4 1. General Methods Reagents and solvents -[(dimethylamino)-1-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]--methylmethanaminium hexafluorophosphate -oxide (ATU) and α-fmoc protected amino acids were obtained from Iris Biotech Gmb. Side-chain protecting groups used for the amino acids were Fmoc-Ala-, Fmoc- Arg(Pbf)-, Fmoc-Asn(Trt)-, Fmoc-Asp(tBu)-, Fmoc-Gln(Trt)-, Fmoc-Glu(tBu)-, Fmoc- Gly-, Fmoc-is(Trt)-, Fmoc-Ile-, Fmoc-Leu-, Fmoc-Lys(Boc)-, Fmoc-Met-, Fmoc-Phe-, Fmoc-Pro-, Fmoc-Ser(tBu)-, Fmoc-Thr(tBu)-, Fmoc-Tyr(tBu)-, Fmoc-Val-, Fmoc- Cys(StBu)- or Fmoc-Cys(Trt)-. Synthesis of bis(2-sulfanylethyl)aminotrityl polystyrene (SEA PS) resin was carried out as described elsewhere.(1) Rink-PEG-PS resin (ovasyn TGR) was obtained from ovabiochem. 4-mercaptophenylacetic acid (MPAA), 3-mercaptopropionic acid (MPA), tris(2- carboxyethyl)phosphine hydrochloride (TCEP) were purchased from Sigma-Aldrich. All other reagents were purchased from Acros rganics or Merck and were of the purest grade available. Peptide synthesis grade,-dimethylformamide (DMF), dichloromethane (C 2 Cl 2 ), diethylether (Et 2 ), acetonitrile (C 3 C), heptane, LC MS-grade acetonitrile (C 3 C,.1% TFA and C 3 C,.1% formic acid), LC MS-grade water ( 2,.1% TFA and 2,.1% formic acid),,diisopropylethylamine (DIEA), acetic anhydride (Ac 2 ) were purchased from Biosolve and Fisher- Chemical. Trifluoroacetic acid (TFA) was obtained from Biosolve. Water was purified with a Milli-Q Ultra Pure Water Purification System. Analyses The reactions were monitored by analytical LC MS (Waters 2695 LC/ZQ 2 quadripole) on an reverse phase column. The column, eluent system and gradient used are indicated in the figure legends. The column eluate was monitored by UV at 215 nm and by evaporative light scattering (ELS, Waters 2424). The peptide masses were measured by on-line LC MS: Ionization mode: ES+, range 35 24, capillary voltage 3 kv, cone voltage 3 V, extractor voltage 3 V, RF lens.2 V, source temperature 12 C, dessolvation temperature 35 C. Samples were prepared using 1 µl aliquots of the reaction mixtures. The aliquots were quenched by adding 9 µl of 1% aqueous TFA, extracted with Et 2 to remove MPAA or MPA before analysis. MALDI-TF mass spectra were recorded with a BrukerAutoflex Speed mass spectrometer. The matrix used for the analysis is indicated in the figure legends. igh resolution LC-MS analysis Reverse-phase high performance liquid chromatography was performed using an UltiMate 3 RSLC LC system (Thermo Scientific). Samples (.4 μg each) were injected onto a XBridge TM BE 3 C18 column (3.5 μm particle size, 1 15 mm, Waters) set at 5 C. Elution was performed at a flow rate of 5 µl/min using.1% aqueous formic acid (FA) for mobile phase A and a gradient of acetonitrile containing.1% FA for mobile phase B which raised from 2% to 8% in 15 min. The liquid chromatography flow was split and infused at 5 nl/min into a Xevo G2-XS Tof mass spectrometer (Waters) by a TriVersa anomate robot (Advion). S4

5 The mass spectrometer was run in sensitivity positive ion mode with source parameters as follows: capillary voltage, 3. kv; sampling cone voltage, 3. V; source offset 8 V; source temperature, 8 C. Acquisitions were performed on the mass range 4 2 with a 1 s scan time. Calibration was performed using the singly charged ions produced by a solution of sodium iodide (2 μg/μl) and cesium iodide (5 ng/μl) in 2-propanol/ water (1/1). Data analysis was performed with MassLynx 4.1 (Waters). The protein peak was deconvoluted by the MassLynx function Transform. S5

6 2. Peptide synthesis Synthesis of peptides 1 and 2 The preparation of SUM-1 (2-51)-SEA off, i.e. the precursor of thioester peptide 1, and of Cys(StBu)- SUM-1 (52-97)-SEA off peptide 2 have been described in detail elsewhere.(2, 3) owever, the protocol for their synthesis and the yield have been significantly improved. The novel protocols are therefore described below. The sequence for SUM-1 (2-51) is: SDQEAKPSTE DLGDKKEGEY IKLKVIGQDS SEIFKVKMT TLKKLKESY The sequence for SUM-1 (52-97) is: C(StBu)QRQGVPMS LRFLFEGQRI ADTPKELG MEEEDVIEVY QEQTGG The first amino acid was coupled manually. The rest of the sequence was assembled using an automated Fmoc SPPS synthesizer. Coupling of the first amino acid residue to the SEA ChemMatrix resin The preparation the SEA ChemMatrix resin and of has been described in detail elsewhere.(2, 3) S Trt S Trt SEA ChemMatrix resin Fmoc-Aa- ATU/DIEA DMF R Fmoc- S Trt S Trt R = : Fmoc-Gly-SEA ChemMatrix resin R = p-phc 2 (Tyr) : Fmoc-Tyr(tBu)-SEA ChemMatrix resin Scheme S1 SEA ChemMatrix resin (.15 mmol/g) (333 mg, 49.9 µmol for SUM-1 (2-51) and 371 mg, 55.6 µmol for SUM-1 (52-97)) was conditioned in DMF (3 2 min, 3 ml) in a manual SPPS glass reactor. Fmoc-Aa- (148 mg for Fmoc-Gly-,.497 mmol; 23 mg for Fmoc-Tyr(tBu)-,.5 mmol) was dissolved in DMF (1 ml). -[(dimethylamino)-1-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-methylmethanaminium hexafluorophosphate -oxide (ATU, 19 mg,.499 mmol) was dissolved in the minimal volume of DMF and added to the amino acid solution. Finally, DIEA (174 µl, 1.34 mmol) was added to the above solution to start the activation of the amino acid. This solution was agitated for 1 min and then added to the resin which was shaken during 1 h 3 min at room temperature. The resin was subsequently washed with DMF (5 2 min, 3 ml). The chloranil assay was negative. The resin was subsequently acetylated with a mixture of acetic anhydride/diea/dmf : 1/5/85 by vol (2 3 ml, 2 min and then 2 min) before being washed successively with DMF (3 2 min, 3 ml), C 2 Cl 2 (3 2 min, 3 ml) and diethylether (3 2 min, 3 ml). The resin was then dried in vacuo during 3 h. S6

7 The loading of the resin was determined by UV quantification at 29 nm of the dibenzofulvenepiperidine adduct formed by treating aliquots of the resin with piperidine (2% by vol in DMF). We found.14 mmmol/g for Fmoc-Gly-SEA ChemMatrix resin and.157 mmol/g for Fmoc-Tyr(tBu)-SEA ChemMatrix resin. Automated solid phase peptide synthesis The peptide elongation step was performed using an automated column peptide synthesizer using standard Fmoc-SPPS protocols (.1 mmol scale). The amino acids (1 equiv) were activated using ATU (1 equiv)/diea (2 equiv) in DMF. Each amino acid was coupled twice. The peptidyl resin was acetylated with Ac 2 /DIEA in DMF after each double coupling. Deprotection and cleavage step The peptidyl resins (.1 mmol scale) were deprotected and cleaved in a mixture of TFA/triisopropylsilane (TIS)/dimethylsulfide (DMS)/thioanisole/ 2 : 9/2.5/2.5/2.5/2.5 by vol (1 ml) during 1 h 3 min (3 times) for SUM-1 (2-51), and in a mixture of TFA/triisopropylsilane (TIS)/dimethylsulfide (DMS)/thioanisole/ 2 /thiophenol : 82.5/2.5/5/2.5/2.5/2.5/2.5 by vol (1 ml) during 1 h 3 min (3 times) for SUM-1 (52-97). The crude peptides were precipitated in an ice-cold mixture of Et 2 /n-heptane : 1/1 by vol to give 463 mg (63% crude) of SUM-1 (2-51)-SEA on peptide and 164 mg (53% crude) of SUM-1 (52-97)-SEA on peptide. xidation and purification of SEA off peptide segments The crude SEA on peptide segments ( 9 µmol) were dissolved in Ac/ 2 : 1/4 by vol (final peptide concentration.9 mm). Iodine solution (2 mm in DMS) was added in dropwise until the appearance of a yellow color. After 3 s, dithiothreitol (DTT, 65 mm in water) was added dropwise until the disappearance of the yellow color to quench the excess of iodine. nce DTT was added, the mixture was immediately purified by reversed-phase PLC to give the corresponding SEA off peptide segments. Semi-preparative PLC conditions for SEA off peptide segments: C3 Zorbax column (5 µm, 3 Å, mm, Agilent), eluent A water containing.1% of TFA, eluent B C 3 C/water : 4/1 by vol containing.1% of TFA, gradient: -2% B in 5 min, then 2-5% B in 6 min, 65 C, flow rate 6 ml/min, UV detection at 215 nm. Yield for SUM-1 (2-51)-SEA off : 5 mg of crude product furnished (13.6 mg, 13.6%) of SUM-1 (2-51)- SEA off peptide. Yield for SUM-1 (52-97)-SEA off 2: mg of crude product furnished (43.8 mg, 14%) of SUM-1 (52-97)-SEA off peptide 2. S7

8 SDQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIFKVKMTTLKKLKES SUM-1 (2-51)-SEA off S S A) Intensity (AU) Time (min) B) 1 Intensity (AU) [M+4] [M+7] [M+8] [M+5] [M+6] [M+9] S8

9 C) 4 x1 Intensity (AU) Figure S1. Analysis of SUM-1 (2-51)-SEA off peptide. LC-MS analysis XBridge BE3 C µm 4.6mm 15 mm, 5 C. Flow 1 ml/min, eluent A.1% trifluoroacetic acid in water, eluent B.1% trifluoroacetic acid in 8% aqueous acetonitrile. Gradient from % buffer B to 1% buffer B in 3 min. A) PLC trace (light scattering detection) B) MS trace Calculated for M (average mass) , observed after deconvolution. C) MALDI-TF analysis of SUM-1 (2-51)-SEA off peptide. Matrix: Sinapinic acid Calculated for [M+] + (monoisotopic mass) , observed S9

10 C(StBu)QRQGVPMSLRFLFEGQRIADTPKELGMEEEDVIEVYQEQTG SUM-1 (52-97)-SEA off 2 S S A) 1. Intensity (AU) Time (min) B) 1 [M+4] [M+5] [M+6] [M+3] S1

11 C) x1 4. Intensity (AU) Figure S2. Analysis of SEA off peptide segment 2. LC-MS analysis XBridge BE3 C µm 4.6mm 15 mm, 5 C. Flow 1 ml/min, eluent A.1% trifluoroacetic acid in water, eluent B.1% trifluoroacetic acid in 8% aqueous acetonitrile. Gradient from % buffer B to 1% buffer B in 3 min. A) PLC trace (light scattering detection) B) MS trace A) PLC trace (light scattering detection). B) MS trace for SEA off peptide segment 2. Calculated for [M+] + (average mass) , observed after deconvolution. C) MALDI-TF analysis of SEA off peptide segment 2. Matrix: Sinapinic acid Calculated for [M+] + (average mass) , observed Synthesis of thioester 1 by thiol-sea exchange SDQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIFKVKMTTLKKLKES SUM-1 (2-51)-SEA off S S C 2 S p 4, TCEP SDQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIFKVKMTTLKKLKES Peptide thioester 1 S C 2 Scheme S2. SEA-thiol exchange reaction for the preparation of peptide thioester 1. The reaction was carried out under nitrogen atmosphere. Tris(2-carboxylethyl)phosphine hydrochloride (TCEP.Cl, 346 mg, 8. mmol) was dissolved in.2 M p 7.3 sodium phosphate S11

12 buffer (15.1 ml). SUM-1 (2-51)-SEA off peptide (36.9 mg, 5.3 µmol) was dissolved in the above solution (15.1 ml). Then, 3-mercaptopropionic acid (MPA, 755 µl) was added and the p of the reaction mixture was adjusted to 4. by addition of 6 M a. The reaction was agitated at 37 C for 48 h and then acidified with 1% aqueous TFA (51 µl). The mixture was extracted with diethylether (3 2 ml) to remove the excess of MPA and immediately purified by RP-PLC using a C18 Xbridge BE3 prep column (1 25 mm, 5 µm) to give mg (4%) of peptide thioester 1. Gradient used for the PLC purification: eluent A water containing.1% of TFA, eluent B C 3 C/water : 4/1 by vol containing.1% of TFA, gradient: -25% B in 5 min, then 25-5% B in 6 min, flow rate 6 ml/min, UV detection at 215 nm. SDQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIFKVKMTTLKKLKES Peptide thioester 1 S C 2 A) Time (min) S12

13 B) 1 Intensity (AU) [M+4] [M+7] [M+8] [M+6] [M+5] C) 8 Intensity (AU) Figure S3. Analysis of peptide thioester 1. LC-MS analysis XBridge BE3 C µm 4.6mm 15 mm, 5 C. Flow 1 ml/min, eluent A.1% trifluoroacetic acid in water, eluent B.1% trifluoroacetic acid in 8% aqueous acetonitrile. Gradient from % buffer B to 1% buffer B in 3 min. A) PLC S13

14 trace (light scattering detection). A) PLC trace (light scattering detection) of peptide thioester 1. B) MS trace, calculated for M (average mass) , observed after deconvolution. C) MALDI- TF analysis of peptide thioester 1. Matrix alpha-cyano-4-hydroxy-cinnamic acid: Calculated for [M+] + (monoisotopic mass) , observed Synthesis of p53 peptide 4a P53 peptide 4a was synthesized using an automated column peptide synthesizer using standard Fmoc-SPPS protocols (2.5 mmol, ovasyn TGR resin,.25 mmol/g). The amino acids (1 equiv) were activated using ATU (1 equiv)/diea (2 equiv) in DMF. Each amino acid was coupled twice. The peptidyl resin was acetylated with Ac2/DIEA in DMF after each double coupling. Lysine 386 was coupled as the Fmoc-Lys(Mtt)- derivative. The -terminal Fmoc group was deprotected manually using 2% piperidine in DMF (1 x 1min then 1 x 1min). 1 The resin was then treated with (Boc) 2 (3 mg ; 1.37 mmol, 15 equiv) solubilized in DMF (5 ml) for 3 min at rt to protect the -terminal amino group. The Mtt group on Lys 386 residue was deprotected selectively by washing the peptidyl resin with 1% TFA in C 2 Cl 2 (15 x 2min). The resin was then neutralized with 5% DIEA in DMF. Fmoc-Cys(Trt)- (293 mg,.5 mmoles), and PyBop (26 mg,.499 mmoles) were solubilized in DMF (5 ml) and added to the peptidyl resin together with DIEA (175 µl, 1. mmole). The resin was agitated for 4 min, and then washed with DMF (3 x 2 min), C 2 Cl 2 (3 x 2 min), diethyl ether (2 x 2 min) and dried in vacuo. The peptidyl resin was finally deprotected and cleaved in TFA/TIS/EDT/ 2 : 9/5/2.5/2.5 by vol (1 ml) for 2 h 3. The crude peptide was precipitated in ice-cold heptane /diethylether : 1/1 by vol, solubilized in deionized water and lyophilized to yield 21 mg of crude peptide 4a. The peptide was purified directly by PLC to give 26.2 mg (6 % overall) of peptide 4a Preparative PLC conditions for peptide 4a: XBridge BE3 C18 (5 µm, 3 Å, 1 25 mm) column, eluent A water containing.1% of TFA, eluent B C 3 C/water : 4/1 by vol containing.1% of TFA, gradient: -1%B in 2 min, then 1-25% B in 6 min, flow rate 6 ml/min, 5 C, UV detection at 215 nm. 1 The -terminal Fmoc group was partially removed upon standing in the synthesis column overnight. S14

15 2 S C -SLKSKKGQSTSRKKLMF TEGPDSD- 2 p53 peptide 4a A) Intensity (AU) B) Time (min) 1 Intensity (AU) [M+3] [M+4] S15

16 C) x Intensity (AU) Figure S4. Analysis of peptide 4a. LC-MS analysis XBridge BE3 C µm 4.6mm 15 mm, 5 C. Flow 1 ml/min, eluent A.1% trifluoroacetic acid in water, eluent B.1% trifluoroacetic acid in 8% aqueous acetonitrile. Gradient from % buffer B to 1% buffer B in 3 min. A) PLC trace (light scattering detection). A) PLC trace (light scattering detection).b) MS trace, calculated for M (average mass) , observed after deconvolution. C) MALDI-TF analysis. Matrix: alpha-cyano-4-hydroxy-cinnamic acid. Calculated for [M+] + (monoisotopic mass) , observed Synthesis of p53 peptide 4b Peptide 4b was synthesized using an automated column peptide synthesizer using standard Fmoc- SPPS protocols (.5 mmol, ovasyn TGR resin,.25 mmol/g). The amino acids (1 equiv) were activated using ATU (1 equiv)/diea (2 equiv) in DMF. Each amino acid was coupled twice. The peptidyl resin was acetylated with Ac2/DIEA in DMF after each double coupling. Lysine 386 was introduced manually as the -Fmoc thiazolidine δ-mercaptolysine derivative,(4) which was kindly provided by Ashraf Brik s lab. For this, -Fmoc thiazolidine δ-mercaptolysine derivative (38 mg, 74 µmol, 1.5 equiv) was preactivated with ATU (17.5 mg, 46 µmol, 1.5 equiv)/diea (26 µl, 149 µmol, 3 equiv) in DMF (.5 ml) during 1 min and then added to the peptidyl resin. The resin was agitated for 1 h 3 and then washed, DMF (3 2 min). The rest of the peptide was assembled using the automated peptide synthesizer. The resin was then washed with DMF (3 x 2 min), C 2 Cl 2 (3 x 2 min), diethyl ether (2 x 2 min) and dried in vacuo. The peptidyl resin was finally deprotected and cleaved in TFA/TIS/EDT/ 2 : 92.5/2.5/2.5/2.5 by vol (1 ml) for 1 h 3. The crude peptide was precipitated in ice-cold heptane /diethylether : 1/1 by vol, solubilized in deionized water and lyophilized to yield 14 mg (68% crude) of crude peptide. S16

17 2 S The thiazolidine group was removed by treated the crude peptide (5 mg) with - methylhydroxylamine (.1 M final concentration in.5 M ammonium acetate, p 4). After 3 h of stirring, the peptide was purified directly by PLC to give 13.9 mg (19 % overall) of peptide 4b. Preparative PLC conditions for peptide 4b: XBridge BE3 C18 (5 µm, 3 Å, 2 1 mm) column, eluent A water containing.1% of TFA, eluent B C 3 C/water : 4/1 by vol containing.1% of TFA, gradient: 5-25% B in 25 min, flow rate 25 ml/min, UV detection at 215 nm, 5 C. A) SLKSKKGQSTSRKKLMF TEGPDSD- 2 p53 peptide 4b Intensity (AU) Time( min) S17

18 B) 1 [M+4] [M+3] [M+5] [M+2] C) Figure S5. Analysis of peptide 4b. LC-MS analysis XBridge BE3 C µm 4.6mm 15 mm, 5 C. Flow 1 ml/min, eluent A.1% trifluoroacetic acid in water, eluent B.1% trifluoroacetic acid in 8% aqueous acetonitrile. Gradient from % buffer B to 1% buffer B in 3 min. A) PLC trace (light scattering detection). B) MS trace, calculated for M (average mass) , observed after deconvolution. C) MALDI-TF analysis. Matrix: alpha-cyano-4-hydroxy-cinnamic acid. Calculated for [M+] + (monoisotopic mass) , observed S18

19 Synthesis of p53 SUM-1 peptide protein conjugates ne-pot synthesis of p53 SUM-1 peptide conjugate 5a SDQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIFKVKMTTLKKLKES Peptide 1 S C 2 C(StBu)QRQGVPMSLRFLFEGQRIADTPKELGMEEEDVIEVYQEQTG Peptide 2 S S one-pot process step 1 MPAA, p 7.2, Gdn.Cl 6 M SDQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIFKVKMTTLKKLKESYCQRQGVPMSLRFLFEGQRIADTPKELGMEEEDVIEVYQEQTG S SUM-1 (2-97)-SEA off 3 S S one-pot process step 2 TCEP, p 5.5 p53 peptide 4a S S 367 LKSKKGQSTSRKKLMFKTEGPDS S SDQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIFKVKMTTLKKLKESYCQRQGVPMSLRFLFEGQRIADTPKELGMEEEDVIEVYQEQTG S p53 SUM-1 conjugate 5a S 367 LKSKKGQSTSRKKLMFKTEGPDS Scheme S3. ne-pot synthesis of p53 SUM-1 peptide conjugate 5a. The whole process was carried under nitrogen atmosphere. First ligation step (CL): A solution of 4-mercaptophenylacetic acid (MPAA, 26.9 mg,.159 mmol) in 6 M guanidinium chloride/.1 M p 7. sodium phosphate buffer was prepared (.8 ml). Peptide thioester 1 (8.31 mg, 1.13 µmol) and SEA off peptide segment 2 (7.1 mg, 1.13 µmol) were dissolved in the above solution (379 µl) and the p was adjusted to 7.2 by addition of aqueous a 6. The reaction was agitated for 6 h at 37 C. Second ligation step (SEA ligation): Then, a solution of TCEP (24.14 mg, 84.2 µmol) and MPAA (14.16 mg, 84.2 µmol) in 6 M guanidinium hydrochloride/.1 M p 7. sodium phosphate buffer (42 µl) was prepared and used to dissolve p53 peptide 4a (9.52 mg, 2.27 µmol). The p of this solution was 4.1. The peptide solution was then added to the above reaction mixture and the p was adjusted to 5.6 by addition of aqueous a (1 µl).the final peptide concentration was 1.5 mm. The reaction was agitated for 36 h and then diluted with water (3 ml), acidified to p 3 by adding 1% aqueous TFA and extracted with diethylether (3 2 ml) to remove the excess of MPAA. The mixture was immediately purified by PLC to yield 7.45 mg (38%) of p53 SUM-1 conjugate 5a. S19

20 Semi-preparative PLC conditions: C3 Zorbax column (5 µm, 3 Å, mm, Agilent), detection at 215 nm, flow rate 6 ml/min, eluent A water containing.1% of TFA, eluent B C 3 C/water : 4/1 by vol containing.1% of TFA, gradient -5% B in 3 min, 5 C. Characterization of p53 SUM-1 conjugate 5a S SDQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIFKVKMTTLKKLKESYCQRQGVPMSLRFLFEGQRIADTPKELGMEEEDVIEVYQEQTG S p53 SUM-1 conjugate 5a S 367 LKSKKGQSTSRKKLMFKTEGPDS A) Intensity (light scattering, AU) 5a 7. 1 Intensity (AU) A SUM-1 (2-97)-Cys 6. A SLKSKKGQSTSRKKLMFKTEGPDSD- 2 M calcd A obs ± A A A A A A Crude ligation mixture after 36 h (second step) a Time (min) S2

21 B) Intensity (AU) Time (min) C) 1 [M+19] [M+2] [M+18] [M+21] [M+17] [M+16] [M+22] [M+23] [M+15] [M+14] [M+13] S21

22 D) 3 [M+] [M+2] E) F) Intensity (AU) a deconvoluted M Intensity (AU) 5a Theoretical profile resolution M S22

23 Figure S6. Analysis of p53 SUM-1 conjugate 5a. A) LC-MS analysis of the crude one-pot mixture, PLC trace (light scattering detection). XBridge BE3 C µm 4.6mm 15 mm, 5 C. Flow 1 ml/min, eluent A.1% formic acid in water, eluent B.1% formic acid in 8% aqueous acetonitrile. Gradient from % buffer B to 5% buffer B in 3 min. B) LC-MS analysis of the purified conjugate 5a. C) MS trace, calculated for M (average mass) , observed after deconvolution. D & E) MALDI-TF analysis. Matrix: sinapinic acid, calculated for [M+] + (average mass) , observed F) RMS reconstructed mass and theoretical profile for C S 6 at resolution 35. MALDI-TF in source fragmentation of p53 SUM-1 conjugate 5a. Proof of structure A) B) S23

24 C) Abs. Int. * 1 c K P S T E D D K K E G E y C* G G c 6 c 7 c 8 c 9 c 1c 11 c 13c 14 c 15 c 16 c 17 c 18 c y y 2y D) Abs. Int. * 1 y C* G G T Q E Q y y y y y y y S24

25 2 S E) S D Q E A K P S T E D L G D K K E G E Y I K L K V I G Q D S S E I F K V K M T T L K K L K E S Y C Q R Q G V P M S L R F L F E G Q R I A D T P K E L G M E E E D V I E V Y Q E Q T G G S C* S L K S K KGQS T S RK K L MF K T EGP DSD 2 Figure S7. In source MALDI-TF sequencing using 2,5-dihydroxybenzoic acid as matrix (positive reflector mode). Ions corresponding to the -terminal part of SUM-1 domain (A) or target peptide (B) could be identified. The y ions shown in (C-E) show the formation of the peptide bond between the C-terminal Gly residue of SUM-1 protein and the Cys residue of the target peptide. C* is for the y ion at which corresponds to the p53 peptide plus the C-terminal Cys residue. ne-pot synthesis of p53 SUM-1 peptide conjugate 5b SDQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIFKVKMTTLKKLKES Peptide 1 S C 2 C(StBu)QRQGVPMSLRFLFEGQRIADTPKELGMEEEDVIEVYQEQTG Peptide 2 S S one-pot process step 1 MPAA, p 7.2, Gdn.Cl 6 M SDQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIFKVKMTTLKKLKESYCQRQGVPMSLRFLFEGQRIADTPKELGMEEEDVIEVYQEQTG S SUM-1 (2-97)-SEA off 3 S S one-pot process step 2 TCEP, p 5.5 SLKSKKGQSTSRKKLMF TEGPDSD- 2 p53 peptide 4b S SDQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIFKVKMTTLKKLKESYCQRQGVPMSLRFLFEGQRIADTPKELGMEEEDVIEVYQEQTG S p53 SUM-1 conjugate 5b Scheme S4. ne-pot synthesis of p53 SUM-1 peptide conjugate 5b. SLKSKKGQSTSRKKLMF TEGPDSD- 2 The one-pot assembly was performed in an inert atmosphere (nitrogen). S25

26 First ligation step (CL): A solution of 4-mercaptophenylacetic acid (MPAA, mg,.2 mmol) in 6 M guanidinium chloride/.1 M p 7. sodium phosphate buffer was prepared (1 ml). Peptide thioester 1 (9.33 mg, 1.27 µmol) and SEA off peptide segment 2 (7.88 mg, 1.27 µmol) were dissolved in the above solution (426 µl) and the p was adjusted to 7.35 by addition of aqueous 6 a. The reaction was agitated for 3 h at 37 C. Second ligation step (SEA ligation): Then, a solution of TCEP (32.68 mg,.114 mmol) and MPAA (19.17 mg,.114 mmol) in 6 M guanidine hydrochloride/.1 M p 7. sodium phosphate buffer (57 µl) was prepared and used to dissolve p53 peptide 4b (6.48 mg, 1.53 µmol, 255 µl of the above solution). The p of this solution was 3.8. The p53 peptide solution was then added to the above reaction mixture and the p was adjusted to 5.57 by addition of aqueous 6 a (2 µl). The final peptide concentration was 1.9 mm. The reaction was agitated for 12 h and then diluted with water (3 ml), acidified to p 3 by adding 1% aqueous TFA and extracted with diethylether (3 2 ml) to remove the excess of MPAA. The mixture was immediately purified by PLC to yield 5.84 mg of p53 SUM-1 conjugate 5b (27%). PLC conditions for the purification: C3 Zorbax column (5 µm, 3 Å, mm, Agilent), detection at 215 nm, flow rate 6 ml/min, 5 C, eluent A water containing.1% of TFA, eluent B C 3 C/water : 4/1 by vol containing.1% of TFA, gradient -5% B in 3 min. S26

27 Characterization of p53 SUM-1 conjugate 5b A) 8. Intensity (light scattering, AU) b b 1.. B) Time (min) Intensity (AU) Time (min) S27

28 C) 1 [M+16] [M+15] [M+17] [M+18] [M+19] [M+2] [M+14] [M+13] [M+21] [M+12] [M+11] D) 15 Intensity (AU) [M+] [M+2] S28

29 E) Intensity (AU) F) Intensity (AU) b deconvoluted mass Intensity (AU) Theoretical profile for M C S M Figure S8. Analysis of p53 SUM-1 conjugate 5b. A) LC-MS analysis of the crude one-pot mixture after 12 h for the second step, PLC trace (light scattering detection). C3 Zorbax column (5 µm, 3 Å, mm, Agilent), 5 C. Flow 1 ml/min, eluent A.1% formic acid in water, eluent B.1% formic acid in 8% aqueous acetonitrile. Gradient from % buffer B to 5% buffer B in 3 min. B) LC- MS analysis of the purified conjugate 5b. C) MS trace, calculated for M (average mass) , observed after deconvolution. D & E) MALDI-TF analysis. Matrix: sinapinic acid, calculated S29

30 for [M+] + (average mass) , observed F) RMS reconstructed mass and theoretical profile for C S 6 at resolution 35. MALDI-TF in source fragmentation of p53 SUM-1 conjugate 5b. Proof of structure A) Fragmentation of SUM-1 domain SDQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIFKVKMTTLKKLKESYCQRQGVPMSLRFLFEGQRIADTPKELGM Abs. Int. c K L K V I G Q D S S E I F K V K M T T L K K L K E S Y C Q R c c c 27 2 c 21c 22c c 25 1 c 24 c 26 5 c 3 c 31 c 32 c 33 c 35 c 34 c 36 c 37 c 38 c 39 c 4 c 41 c 42 c 43 c 44 c 45 c 46 c 47 c 48 c 49 c 5 c 51 c 52 c Abs. Int. * 1 c E D L G D K K E G E Y I K c 9 c 1 3 c 11 c 13 2 c 14 c 15 1 c 12 c 16 c 17 c 18 c 19 c 2 c S3

31 B) P53 SLKSKKGQSTSRKKLMFJTEGPDSD Abs. Int. * 1 c S T S R K K L M F 9 8 c 13 7 c 14 6 c c 1 c 11 c 12 c 15 3 c 16 2 c 17 1 c 18 c C) Abs. Int. y G* G T Q E Q Y V E D y 1 y 2 y 3 y 12 y 11 y 21 1 y 4 y 5 y 6 y 7y 8 y Abs. Int. * 1 c V I G Q D S S E y G* G T Q E G c c 27 c y y c 3 y 3.8 c 31.6 c 25.4 c y S31

32 D) S SDQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIFKVKMTTLKKLKESYCQRQGVPMSLRFLFEGQRIADTPKELGMEEEDVIEVYQEQTG G* 39.4 S SLKSKKGQSTSRKKLMF TEGPDSD- 2 Figure S9. In source MALDI-TF sequencing using 2,5-dihydroxybenzoic acid as matrix (positive reflector mode). C ions corresponding to the -terminal part of SUM-1 domain (A) or target peptide (B) could be identified. Importantly, the y ions detected in (C) show the formation of the peptide bond between the C-terminal Gly residue of SUM-1 protein and the target peptide (D). G* is for the y ion at which corresponds to the p53 peptide plus the C-terminal Gly residue. Synthesis of conjugate 6a by selective desulfurization of conjugate 5a Desulfurization of conjugate 5a in native conditions Conjugate 5a (3,4 mg, 2.4 µmol, 1 mm final concentration) was dissolved in a solution of TCEP (57.31 mg/ml, 2 mm final concentration), 2,2'-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride (VA- 44,.43 mg/ml, 1.33 mm final concentration) and reduced glutathione (6.15 mg/ml, 2 mm final concentration) in.1 M p 7.2 ammonium phosphate buffer (244 µl). The desulfurization was carried out at 25 C and monitored by MALDI-TF mass spectrometry. After 14 h, the reaction product was purified by PLC to yield 662 µg (19 %) of pure conjugate 6a. PLC conditions: C3 Zorbax 3 Å column, detection at 215 nm, flow rate 6 ml/min, eluent A water containing.1% of Cl, eluent B C 3 C/water : 6/4 by vol containing.1% of Cl, gradient -8% B in 6 min. S32

33 a) Intensity (AU) p53 SUM-1 conjugate 5a b) Denaturing 1 h p53 SUM-1 conjugate 7a c) ative 1 h d) ative 5 h ative 14 h p53 SUM-1 conjugate 6a e) Figure S1. Desulfurization of synthetic p53 SUM-1 conjugate 5a (VA mm, TCEP 2 mm, GS 2 mm, 25 C, p 7.2). MALDI-TF analysis of the desulfurization reaction using sinapinic acid as matrix (a: starting conjugate 5a, b: 6 M Gdn.Cl, c-e: native conditions). S33

34 Characterization of conjugate 6a A) Intensity (light scattering, AU) Time B) 1 [M+2] [M+18] 18+ [M+21] [M+18] [M+22] [M+23] [M+24] [M+17] [M+16] [M+15] [M+14] [M+13] S34

35 C) 2 Intensity (AU) D) 6a deconvoluted mass theoretical profile for 6a resolution mass Figure S11. Analysis of p53 SUM-1 conjugate 6a. A) LC-MS analysis, PLC trace (light scattering detection). ). C3 Zorbax column (5 µm, 3 Å, mm, Agilent), 5 C. Flow 1 ml/min, eluent A.1% formic acid in water, eluent B.1% formic acid in 8% aqueous acetonitrile. Gradient from % buffer B to 1% buffer B in 3 min. B) MS trace, calculated for M (average mass) observed S35

36 after deconvolution. C) MALDI-TF analysis. Matrix: sinapinic acid. Calculated for [M+] + (average mass) , observed (linear positive mode).d) RMS analysis, reconstructed mass compared with the theoretical profile for C S 5 at resolution 35. MALDI-TF in source fragmentation of p53 SUM-1 conjugate 6a. Proof of structure A) Abs. Int. * 1 c D L G D K K E G E K L S y A* G G c 1 2 c 11 c 13 1 c 12 c 14 c 15 c 18 c 16 c 17 c 19 c 21 c 22 c 23 y 1 y c 2y 29c B) S36

37 C) Abs. Int. c V I G Q E I F K V K M T T L y A* G G T Q E Q Y V E I V D y y 2 c 28 y 3 c 34 4 c 27 y 7 c 37 c 42 y c 31 c 35 c 38 3 c 33 y 13 y 4 c 41 y 5 y 6 2 c 26 y 8 y 9c 36 c 25 c 32 y 1 c 39 c 24 y 11 c c 43 Abs. Int. c G Q E y A* G G T Q E A y c 28 y 2 y c 27 y 4 c 31 y Figure S12. In source MALDI-TF sequencing of conjugate 6a using 2,5-dihydroxybenzoic acid as matrix (positive reflector mode). C ions corresponding to the -terminal part of SUM-1 domain (A) or target peptide (B) could be identified. Importantly, the y ions detected in (C) show the formation of the peptide bond between the C-terminal Ala residue of SUM-1 protein and the target peptide (D). A* is for the y ion at which corresponds to the p53 peptide plus the C-terminal Ala residue. S37

38 Desulfurization of conjugate 5a in denaturing conditions. Preparation of 7a Peptide 5a (5 µg) was dissolved in 6 M Gdn.Cl.1 M p 7.2 ammonium phosphate buffer containing TCEP (2 mm final concentration), VA-44 (1.33 mm final concentration) and GS (2 mm final concentration). The desulfurization was carried out at 25 C and monitored by MALDI-TF mass spectrometry. After 1 h, conjugate 7a was desalted by PLC and subjected to alkylation/enzymatic digestion for MS analysis. Alkylation of 5a, 6a and 7a with iodoacetamide, enzymatic cleavage and analysis of the peptide fragments by MALDI-TF MS or LC-MS The conjugates 5a, 6a and 7a (fully desulfurized conjugate from 5a) (~2 µg) were alkylated with iodoacetamide (2 µl, 1mg/mL in 6 M Gdn.Cl.1 M p 7.2 ammonium phosphate buffer) for 3 min at 25 C. The alkylation step was monitored by MALDI-TF mass spectrometry as shown below. This experiment shows that the conjugate 5a is dialkylated, while the conjugate 6a is monoalkylated. As expected, conjugate 7a resulting from the desulfurization in denaturing conditions was not alkylated Before treatment After treatment Desulfurization in Denaturing conditions 7a monoalkylation Before treatment After treatment Desulfurization in native conditions 6a Before treatment dialkylation After treatment 5a Figure S13. Monitoring of the alkylation step by MALDI-TF mass spectrometry (matrix: sinapinic acid). This experiment shows that the conjugate 7a obtained by desulfurization in denaturing conditions is not alkylated due to the absence of thiol groups, while conjugate 6a obtained by desulfurization in native conditions is only alkylated once (no peak at 1475 was observed that S38

39 could be due to a lack of selectivity during the desulfurization step). The control experiment with conjugate 5a results in the incorporation of two carbamidomethyl groups, as expected. The samples were subsequently diluted tenfold with.1 M p 7.2 ammonium phosphate buffer and digested overnight at room temperature with of EndoGluC protease (Staphylococcus aureus protease V8, 1 µg, 5% by weight). The formed peptide fragments were analyzed by MALDI-TF MS and LC-MS. A) SYCQRQGVPMSLRFLFE endoglu 1 (Cys52) Calcd (monoisotopic) SYAQRQGVPMSLRFLFE endoglu 1 (Ala52) Calcd (monoisotopic) VYQEQTG S VYQEQTG SLKSKKGQSTSRKKLMFKTEGPD endoglu 2 (Cys98) Calcd (monoisotopic) SLKSKKGQSTSRKKLMFKTEGPD endoglu 2 (Ala98) Calcd (monoisotopic) D) B) Desulfurization in denaturing conditions 7a EndoGlu 1 (Ala 52) EndoGlu 2 (Ala 98) C) Desulfurization in native conditions Conjugate 6a EndoGlu 1 (Cys 52) native EndoGlu 2 (Ala 98) D) Conjugate 5a EndoGlu 1 (Cys 52) Temoin EndoGlu 2 (Cys 98) Figure S14. Analysis of the EndoGluC protease digest by MALDI-TF mass spectrometry (matrix: alpha cyanno). Two types of peptide fragments were identified that allow documenting the presence S39

40 of a Cys residue (or Ala) at position 52 within the SUM-1 domain, and 98 in the linker region between SUM-1 and the p53 peptide (A). The conjugate 7a produced by desulfurization in denaturing conditions showed an Ala residue in both positions (B), while the conjugate 6a produced by desulfurization in native conditions showed a Cys residue in position 52, and an Ala residue in position 98 (C). We noticed also in Fig S12C the presence of a minor peak at 2143 showing that a small proportion of Cys52 is nevertheless converted into Ala52. In the control experiment with conjugate 5a, Cys was found at both positions, as expected (D). Intensity (light scattering, AU) 28. EndoGlu 2 (Ala 98) EndoGlu 2 (Cys 98) Desulfurization in denaturing conditions 7a Desulfurization in native conditions Conjugate 6a Conjugate 5a Time (min) Figure S15. Analysis of the EndoGluC protease digests by LC-MS. EndoGlu 2 fragments corresponding to the linker region between SUM-1 domain and p53 peptide. LC-MS analysis, PLC trace (light scattering detection). Column xbridge tm BE3 C18 (3.5 µm, 3 Å, mm, Agilent), 5 C. Flow 1 ml/min, eluent A.1% trifluoroacetic acid in water, eluent B.1% trifluoroacetic acid in 8% aqueous acetonitrile. Gradient from % buffer B to 5% buffer B in 3 min. S4

41 Intensity (light scattering, AU) 18. EndoGlu 1 (Ala 52) EndoGlu 1 (Cys 52) Desulfurization in denaturing conditions 7a Desulfurization in native conditions Conjugate 6a Conjugate 5a Time (min) Figure S16. Analysis of the EndoGluC protease digests by LC-MS. EndoGlu 1 fragments corresponding to the central region of the SUM-1 domain. These chromatograms confirm the selectivity of the desulfurization reaction in native conditions. The EndoGlu 1 (Ala52) fragment that has been detected in Fig. S12C (desulfurization in native conditions, conjugate 5c) is barely detected. LC-MS analysis, PLC trace (light scattering detection). Column xbridge tm BE3 C18 (3.5 µm, 3 Å, mm, Agilent), 5 C. Flow 1 ml/min, eluent A.1% trifluoroacetic acid in water, eluent B.1% trifluoroacetic acid in 8% aqueous acetonitrile. Gradient from % buffer B to 5% buffer B in 3 min. Synthesis of conjugate 6b by selective desulfurization of conjugate 5b Desulfurization of conjugate 5b in native conditions Conjugate 5b (1.15 mg,.67 µmol,.58 mm final concentration) was dissolved in a solution of TCEP (57.31 mg/ml, 2 mm final concentration), 2,2'-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride (VA-44,.43 mg/ml, 1.33 mm final concentration) and reduced glutathione (3.7 mg/ml, 1 mm final concentration) in.1 M p 7.2 ammonium phosphate buffer (1.15 ml). The desulfurization was carried out at 25 C and monitored by MALDI-TF mass spectrometry. After 3 h, the reaction product was purified by PLC to yield.94 mg (8% yield) of pure conjugate 6b. PLC conditions: C3 Zorbax column (5 µm, 3 Å, mm, Agilent), 5 C, detection at 215 nm, flow rate 6 ml/min, eluent A.1% trifluoroacetic acid in water, eluent B.1% trifluoroacetic acid in 8% aqueous acetonitrile. Gradient from 2% buffer B to 4% buffer B in 6 min. S41

42 Characterization of conjugate 6b A) Intensity (light scattering, AU) Conjugate 6b Time (min) B) 1 A A A A A A Conjugate 6b % A A A A A Deconvoluted ± S42

43 C) Conjugate 5d [M+] [M+2] D) Intensity (AU) b Deconvoluted mass Intensity (AU) Theoretical profile for 6b M M Figure S17. Analysis of p53 SUM-1 conjugate 6b. A) LC-MS analysis, PLC trace (light scattering detection). Zorbax C3 (5 µm, 4.6mm 15 mm), 5 C. Flow 1 ml/min, eluent A.1% formic acid in water, eluent B.1% formic acid in 8% aqueous acetonitrile. Gradient from % buffer B to 5% buffer B in 3 min. B) MS trace, calculated for [M] (average mass) , observed /- 2.2 after deconvolution. C) MALDI-TF analysis. Matrix: sinapinic acid, calculated for [M+] + (average S43

44 mass) , observed D) RMS analysis, reconstructed mass compared with theoretical profile for C S 5 at resolution 35. MALDI-TF in source fragmentation of p53 SUM-1 conjugate 6b. Proof of structure A) Abs. Int. * 1 c T S R K K L M F 12 c 13 1 c c 1 c 11 c 12 c 15 4 c 16 2 c 17 c 18 c S44

45 B) Abs. Int. * 1 c D L G D K K E G E Y I K L K V I G Q y G* G c 1 4 c 11 c 13 c 14 c 15 2 c 12 c 16 c 18 c 17 c 19 c 2 c 21 c 22 c 23 c 24c 25 c 26 c 27 c y 28 1 y 2 c 3 c Abs. Int. * 1 c V I G Q D S S E F K V K M T T L y G* G T Y V E I V D M y c c c 27 y 2 c 3 c 31 y 3.5 c 24 c 25 c c 42 c y 35 8 c 34 c 37 y c c 39 c y 36 9 c 4 c 32 c c 43 y 6 y y y 7 y y 1 a C) Abs. Int. * 1 c V I G Q D S S E y G* G T y 1 G c c 25 c c c 28 y 2 y 3 c 3 c 31 c S45

46 D) S SDQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIFKVKMTTLKKLKESYCQRQGVPMSLRFLFEGQRIADTPKELGMEEEDVIEVYQEQTG G* b SLKSKKGQSTSRKKLMF TEGPDSD- 2 Figure S18. In source MALDI-TF sequencing of conjugate 6b using 2,5-dihydroxybenzoic acid as matrix (positive reflector mode). Ions corresponding to the -terminal part of target p53 peptide (A) or SUM-1 domain (B) could be identified. The ions found in (C) show the formation of the peptide bond between the C-terminal Gly residue of SUM-1 protein and the Lys 2 residue of the target p53peptide. G* is for the y ion at which corresponds to the p53 peptide plus the C-terminal Gly residue (D). Alkylation of conjugates 5b and 6b with iodoacetamide, enzymatic cleavage and analysis of the peptide fragments by MALDI-TF MS or LC-MS The conjugates (~2 µg) were treated with iodoacetamide (2 µl, 1mg/mL in 6 M Gdn.Cl.1 M p 7.2 ammonium phosphate buffer) for 3 min at 25 C. The alkylation step was monitored by MALDI- TF mass spectrometry as shown below. This experiment shows that conjugate 5b is dialkylated, while the conjugate 6b obtained by selective desulfurization is monoalkylated Conjugate 6b Before treatment Intensity(AU) alkylation Conjugate 6b Conjugate 5b After treatment Before treatment 2 alkylations Conjugate 5b After treatment Figure S19. Monitoring of the alkylation step by MALDI-TF mass spectrometry (matrix: sinapinic acid). This experiment shows that the conjugate 6b obtained by selective desulfurization in native conditions is only alkylated once (no peak at was observed that could be due to a lack of S46

47 selectivity). The control experiment with conjugate 5b results in the incorporation of two carbamidomethyl groups, as expected. The alkylated samples were subsequently diluted tenfold with.1 M p 7.2 ammonium phosphate buffer and digested with EndoGluC protease (Staphylococcus aureus protease V8, 1 µg, 5% w/w) overnight at room temperature. The formed peptide fragments were identified by MALDI-TF MS and LC-MS. A) SYCQRQGVPMSLRFLFE endoglu 1 (Cys52) Calcd (monoisotopic) SYAQRQGVPMSLRFLFE endoglu 1 (Ala52) Calcd (monoisotopic) VYQEQTG VYQEQTG SC 2 C 2 SLKSKKGQSTSRKKLMFKTEGPD endoglu 2 (acetamidomethyl thiolysine) Calcd (monoisotopic) SLKSKKGQSTSRKKLMFKTEGPD endoglu 2 (lysine) Calcd (monoisotopic) B) endoglu1 (Cys52) endoglu 2 (lysine) Conjugate 6b endoglu1 (Cys52) Conjugate 5b endoglu 2 (acetamidomethyl thiolysine) S47

48 Figure S2. Analysis of the EndoGluC protease digests by MALDI-TF mass spectrometry (matrix: alpha-cyano-4-hydroxy-cinnamic acid). Two types of peptide fragments were identified that allow documenting the presence of a Cys residue at position 52 within the SUM-1 domain, and the presence of thiolysine or lysine at position 2 of the p53 peptide in 5b and 6b respectively (A). The conjugate 6b produced by selective desulfurization in native conditions showed a lysine residue in position 2, and an cysteine residue in position 52 (B). VYQEQTG SC 2 C 2 VYQEQTG SLKSKKGQSTSRKKLMFKTEGPD endoglu 2 (acetamidomethyl thiolysine) A) Intensity (light scattering, AU) Co-injection SLKSKKGQSTSRKKLMFKTEGPD endoglu 2 (lysine) B) Conjugate 5b C) Conjugate 6b Time (min) Figure S21. Analysis of the EndoGluC protease digests by LC-MS (p53 SUM-1 conjugates 5b and 6b). EndoGlu 2 fragments corresponding to the linker region between SUM-1 domain and p53 peptide. LC-MS analysis, PLC trace (light scattering detection). Column xbridge tm BE3 C18 (3.5 µm, 3 Å, mm, Agilent), 5 C. Flow 1 ml/min, eluent A.1% trifluoroacetic acid in water, eluent B.1% trifluoroacetic acid in 8% aqueous acetonitrile. Gradient from % buffer B to 5% buffer B in 3 min. S48

49 EndoGlu 1 (Cys52) EndoGlu 1 (Ala52) A) Co-injection B) p53 SUM-1 conjugate 6b C) p53 SUM-1 conjugate 7b Time Figure S22. Analysis of the EndoGluC protease digests by LC-MS (p53 SUM-1 conjugates 6b and 7b). EndoGlu 1 fragments corresponding to the central region of the SUM-1 domain. These chromatograms confirm the high selectivity of the desulfurization reaction in native conditions. S49

50 Preparation of SUM-1 proteins 8 and 9 The recombinant hsum-1 protein was purchased from Enzo life sciences (ref: ALX C55). The sequence of this protein is: GSMSDQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIFKVKMTTLKKLKESYCQRQGVPMSLRFLFEGQRIAD TPKELGMEEEDVIEVYQEQTGG This protein corresponds to hsum with an GS dipeptide extension at the -terminus. MALDI-TF characterization of commercial hsum-1 (mixed disulfide with β-mercaptoethanol) Fig. S23. MALDI-TF analysis of commercial hsum-1. Matrix: sinapinic acid. Calculated for [M+] + (average mass) , observed (linear positive mode). Preparation of SUM-1 protein 8 Recombinant SUM-1 protein (5 µg) was dissolved in 6 M Gdn.Cl.1 M p 7.2 ammonium phosphate buffer containing TCEP (.5 mg/ml, 1.7 mm). The reaction was carried out at 25 C and monitored by MALDI-TF mass spectrometry. After 1 h, the sample was purified by RP-PLC to yield 274 µg (55%) of SUM-1 protein 8. Preparative PLC conditions: XBridge BE3 C18 (5 µm, 3 Å, 1 3 mm) column, eluent A water containing.1% of TFA, eluent B C 3 C/water : 4/1 by vol containing.1% of TFA, gradient: - 1% B in 1 min, flow rate 6 ml/min, UV detection at 215 nm, 5 C. The purified fractions were pooled, frozen at -2 C and lyophilized. S5

51 A) Intensity (light scattering, AU) hsum Time (min) B) 1 A A A A A A A A A S51

52 C) 25 Intensity (AU) hsum D) hsum-1 8 Theoretical profile Experimental profile deconvoluted Fig. S24. Characterization of hsum-1 protein 8. A) LC trace from LC-MS analysis: C3 Zorbax column (5 µm, 3 Å, 4.6 mm 15 mm), 5 C. Flow 1 ml/min, eluent A.1% trifluoroacetic acid in water, S52