1. Supporting Information Identification of Two Novel Types of Protein Tryptophan Modifications Shuzhen Zheng, † Kai Zhang,*,†, ‡ Shanshan Tian, ‡ Xiwen He, † Yukui Zhang*, †, § † Department of Chemistry, Nankai University, Tianjin 300071, P.R. China ‡ Department of Biochemistry and Molecular biology & Tianjin Key Laboratory of Medical Epigenetics, Tianjin Medical University, Tianjin 300070, P.R. China § National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, P.R. China

2. Experimental Section Preparation and Separation of Protein Kinase. Expression and purification of protein kinase were carried out as described [9]. Briefly, protein kinase genes were cloned into a high-copy URA3 expression vector which produced GST fusion protein. Thirty yeast expression stains containing plasmids of interest in which the correct yeast ORFs were fused in-frame into GST were isolated. The proteins were overexpressed in yeast, and were purified from yeast extracts using glutathione-agarose beads. The recombinant fusion proteins were eluted from GST-agarose beads in a sample-loading buffer (20Mm Tris-HCL (PH 6.8), 3% glycerol, 2% β-mercaptoethanol, 1%SDS, 0.03% brompherol blue). The supernatants were collected after centrifugation and dried down to 30ul in a Speed-Vac prior to SDS-PAGE. The purified GST-kinases were further resolved by SDS-PAGE and visualized by colloidal coomassie staining. SDS-PAGE separation and in-gel digestion The proteins were resolved in a 15% SDS-PAGE gel and visualized by colloidal coomassie blue staining. Gel bands of interest were excised and subjected to in-gel digestion as described previously [13].Briefly, the gel bands were sliced into small pieces (~1 mm 3 ) and destained with 25 mM ammonium bicarbonate in ethanol/water (50:50, v/v). The destained gel pieces were washed in an acidic buffer (acetic acid/ethanol/water, 10:50:40, v/v/v) three times for 1 h each time, and in water two times for 20 min each time. The gel pieces were dehydrated in acetonitrile and dried in a Speed-Vac (Thermo Fisher, Waltham, MA). Two hundred nanograms of porcine modified trypsin (Promega, Madison, WI) in 50 mM ammonium bicarbonate was added to the dried gels and incubated overnight at 37 °C. Tryptic peptides were sequentially extracted from the gel pieces with 50% acetonitrile (acetonitrile/water/TFA, 50:45:5, v/v/v) and 75% acetonitrile (acetonitrile/water/TFA, 75:24:1, v/v/v). The peptide extracts were pooled and dried in a Speed-Vac. The peptide extracts were desalted using a µ-C18 Zip-tip before HPLC/MS/MS analysis.

3. Lysozyme in-gel digestion. The 100 pM lysozyme sample was divided in 2 aliquots × 10 uL, of which one was incubated for 10 min at 95 ℃ with 10 uL sample loading buffer containing (20 mM Tris-HCL (pH 6.8), 3% glycerol, 1% SDS, 0.03% bromophenol blue, and 2% β-mercaptoethanol) and the other was performed as above except for β- mercaptoethanol. The treated lysozyme samples were separated by SDS-PAGE. SDS-PAGE gels were stained with Coomassie Simply Blue solution. Gel bands of interest were excised and subjected to in-gel digestion as described previously [9]. Lysozyme in-solution digestion. The 100 pM lysozyme was dissolved in 50 uL of 100 mM ammonium bicarbonate. The sample was reduced for 45 min at 56 ℃ with 50 mM DTT, following alkylation for 30 min at room temperature in the dark. Trypsin as an enzyme: substrate ratio of 1:50, and sufficient ammonium carbonate to maintain a pH of 7.4 was added to the reduced and alkylated sample. Incubate the digestion overnight at 37 ℃ and stop the reaction by adding 10 uL 0.1% TFA to make the pH of the solution below pH 3. The mimic experiment. The procedural details were as follows: (1) dissolved 1.5 mg synthetic peptide (TGEYTGLPEEWEK) in 1.5 mL deionized water and then divided the stock solution in 2 aliquots × 500 uL; (2) slowly drip the 30% H 2 O 2 100 uL into one of the above solutions, stirring reaction for 2 h in room temperature; (3) for the other, slowly drip the 30% H 2 O 2 200 uL into the solution, stirring reaction for 2 h at room temperature and continue slowly drip β-mercaptoethanol 100 uL into the solution, stirring reaction for 2 h at room temperature. The products were detected by high resolution MS. MS spectra of peptide TGEYTGLPEEWEK and reaction products were shown in Figure S-3.

4. Mass spectrometry and data analysis. The resulting peptides were cleaned with C18 ZipTips and dissolved in 10 µL of HPLC buffer A (0.1% (v/v) formic acid in water), and 2 µL was injected into a Nano-LC system (EASY-nLC 1000, Thermo Fisher Scientific, Waltham, MA). Peptides were separated on a homemade capillary HPLC column (100-mm length×75-µm inner diameter) containing Jupiter C12 resin (4-µm particle size, 90-Å pore diameter, Phenomenex, St. Torrance, CA) with a 120 min HPLC-gradient from 5 to 90% HPLC buffer B (0.1% formic acid in acetonitrile) at a flow rate of 200 nL/min. The HPLC elute was electrosprayed directly into an LTQ-Orbitrap Discovery mass spectrometer (Thermo Fisher Scientific, Waltham, MA) using a nanospray source. The LTQ-orbitrap mass spectrometer was operated in a data-dependent mode with resolution R = 60,000 at m/z 400. Full scan MS spectra from m/z 300 – 2000 were acquired in the Orbitrap. The ten most intense ions were sequentially isolated in the linear ion trap and subjected to collision-activated dissociation (CAD) with a normalized energy of 35%. The exclusion duration for the data-dependant scan was 36 sec, the repeat count was 2, and the exclusion window was set at ±2 Da. AGC settings were 1E6 for full scan Orbitrap analysis, 1E4 for MSn scan in the ion trap and 6E4 for MSn scan in the Orbitrap. Signal threshold for CID acquisition was set at 5000. The resulting MS/MS data were searched against the Saccharomyces cerevisiae entries of NCBInr protein sequence database using Proteome Discoverer (v1.4) with an overall false discovery rate (FDR) for peptides of less than 1%. Trypsin was specified as the proteolytic enzyme, and up to 2 missed cleavage sites per peptide were allowed. Carbamidomethylation of cysteine was set as a fixed modification and hydroxylation, di-hydroxylation, kynurenine, +92 (91.9927) Da and +108 (107.9876) Da of tryptophan as variable modifications.

5. Table S-1. A list of peptides with a series of mass shift at tryptophan identified in yeast kinases △ m/z ModificationCorresponding product(s)Reference +4+O-CKynurenine[7, 8] +16+OHydroxytryptophan[11, 12] +32+2ONFK/dihydroxytryptophan[7, 8, 11, 12] +12+CTetrahydro-β -carboline[10] +28+2O-4H β-Unsaturated-2,4-bis- tryptophandione [13] +48+3OHydroxyformylkynurenine[14,15] +64+4ODihydroxyformylkynurenine[15] +92 unknown +108 unknown

6. Table S-2 Tryptophan modified peptides identified in yeast kinases Gene nameModified peptideMod.AAMass shift (+ΔDa) Specturm Num. Retention time (min.) Sps1pADIWSLGITTYELLKW 0676.04 32170.58 108478.12 GKEFWNFESTRW 0347.51 108149.12 Kkq8pIFQWEPRW0346.89 32137.63 108149.38 LLDMQWMKW0244.31 16140.41 108246.38 CDC15HVWINSTENVKW01750.80 16111.79 32145.56 92110.51 1083635.76 LSSYAPWSFEK W0450.49 4148.34 108351.65 TAAFIWKW0244.38 92143.05 108247.27 Ste20pTGEYTGLPEEWEKW0144.70 32139.53 92142.01 108345.64 Kdx1pGYITSIWYKW0245.99 16343.49 32140.32 92142.27 108247.67 PKA1NHPWFKW0346.63 1628.74 3217.44 9228.69 108513.83 EVVWEKW01025.17 92110.63 Nnk1pGNYELWSSPDAILTQNKW0244.27 4242.99 16342.58 108245.06 EVAIWRW0231.98 32411.81 108234.79

7. 300400500600700800900100011001200130014001500 m/z 0 100 Relative Abundance 260.09 981.48 796.40 1169.54 1542.65 867.43 626.28 1340.59 1186.55 1112.51 884.44 491.27 998.49 300.19 1357.60 610.24 415.21 1455.61 213.16 b2 y4 2+ -NH 3 b3 b4 397.21 b4-H 2 O 444.24 b9 2+ y7 2+ -NH 3 b11 2+ -H 2 O b7 2+ -NH 3 679.73 y11 2+ y5-NH 3 y6-NH 3 y6 886.91 [M+2H] 2+ y7-NH 3 y7 y8-NH 3 1129.49 y8 y9-NH 3 y9 1237.55 b11 1283.56 y10-NH 3 y11-NH 3 y11 y12-NH 3 y13-NH 3 886.9887.1887.3887.5887.7887.9 0 100886.9099 887.4135 887.9120 [M+2H] ++ =886.9099 W* = W + 92 W** = W + 4 (kynurenine) 813.37 y5 Relative Abundance m/z Figure S-1a. An in vitro-modified peptide with a mass shift of +92 at tryptophan in lysozyme which was resolved by SDS-PAGE and subjected to in-gel digestion.

8. 200300400500600700800900100011001200 m/z 0 100 290.11 449.22 308.12 548.28 1013.40 717.30 956.37 403.20 1213.51 1126.48 478.22 842.33 221.09 201.12 y2-NH 3 b2 289.19 y3 b3-H 2 O b3 b4-H 2 O y4 b5 y5 600.26 b10 2+ -NH3 [M+2H] 2+ y6 y7 y8 y9 y10 m/z 100 [M+2H] ++ =717.3039 W* = W + 108 Cc = C + Carbamidomethyl 717.3717.5717.7717.9718.1718.3 0 100 717.3039 717.8056 718.3059 Relative Abundance m/z Figure S-1b. An in vitro-modified peptide with a mass shift of +108 at tryptophan in lysozyme which was resolved by SDS-PAGE and subjected to in-gel digestion.

9. (a) W + 92 Da (b) W + 108 Da Figure S-2. Three dimensional views of the W+92 Da (a) and W+108Da (b) by using ChemBio3D ultra 11.0. Gray, carbon; white, hydrogen; red, oxygen; yellow, sulfur; blue, nitrogen; pink, lone pair.

10. Figure S-3. (a) MS of TGEYTGLPEEWEK, (b) MS of product of TGEYTGLPEEWEK with H 2 O 2, (c) MS of product of TGEYTGLPEEWEK with H 2 O 2 and β-mercaptoethanol. (a) (b) (c)

11. MS/MS spectra of tryptophan-modified peptides

12. Sequence: ADIWSLGITTYELLK, Charge: +2, Monoisotopic m/z: 862.30939 Da, q-Value: 0, PEP: 0.0007088, MH+: 1723.61150 Da, RT: 76.04 min

13. Sequence: ADIWSLGITTYELLK, W4-W+32 (31.98928 Da) Charge: +2, Monoisotopic m/z: 877.75610 Da, q-Value: 0, PEP: 0.00227, MH+: 1754.50493 Da, RT: 70.58 min

14. Sequence: ADIWSLGITTYELLK, W4-W+108 (107.98757 Da) Charge: +2, Monoisotopic m/z: 915.81860 Da, q-Value: 0, PEP: 0.0001112, MH+: 1830.62993 Da, RT: 78.12 min

15. Sequence: GKEFWNFESTR, Charge: +3, Monoisotopic m/z: 467.55585 Da, q-Value: 0, PEP: 0.006215, MH+: 1400.65299 Da, RT: 47.51 min

16. Sequence: GKEFWNFESTR, W5-W+108 (107.98757 Da) Charge: +3, Monoisotopic m/z: 503.54074 Da, q-Value: 0, PEP: 0.01891, MH+: 1508.60767 Da, RT: 49.12 min

17. Sequence: IFQWEPR, Charge: +2, Monoisotopic m/z: 488.23187 Da, q-Value: 0, PEP: 0.02498, MH+: 975.45647 Da, RT: 46.89 min

18. Sequence: IFQWEPR, W4-W+32 (31.98928 Da) Charge: +2, Monoisotopic m/z: 504.17764 Da, q-Value: 0, PEP: 0.01306, MH+: 1007.34801 Da, RT: 37.63 min

19. Sequence: IFQWEPR, W4-W+108 (107.98757 Da) Charge: +2, Monoisotopic m/z: 542.17847 Da, q-Value: 0, PEP: 0.0003571, MH+: 1083.34966 Da, RT: 49.38 min

20. Sequence: LLDMQWMK, M4-Oxidation (15.99492 Da), M7-Oxidation (15.99492 Da) Charge: +2, Monoisotopic m/z: 548.74713 Da, q-Value: 0, PEP: 0.00008067, MH+: 1096.48699 Da, RT: 44.31 min

21. Sequence: LLDMQWMK, M4-Oxidation (15.99492 Da), W6-W+16 (15.99491 Da), M7-Oxidation (15.99492 Da) Charge: +2, Monoisotopic m/z: 556.72192 Da, q-Value: 0, PEP: 0.01346, MH+: 1112.43657 Da, RT: 40.41 min

22. Sequence: LLDMQWMK, M4-Oxidation (15.99492 Da), W6-W+108 (107.98757 Da), M7-Oxidation (15.99492 Da) Charge: +2, Monoisotopic m/z: 602.79639 Da, q-Value: 0.007, PEP: 0.1403, MH+: 1204.58550 Da, RT: 46.38 min

23. Sequence: HVWINSTENVK, Charge: +2, Monoisotopic m/z: 664.02179 Da, q-Value: 0, PEP: 2.367E-09, MH+: 1327.03630 Da, RT: 50.80 min

24. Sequence: HVWINSTENVK, W3-W+16 (15.99491 Da) Charge: +2, Monoisotopic m/z: 671.83594 Da, q-Value: 0, PEP: 0.001534, MH+: 1342.66460 Da, RT: 11.79 min

25. Sequence: HVWINSTENVK, W3-W+32 (31.98928 Da) Charge: +2, Monoisotopic m/z: 679.83020 Da, q-Value: 0, PEP: 0.01091, MH+: 1358.65312 Da, RT: 45.56 min

26. Sequence: HVWINSTENVK, W3-W+92 (91.99265 Da) Charge: +2, Monoisotopic m/z: 709.82141 Da, q-Value: 0, PEP: 2.13E-08, MH+: 1418.63555 Da, RT: 10.51 min

27. Sequence: HVWINSTENVK, W3-W+108 (107.98757 Da) Charge: +2, Monoisotopic m/z: 717.94366 Da, q-Value: 0, PEP: 1.346E-10, MH+: 1434.88005 Da, RT: 35.76 min

28. Sequence: LSSYAPWSFEK, Charge: +2, Monoisotopic m/z: 658.20361 Da, q-Value: 0, PEP: 0.000003535, MH+: 1315.39995 Da, RT: 50.49 min

29. Sequence: LSSYAPWSFEK, W7-W+4 (3.99492 Da) Charge: +2, Monoisotopic m/z: 659.84607 Da, q-Value: 0, PEP: 0.008041, MH+: 1318.68486 Da, RT: 48.34 min

30. Sequence: LSSYAPWSFEK, W7-W+108 (107.98757 Da) Charge: +2, Monoisotopic m/z: 711.86542 Da, q-Value: 0, PEP: 0.00001094, MH+: 1422.72356 Da, RT: 51.65 min

31. Sequence: TAAFIWK, Charge: +2, Monoisotopic m/z: 418.89261 Da, q-Value: 0, PEP: 7.339E-09, MH+: 836.77794 Da, RT: 44.38 min

32. Sequence: TAAFIWK, W6-W+92 (91.99265 Da) Charge: +2, Monoisotopic m/z: 464.95374 Da, q-Value: 0, PEP: 0.0004504, MH+: 928.90019 Da, RT: 43.05 min

33. Sequence: TAAFIWK, W6-W+108 (107.98757 Da) Charge: +2, Monoisotopic m/z: 472.83185 Da, q-Value: 0, PEP: 0.0106, MH+: 944.65642 Da, RT: 47.27 min

34. Sequence: TGEYTGLPEEWEK, Charge: +2, Monoisotopic m/z: 769.81067 Da, q-Value: 0, PEP: 0.000003181, MH+: 1538.61406 Da, RT: 44.70 min

35. Sequence: TGEYTGLPEEWEK, W11-W+16 (15.99491 Da) Charge: +2, Monoisotopic m/z: 777.83533 Da, q-Value: 0, PEP: 0.0001211, MH+: 1554.66338 Da, RT: 42.88 min

36. Sequence: TGEYTGLPEEWEK, W11-W+32 (31.98928 Da) Charge: +2, Monoisotopic m/z: 785.82727 Da, q-Value: 0, PEP: 0.001187, MH+: 1570.64726 Da, RT: 39.53 min

37. Sequence: TGEYTGLPEEWEK, W11-W+108 (107.98757 Da) Charge: +2, Monoisotopic m/z: 823.96204 Da, q-Value: 0, PEP: 0.004568, MH+: 1646.91680 Da, RT: 45.64 min

38. Sequence: GYITSIWYK, W7-W+16 (15.99491 Da) Charge: +2, Monoisotopic m/z: 573.79150 Da, q-Value: 0, PEP: 0.0002069, MH+: 1146.57573 Da, RT: 43.49 min

39. Sequence: GYITSIWYK, W7-W+32 (31.98928 Da) Charge: +1, Monoisotopic m/z: 1162.37427 Da, q-Value: 0, PEP: 0.02259, MH+: 1162.37427 Da, RT: 40.82 min

40. Sequence: GYITSIWYK, W7-W+92 (91.99265 Da) Charge: +2, Monoisotopic m/z: 611.82831 Da, q-Value: 0, PEP: 0.0006818, MH+: 1222.64934 Da, RT: 42.27 min

41. Sequence: GYITSIWYK, W7-W+108 (107.98757 Da) Charge: +2, Monoisotopic m/z: 619.87146 Da, q-Value: 0, PEP: 0.04319, MH+: 1238.73564 Da, RT: 47.67 min

42. Sequence: NHPWFK, Charge: +2, Monoisotopic m/z: 414.94394 Da, q-Value: 0.002, PEP: 0.03391, MH+: 828.88060 Da, RT: 46.63 min

43. Sequence: NHPWFK, W4-W+16 (15.99491 Da) Charge: +2, Monoisotopic m/z: 422.87964 Da, q-Value: 0.008, PEP: 0.2435, MH+: 844.75200 Da, RT: 8.74 min

44. Sequence: NHPWFK, W4-W+32 (31.98928 Da) Charge: +2, Monoisotopic m/z: 430.96167 Da, q-Value: 0, PEP: 0.01088, MH+: 860.91606 Da, RT: 7.44 min

45. Sequence: NHPWFK, W4-W+92 (91.99265 Da) Charge: +2, Monoisotopic m/z: 460.91656 Da, q-Value: 0, PEP: 0.003853, MH+: 920.82585 Da, RT: 8.69 min

46. Sequence: NHPWFK, W4-W+108 (107.98757 Da) Charge: +2, Monoisotopic m/z: 468.75671 Da, q-Value: 0, PEP: 0.004114, MH+: 936.50615 Da, RT: 13.83 min

47. Sequence: EVVWEK, Charge: +1, Monoisotopic m/z: 789.37073 Da, q-Value: 0, PEP: 0.004879, MH+: 789.37073 Da, RT: 25.17 min

48. Sequence: EVVWEK, W4-W+92 (91.99265 Da) Charge: +2, Monoisotopic m/z: 441.24750 Da, q-Value: 0.005, PEP: 0.0896, MH+: 881.48772 Da, RT: 10.63 min

49. Sequence: GNYELWSSPDAILTQNK, Charge: +2, Monoisotopic m/z: 968.88708 Da, q-Value: 0, PEP: 3.068E-08, MH+: 1936.76689 Da, RT: 44.27 min

50. Sequence: GNYELWSSPDAILTQNK, W6-W+4 (3.99492 Da) Charge: +2, Monoisotopic m/z: 970.41565 Da, q-Value: 0, PEP: 0.0002783, MH+: 1939.82402 Da, RT: 42.99 min

51. Sequence: GNYELWSSPDAILTQNK, W6-W+16 (15.99491 Da) Charge: +2, Monoisotopic m/z: 976.39929 Da, q-Value: 0, PEP: 1.034E-07 MH+: 1951.79131 Da, RT: 42.58 min

52. Sequence: GNYELWSSPDAILTQNK, W6-W+108 (107.98757 Da) Charge: +2, Monoisotopic m/z: 1022.79199 Da, q-Value: 0, PEP: 5.757E-10, MH+: 2044.57671 Da, RT: 45.06 min

53. Sequence: EVAIWR, Charge: +2, Monoisotopic m/z: 387.23477 Da, q-Value: 0, PEP: 0.003235, MH+: 773.46227 Da, RT: 31.98 min

54. Sequence: EVAIWR, W5-W+32 (31.98928 Da) Charge: +2, Monoisotopic m/z: 403.26974 Da, q-Value: 0, PEP: 0.001706 MH+: 805.53221 Da, RT: 11.81 min

55. Sequence: EVAIWR, W5-W+108 (107.98757 Da) Charge: +2, Monoisotopic m/z: 441.28604 Da, q-Value: 0, PEP: 0.001758, MH+: 881.56481 Da, RT: 34.79 min