Supplementary material

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-Supplementary material

826.5: b₆ b₅ b₄ a₆ a₅ y₂ y₇2+ b₇2+ b₆2+ y₃/a₆2+ y₄ b₂ Dac_{R V Y I H P F} b2 b4b5b6b7 y2 y4 y7 y3 235.1 263.1 364.3 413.8 466.3 535.9 576.4 669.5 689.5 782.6 798.6 827.5 0.0 0.5 1.0 1.5 2.0 x105 200 300 400 500 600 700 800 900 1000 1100 m/z

Figure S-1: MS/MS spectrum of doubly-charged AII1 (m/z 545.0, 31.7 min) from the sample

of acetylated angiotensin II without subsequent treatment in a boiling water bath. The acetylated N-terminal residue is marked with “ac”. The b6 ion (m/z 826.5), most intense ion of

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2 / 15 -868.5: b₆ y₂ b₅ a₆ a₅ b₄ y₃ y₅ y₄ b₃ b₇2+ y₆ b₂ b₆2+ a₆2+ y₇2+ Dac_{R V Y}ac_{I H P F} b2b3 b4b5b6b7 y2 y3 y4 y5 y6 y7 263.1 364.2 434.7 487.3 556.9 618.4 711.5 731.5 824.5 840.5 869.5 0.0 0.5 1.0 1.5 x106 200 300 400 500 600 700 800 900 1000 1100 m/z

of acetylated angiotensin II without subsequent treatment in a boiling water bath. The acetylated residues are marked with “ac”. The b6 ion (m/z 868.5), most intense ion of the

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3 / 15 -y₂ y₃ y4 y₅ y₆ y₇2+ 910.6: b₆ b₃/b₆2+ b₅ a₆ a₅ b₄ b₂ 235.2 263.1 356.0 381.8 400.3 455.8478.8 508.3 532.4 578.0 599.3 616.4 660.4689.6 745.5 773.4 817.6 866.7 882.5 911.6 0 1000 2000 3000 4000 5000 200 300 400 500 600 700 800 900 1000 1100 m/z Dac_Rac_{V Y}ac_{I H P F} b2 b3 b4b5b6 y2 y3 y4 y5 y6 y7 R1 N H R2 O N H N H NH2 R1 N H R2 O N H N H NH O Arginine Acetylated arginine

of acetylated angiotensin II without subsequent treatment in a boiling water bath. The acetylated residues are marked with “ac”. The b6 ion (m/z 910.6), most intense ion of the

spectrum, is truncated (ion counts: 1.5 × 104). We propose a structure of acetylated arginine as an insert.

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4 / 15 -868.5: b₆ y₂ b₅ a₆ a₅ b₄ y₃ y4 b₇2+ b₆2+ y₇2+ 235.0 263.1 364.2 434.9 487.3 556.9 618.3 647.4 683.5 711.5 731.5 775.5 809.5 824.5 840.6 869.6 1172.4 0.0 0.5 1.0 1.5 2.0 2.5 200 300 400 500 600 700 800 900 1000 1100 m/z x104 Dac_{R V Y}ac_{I H P F} b4b5b6b7 y2 y3 y4 y7

of acetylated angiotensin II after 15 min of treatment in a boiling water bath. The acetylated residues are marked with “ac”. The b6 ion (m/z 868.5), most intense ion of the spectrum, is

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5 / 15 -b₈ b₇ b₆ a₇ b₈2+ b₅ b₄ b₃ b₂ a₃ a₂ y₈ a₈ y₇ y₆ y₅ y₄ y₃ b₉2+ y₈2+ y₉2+ <E H W Sac_{Y K}ac_{L R P G}_-NH 2 b2b3 b4 b5 b6 b7b8 y3 y4 y5 y6 y7 b9 y8 y9 220.9 249.0 328.2 435.2 504.2 545.3 583.9 611.5 660.6 744.4 774.6 897.5 918.6 1010.6 1089.7 1166.7 0 1 2 3 4 5 200 300 400 500 600 700 800 900 1000 1100 m/z x104

Figure S-5: MS/MS spectrum of doubly-charged K6-LHRH2 (m/z 669.5, 29.3 min) from the

sample of acetylated (D-Lys6)-luteinizing hormone-releasing hormone without subsequent treatment in a boiling water bath. The acetylated residues are marked with “ac”.

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6 / 15 -b₇ b₆ a₇ b₈2+ b₅ b₄ b₃ b₂ a₃ a₂ y₈ y₇ y₆ y₅ y₄ y₃ b₉2+ y₈2+ a₆ y₆- 42 a₈2+ 221.0 249.0 328.2 435.3 566.4 604.9 660.4 681.4 709.4 774.5 816.6 885.6 939.5 1024.6 1052.6 1131.7 0.0 0.5 1.0 1.5 200 300 400 500 600 700 800 900 1000 1100 m/z x105 <E H W Sac_Yac_Kac_{L R P G}_-NH 2 b2b3 b4 b5 b6 b7b8 y3 y4 y5 y6 y7 b9 y8

sample of acetylated (D-Lys6)-luteinizing hormone-releasing hormone without subsequent treatment in a boiling water bath. The acetylated residues are marked with “ac”. The peak with the m/z of y6 minus 42 Da is likely the result of in-source fragmentation of the tyrosyl acetate.

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7 / 15 -249.1 371.3 435.2 483.3 503.9 565.5 605.3 626.3 653.4 682.0 702.3 745.5 859.7 939.5 987.8 1023.9 1052.6 1173.8 0 500 1000 1500 200 300 400 500 600 700 800 900 1000 1100 m/z y₃ b₂ b₃ b₄ b₆ b₇ b₈2+ y₄ y₅ <E H W Sac_Yac_Kac_{L R}ac_{P G}_-NH 2 b2b3 b4 b6 b7b8 y3 y4 y5

sample of acetylated (D-Lys6)-luteinizing hormone-releasing hormone without subsequent treatment in a boiling water bath. The acetylated residues are marked with “ac”.

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8 / 15 -b₇ b₆ a₇ b₈2+ b₅ b₄ b₃ b₂ a₂ y₈ y₇ y₆ y₄ y₃ y₅/b₉2+ y₈2+ b₈ 249.1 328.2 441.4 524.6 562.9 611.5 639.4 667.4 690.5 774.6 855.5 968.6 1047.7 1124.7 0.0 0.2 0.4 0.6 0.8 1.0 1.2 200 300 400 500 600 700 800 900 1000 1100 m/z x105 <E H W S Y Kac_{L R P G}_-NH 2 b2b3 b4b5 b6 b7b8 y3 y4 y5 y6 y7 b9 y8

sample of acetylated (D-Lys6)-luteinizing hormone-releasing hormone after 15 min of treatment in a boiling water bath. The acetylated lysine residue is marked with “ac”.

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9 / 15 -b₇ y₈ b₈ y₇ b₂ a₂ b₃ a₃ y₄ b₄ y₈2+ y₆ a₇ b₈2+ y₃ y₅ b₅ <E H W dA Y Kac_{L R P G}_-NH 2 b2b3 b4 b5 b7b8 y3 y4 y5 y6 y7 y8 221.0 249.0 328.2 407.3 435.2 504.2 553.9 611.5 630.6 667.3 774.5 843.6 950.6 1029.7 1106.7 0.00 0.25 0.50 0.75 1.00 1.25 200 300 400 500 600 700 800 900 1000 1100 m/z N H O O O R1 R2 O N H O O R1 R2 Dehydroalanine (dA) ß-elimination Acetylated serine ×105

Figure S-9: MS/MS spectrum of doubly-charged K6-LHRH1* (m/z 639.4, 29.4 min) from the

sample of acetylated (D-Lys6)-luteinizing hormone-releasing hormone after 60 min of treatment in a boiling water bath. The acetylated lysine residue is marked with “ac”. ‘dA’ stands for ‘dehydroalanine’. The structures of acetylated arginine and dehydroalanine are proposed as an insert.

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-Note S-10: Occurrence in proteins of hydroxyl-containing amino acids in an “activating” position. The occurrence in proteins of His, Ser, Tyr and Thr is respectively 2.2%, 7.1%, 3.2% and 5.7%1. Therefore the occurrence of a His-Xaa-OHaa or OHaa-Xaa-His triad (where OHaa is Ser, Tyr or Thr and Xaa may be any amino acid) is calculated as: 2 × 0.022 × (0.071 + 0.032 + 0.057) = 0.00704. Therefore such a triad may be statistically expected every 0.00704-1 (i.e. about 142) amino acids.

1. Clementi, C.; Carloni, P.; Maritan, A., Protein design is a key factor for subunit-subunit association. Proc Natl Acad Sci U S A 1999, 96, (17), 9616-21.

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11 / 15 -261.2 319.2 381.5 465.8 489.4 558.3 586.4 642.4 699.8 756.2 794.1 832.1 890.2 910.2 946.0 981.7 1031.3 1088.7 1122.1 0.00 0.25 0.50 0.75 1.00 1.25 200 300 400 500 600 700 800 900 1000 1100 m/z L Kac_{P L A Q S H A T K}ac_{H K}ac_{I P I K}ac_Yac_{L E} y2 y3 y4 y6 b2 b3 b15b16 b17 b18 y₂ b₁₈3+ y₃ y4 b₂ b₃ b₁₉3+ b₁₆2+ b₁₇2+ b₁₈2+ y₁₈2+ ×105

Figure S-11: MS/MS spectrum of triply-charged peptide

(LKacPLAQSHATKacHKacIPIKacYacLE, m/z 843.0, 58.0 min) from a sample of acetylated horse heart myoglobin prior to treatment in a boiling water bath. The acetylated residues are marked with “ac”.

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12 / 15 -261.1 332.2 _529.3 603.4 732.5 758.0 775.9 808.5 834.4 877.0 926.3 954.0 976.2 1016.6 1040.0 1077.9 0.25 0.50 0.75 1.00 1.25 x106 200 300 400 500 600 700 800 900 1000 1100 m/z b₃₀3+ /y₃₀3+ b₂₈3+ b₂₇3+ b₂₆3+ b₂₅3+ b₂₄3+ b₂₃3+ b₂₂3+ b₂/y₂ y₃ y₄ y₅ y₆ y₂₈3+ b₂₉3+ /y₂₉3+ b₃₀4+ /y₃₀4+ b₂₉4+ /y₂₉4+ F I S D A I I H V L H S Kac_{H P G D F G A D A Q G A M}ox_{T K}ac_{A L E} y2 y3 y4 y5 y6 b30 b29 b27 b25 b26 b24 b23 b22 y29 y30 y28 b28

Figure S-12: MS/MS spectrum of quadruply-charged peptide

(FISDAIIHVLHSKacHPGDFGADAQGAMoxTKacALE, m/z 845.5, 61.5 min) from a sample of acetylated horse heart myoglobin after 60 min of incubation in a boiling water bath. The acetylated residues are marked with “ac”, and the oxidized methionine is marked with “ox”.

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13 / 15 -×105 261.2 332.1 604.3 713.6 781.7 819.0 842.4 891.2 914.7 938.3 979.6 1054.0 1091.8 0 1 2 3 4 5 6 200 300 400 500 600 700 800 900 1000 1100 m/z b₃₀3+ /y₃₀3+ b₂₉3+ /y₂₉3+ b₃₀4+ /y₃₀4+ b₂₉4+ /y₂₉4+ b₂₈3+ b₂₇3+ b₂₆3+ b₂₅3+ b₂₃3+ b₂₂3+ b₂₁3+ b₂/y₂ y3 b₂₈4+ y₂₈4+ y₆ y₈ F I S D A I I H V L H Sac_Kac_{H P G D F G A D A Q G A M}ox_{T K}ac_{A L E} y2 y3 y8 y6 b30 b29 b27 b25 b26 b23 b22 y29 y30 y28 b28

(FISDAIIHVLHSacKacHPGDFGADAQGAMoxTKacALE, m/z 856.0, 62.6 min) from a sample of acetylated horse heart myoglobin after 60 min of incubation in a boiling water bath. The acetylated residues are marked with “ac”, and the oxidized methionine is marked with “ox”.

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14 / 15 -A E Mox_Kac_{A S E D L K}ac_Kac_{H G T V V L T A L G G I L I K}ac_Kac_Kac_{G H H E A E} y3 y4 y6 y₃₀ y₂₉ y₃₁ b3 290.1 320.2 348.3 365.2 394.4 429.2 461.3 485.3 518.5 546.4_571.3 589.3 615.3 644.4 698.0 737.0 766.2 795.6 826.0 840.7 900.2 933.9 959.0 997.7 1041.0 1069.6 1092.9 1150.3 0 1 2 3 4 200 300 400 500 600 700 800 900 1000 1100 m/z y₃₁3+ y₃₀3+ y₂₉3+ y₂₈3+ y₂₇3+ y₂₆3+ y₂₈ y₂₇y₂₆ y₂₅3+ y₂₅ y₂₂3+ y₂₄ y₂₃ y₂₃3+y243+ y₂₂ b₃/y₃ y₄ y₆ b₄ b₅ b4 b5 5 ×105

(AEMoxKacASEDLKacKacHGTVVLTALGGILIKacKacKacGHHEAE, m/z 949.8, 68.6 min) from a sample of acetylated horse heart myoglobin after 60 min of incubation in a boiling water bath. The acetylated residues are marked with “ac”, and the oxidized methionine is marked with “ox”.

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15 / 15 -327.2 417.3 474.2 513.3 672.4 726.5 828.0 873.5 915.4 941.8 976.9 991.2 1043.7 1142.6 0 1 2 3 4 200 300 400 500 600 700 800 900 1000 1100 m/z b₁₇2+ b₁₆2+ b₁₀ y₃ y₄ b₆ b₁₄2+ y₅ _b 132+ b7 y₆ b₈ y₇ b₉ ac_{G L S D G E W Q Q V L N V W G K}ac_{V E} y3 y4 y5 y6 y7 b9b10 b13b14 b16 b17 b6 b7 b8 ×105

Figure S-15: MS/MS spectrum of doubly-charged peptide

(acGLSDGEWQQVLNVWGKacVE, m/z 1065.0, 81.7 min) from a sample of acetylated horse heart myoglobin after 60 min of incubation in a boiling water bath. The acetylated residues are marked with “ac”.