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 , P.R. China ‡ Department of Biochemistry and Molecular biology & Tianjin Key Laboratory of Medical Epigenetics, Tianjin Medical University, Tianjin , P.R. China § National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian , P.R. China

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.

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 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 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.

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 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 ( ) Da and +108 ( ) Da of tryptophan as variable modifications.

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

Table S-2 Tryptophan modified peptides identified in yeast kinases Gene nameModified peptideMod.AAMass shift (+ΔDa) Specturm Num. Retention time (min.) Sps1pADIWSLGITTYELLKW GKEFWNFESTRW Kkq8pIFQWEPRW LLDMQWMKW CDC15HVWINSTENVKW LSSYAPWSFEK W TAAFIWKW Ste20pTGEYTGLPEEWEKW Kdx1pGYITSIWYKW PKA1NHPWFKW EVVWEKW Nnk1pGNYELWSSPDAILTQNKW EVAIWRW

m/z Relative Abundance b2 y4 2+ -NH 3 b3 b b4-H 2 O b9 2+ y7 2+ -NH 3 b H 2 O b7 2+ -NH y11 2+ y5-NH 3 y6-NH 3 y [M+2H] 2+ y7-NH 3 y7 y8-NH y8 y9-NH 3 y b y10-NH 3 y11-NH 3 y11 y12-NH 3 y13-NH [M+2H] ++ = W* = W + 92 W** = W + 4 (kynurenine) 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.

m/z y2-NH 3 b y3 b3-H 2 O b3 b4-H 2 O y4 b5 y b NH3 [M+2H] 2+ y6 y7 y8 y9 y10 m/z 100 [M+2H] ++ = W* = W Cc = C + Carbamidomethyl 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.

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

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)

MS/MS spectra of tryptophan-modified peptides

Sequence: ADIWSLGITTYELLK, Charge: +2, Monoisotopic m/z: Da, q-Value: 0, PEP: , MH+: Da, RT: min

Sequence: ADIWSLGITTYELLK, W4-W+32 ( Da) Charge: +2, Monoisotopic m/z: Da, q-Value: 0, PEP: , MH+: Da, RT: min

Sequence: ADIWSLGITTYELLK, W4-W+108 ( Da) Charge: +2, Monoisotopic m/z: Da, q-Value: 0, PEP: , MH+: Da, RT: min

Sequence: GKEFWNFESTR, Charge: +3, Monoisotopic m/z: Da, q-Value: 0, PEP: , MH+: Da, RT: min

Sequence: GKEFWNFESTR, W5-W+108 ( Da) Charge: +3, Monoisotopic m/z: Da, q-Value: 0, PEP: , MH+: Da, RT: min

Sequence: IFQWEPR, Charge: +2, Monoisotopic m/z: Da, q-Value: 0, PEP: , MH+: Da, RT: min

Sequence: IFQWEPR, W4-W+32 ( Da) Charge: +2, Monoisotopic m/z: Da, q-Value: 0, PEP: , MH+: Da, RT: min

Sequence: IFQWEPR, W4-W+108 ( Da) Charge: +2, Monoisotopic m/z: Da, q-Value: 0, PEP: , MH+: Da, RT: min

Sequence: LLDMQWMK, M4-Oxidation ( Da), M7-Oxidation ( Da) Charge: +2, Monoisotopic m/z: Da, q-Value: 0, PEP: , MH+: Da, RT: min

Sequence: LLDMQWMK, M4-Oxidation ( Da), W6-W+16 ( Da), M7-Oxidation ( Da) Charge: +2, Monoisotopic m/z: Da, q-Value: 0, PEP: , MH+: Da, RT: min

Sequence: LLDMQWMK, M4-Oxidation ( Da), W6-W+108 ( Da), M7-Oxidation ( Da) Charge: +2, Monoisotopic m/z: Da, q-Value: 0.007, PEP: , MH+: Da, RT: min

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

Sequence: HVWINSTENVK, W3-W+16 ( Da) Charge: +2, Monoisotopic m/z: Da, q-Value: 0, PEP: , MH+: Da, RT: min

Sequence: HVWINSTENVK, W3-W+32 ( Da) Charge: +2, Monoisotopic m/z: Da, q-Value: 0, PEP: , MH+: Da, RT: min

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

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

Sequence: LSSYAPWSFEK, Charge: +2, Monoisotopic m/z: Da, q-Value: 0, PEP: , MH+: Da, RT: min

Sequence: LSSYAPWSFEK, W7-W+4 ( Da) Charge: +2, Monoisotopic m/z: Da, q-Value: 0, PEP: , MH+: Da, RT: min

Sequence: LSSYAPWSFEK, W7-W+108 ( Da) Charge: +2, Monoisotopic m/z: Da, q-Value: 0, PEP: , MH+: Da, RT: min

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

Sequence: TAAFIWK, W6-W+92 ( Da) Charge: +2, Monoisotopic m/z: Da, q-Value: 0, PEP: , MH+: Da, RT: min

Sequence: TAAFIWK, W6-W+108 ( Da) Charge: +2, Monoisotopic m/z: Da, q-Value: 0, PEP: , MH+: Da, RT: min

Sequence: TGEYTGLPEEWEK, Charge: +2, Monoisotopic m/z: Da, q-Value: 0, PEP: , MH+: Da, RT: min

Sequence: TGEYTGLPEEWEK, W11-W+16 ( Da) Charge: +2, Monoisotopic m/z: Da, q-Value: 0, PEP: , MH+: Da, RT: min

Sequence: TGEYTGLPEEWEK, W11-W+32 ( Da) Charge: +2, Monoisotopic m/z: Da, q-Value: 0, PEP: , MH+: Da, RT: min

Sequence: TGEYTGLPEEWEK, W11-W+108 ( Da) Charge: +2, Monoisotopic m/z: Da, q-Value: 0, PEP: , MH+: Da, RT: min

Sequence: GYITSIWYK, W7-W+16 ( Da) Charge: +2, Monoisotopic m/z: Da, q-Value: 0, PEP: , MH+: Da, RT: min

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

Sequence: GYITSIWYK, W7-W+92 ( Da) Charge: +2, Monoisotopic m/z: Da, q-Value: 0, PEP: , MH+: Da, RT: min

Sequence: GYITSIWYK, W7-W+108 ( Da) Charge: +2, Monoisotopic m/z: Da, q-Value: 0, PEP: , MH+: Da, RT: min

Sequence: NHPWFK, Charge: +2, Monoisotopic m/z: Da, q-Value: 0.002, PEP: , MH+: Da, RT: min

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

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

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

Sequence: NHPWFK, W4-W+108 ( Da) Charge: +2, Monoisotopic m/z: Da, q-Value: 0, PEP: , MH+: Da, RT: min

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

Sequence: EVVWEK, W4-W+92 ( Da) Charge: +2, Monoisotopic m/z: Da, q-Value: 0.005, PEP: , MH+: Da, RT: min

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

Sequence: GNYELWSSPDAILTQNK, W6-W+4 ( Da) Charge: +2, Monoisotopic m/z: Da, q-Value: 0, PEP: , MH+: Da, RT: min

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

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

Sequence: EVAIWR, Charge: +2, Monoisotopic m/z: Da, q-Value: 0, PEP: , MH+: Da, RT: min

Sequence: EVAIWR, W5-W+32 ( Da) Charge: +2, Monoisotopic m/z: Da, q-Value: 0, PEP: MH+: Da, RT: min

Sequence: EVAIWR, W5-W+108 ( Da) Charge: +2, Monoisotopic m/z: Da, q-Value: 0, PEP: , MH+: Da, RT: min