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NL:7.73E8 729.0729.5730.0730.5731.0731.5732.0732.5733.0733.5 m/z 0 10 20 30 40 50 60 70 80 90 100 Relative Abundance 729.9020 730.4028 730.9041 731.4056.

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Presentation on theme: "NL:7.73E8 729.0729.5730.0730.5731.0731.5732.0732.5733.0733.5 m/z 0 10 20 30 40 50 60 70 80 90 100 Relative Abundance 729.9020 730.4028 730.9041 731.4056."— Presentation transcript:

1 NL:7.73E8 729.0729.5730.0730.5731.0731.5732.0732.5733.0733.5 m/z 0 10 20 30 40 50 60 70 80 90 100 Relative Abundance 729.9020 730.4028 730.9041 731.4056 731.9074 2+ Precursor ion Figure S1A YVPVSLC(fgly)TPSRAAL (45-58)(C51 as fgly) Theoretical monoisotopic mass = 729.9012 (2+) y 12 2+ 614.5 y6y6 1+ y7y7 y8y8 y 10 1+ y 12 1+ b 12 1+ 845.4 b8b8 1+ b 13 2+ [b 8 -H 2 O] 1+ CID-MS2 (using ion trap) (m/z 729.90, 2+) Figure S1B Y V PVS L C(fgly) T PSRA A L (45-58) b8b8 b 13 y6y6 y 12 y7y7 y8y8 NL:1.30E7 20030040050060070080090010001100120013001400 m/z 0 10 20 30 40 50 60 70 80 Relative Abundance 599.0 827.4 664.5 715.4 1196.6 1000.4 800.5 565.2 895.4743 482.30 235.21256.6 1065.6 360.2 1435.6

2 NL:3.19E7 738.0738.5739.0739.5740.0740.5741.0741.5742.0742.5743.0 m/z 0 10 20 30 40 50 60 70 80 90 100 Relative Abundance 738.9059 739.4073 739.9084 740.4108 2+ Precursor ion Figure S2A YVPVSLCTPSRAAL (45-58)(C51 as free cys) Theoretical monoisotopic mass = 738.8976 (2+) y6y6 1+ [M-H 2 O] 2+ [y 12 -H 2 O] 1+ [b 8 -2H 2 O] 1+ [y 10 -H 2 O] 1+ b2b2 [M-SH 2 ] 2+ [M-SCH 2 ] 2+ [y 12 -H 2 O] 2+ b 13 2+ [b 12 -H 2 O] 2+ b4b4 1+ NL:4.67E5 20030040050060070080090010001100120013001400 m/z 0 10 20 30 40 50 60 70 80 90 100 Relative Abundance 729.9 614.5 827.3 1196.6 1000.6 893.9 460.2 1309.7380.2 263.31418.9 CID-MS2 (using ion trap) (m/z 738.90, 2+) Figure S2B YVPV SLCT PSRAA L (45-58) b8b8 y6y6 y 12 b2b2

3 NL:2.92E7 511.8512.0512.2512.4512.6512.8513.0513.2513.4513.6513.8514.0514.2514.4514.6 m/z 0 10 20 30 40 50 60 70 80 90 100 Relative Abundance 512.2453 512.7463 513.2459 513.7459 2+ Precursor ion Figure S3ARMSRGGCSGL (270-279)(C276 as free cys) Theoretical monoisotopic mass = 512.2448 (2+) [M-H 2 O] 2+ b9b9 b4b4 b3b3 y9y9 1+ b8b8 [b 8 -H 2 O] 1+ b4b4 R MS R GG C S G L (270-279) CID-MS2 (using ion trap) (m/z 512.22, 2+) [b 7 -H 2 O] 1+ [b 6 -H 2 O] 1+ [M-SCH 2 ] 2+ c9c9 y9y9 b3b3 NL:1.10E6 2003004005006007008009001000 m/z 0 10 20 30 40 50 60 70 80 90 100 Relative Abundance 456.0 503.7 447.0 867.5 964.5 488.3 818.2 731.2 875.5 396.1 531.2 266.6 628.3 358.6 189.1 835.5 800.3947.3 704.5 893.9323.2 600.1 1027.7 Figure S3B

4 NL:1.07E8 1270.01271.01272.01273.01274.01275.0 m/z 0 20 40 60 80 100 Relative Abundance 1272.5826 1272.2495 1272.9169 1273.2507 1271.9153 1273.5851 1273.9186 Theoretical monoisotopic mass = 1271.9117 (3+) Monoisotopic mass Precursor ion Figure S4A NL:2.76E5 40050060070080090010001100120013001400150016001700180019002000 m/z 0 10 20 30 40 50 60 70 80 90 100 Relative Abundance 1248.8 1320.9 1443.0 829.9 931.7 1650.3 1184.1 1544.0 1142.0 787.9 1068.7 1375.2 517.6 1501.3 1720.1 1980.7 y 5 1+(P1) [b 11 -2H 2 O] 4+(P1) b 9 1+(P2) [y 16 -H 2 O] 3+(P2) y 13 3+(P1) 1239.8 [y 9 -H 2 O] 2+(P2) y 9 2+(P2) b 13 1+(P2) y 13 2+(P2) b 5 2+(P1) y 15 2+(P2) b 10 2+(P1) 1612.8 y 16 2+(P2) b 18 3+(P2) y 14 2+(P2) y 19 2+(P2) x5 CID-MS2 (using ion trap) (m/z 1272.58, 3+) Figure S4B 3+ b9b9 LRCGKGTTYEGGVRE (P1) (280-294) (C282) FTQGSAHSDTTADPACHASSSL(P2)(381-402) (C396) y9y9 b 18 y 19 b5b5 b 10 y5y5 y 13

5 NL:9.16E5 2004006008001000120014001600 18002000 m/z 0 10 20 30 40 50 60 70 80 90 100 Relative Abundance 1909.0 813.8 1267.4 1097.0 1684.5 451.3 1626.1 1843.9 1122.4 894.5 1298.5 1184.9 746.5 1462.2 1711.8 502.4 1594.8 630.3 995.5 394.3 266.2 1341.8 C 4 1+(P2) C 2 1+(P2) C 3 1+(P2) Z 5 1+(P1) Z 6 1+(P1) C 7 1+(P2) [P1] 2+ [P2] 2+ Z 8 1+(P1) [M-H 2 O] 3+ · · Z 10 2+(P2) C 7 2+(P1) Z 18 2+(P2) [M] 2+ · · · 1901.1 [M-NH 3 ] 2+ · · · Z 14 3+(P2) Z 20 3+(P2) x2 Z 19 2+(P2) C 14 2+(P1) 1777.5 Z 20 2+(P2) LRCGKGTTYEGGVRE (P1) (280-294) (C282) FTQGSAHSDTTADPACHASSSL(P2)(381-402) (C396) c2c2 z 10 c7c7 z 20 c7c7 c 14 z5z5 z8z8 Figure S4C ETD-MS 2 (using ion trap) (m/z 764.95, 5+)

6 FLGIPYSHDQGPCQNLTCFPPATPCDGGCDQGLVPIPLLANLSVEAQPPWLPGLEAR C138C143C150C154 Figure S5 Nested Disulfides with Correct and Scrambled Configurations in a Tryptic Peptide A (correct) FLGIPYSHDQGPCQNLTCFPPATPCDGGCDQGLVPIPLLANLSVEAQPPWLPGLEAR C138C143C150C154 B (scramble 1) FLGIPYSHDQGPCQNLTCFPPATPCDGGCDQGLVPIPLLANLSVEAQPPWLPGLEAR C138C143C150C154 C (scramble 2)

7 FLGIPYSHDQGPCQNLTCFPPATPCDGGCDQGLVPIPLLANLSVEAQPPWLPGLEAR C138C143C150C154 Figure S6 Digestion Strategy for Nested Disulfides A (Lys-C + trypsin) C138C143C150C154 B (Lys-C + trypsin + PNGase F) FLGIPYSH DQGPCQ DLTCFPPATPCDGGC DQGLVPIPLLA DLSVEAQPPWLPGLEAR C138C143C150C154 C (Lys-C + trypsin + PNGase F + Asp-N) FLGIPYSHDQGPCQDLTCFPPATPCDGGCDQGLVPIPLLADLSVEAQPPWLPGLEAR

8 Figure S7 Digestion Strategy for Nested Disulfides FLGIPYSH DQGPCQ DLTCFPPATPCDGGC DQGLVPIPLLA DLSVEAQPPWLPGLEAR C138C143C150C154 Lys-C + trypsin + PNGase F + Asp-N DQGPCQ DLTCFPPATPCDGGC C138C143C150C154 DQGPCQ DLTCFPPATPCDGGC C138 C143C150 C154

9 1494.9 DQGPCQ (p1) (134-139) D L T CFPPATPC D GGC (p2) (140-154) [M+3H] 2+ P1 1+ P2 1+ [M+3H] 3+ ETD-MS2 (using ion trap) (m/z 713.90, 3+) NL:7.67E3 714.5 1070.6 1426.1 647.4 1782.8 1368.8 915.5 1225.5 842.61881.5 283.3517.2429.4 1590.8202.2 Figure S8

10 y6y6 1+ y7y7 y 10 1+ b 11 1+ b 14 1+ b 15 1+ 818.2810 y9y9 1+ b 12 1+ CID-MS 2 (using Orbitrap) (m/z 1061.40, 2+) Theoretical monoisotopic mass = 1060.8955 (2+) DQGPCQDLTCF P PA T PCDGGC (134-154) (C138-C143, C150-C154) Enzyme digestion: Lys-C + Trypsin + PNGase F + Asp-N at pH8 Figure S9A y6y6 y 10 b 11 b 15 400600800100012001400160018002000 m/z 0 10 20 30 40 50 60 70 80 90 100 Relative Abundance 915.3339 z=1 1043.3848 z=2650.1906 z=1 1206.4578 z=1 549.1428 z=1 1471.6019 z=1 1572.6499 z=1 376.7184 z=8 503.0838 z=? 1754.5939 z=1 777.9993 z=? 1858.2398 z=? 1622.5437 z=1 1303.5108 z=1 1130.8780 z=? 699.3681 z=1 1436.4742 z=1 871.4703 z=1 1974.7286 z=1 Precursor ion, 2+ A Scrambled Disulfide

11 Correct Scrambled DQGPCQDLTCFPPATPCDGGC (134-154) DQGPCQ (p1) (134-139) D L T CFPPATPC D GGC (p2) (140-154) Figure S9B Chromatographic Retention Times for Correct and Scrambled Disulfides

12 HRFLGIPYSHDQGPCQNLTCFPPATPCDGGCDQGLVPIPLL (124-164) (C138-C154, C143-C150) b 38 3+ b 39 4+ b 40 4+ 1009.2 b 38 4+ b 37 4+ y 3 1+ y 5 1+ b 11 2+ b 6 1+ b 35 3+ b 36 3+ b 40 3+ b 32 2+ b 35 2+ 1863.1 x20 x5 x20 CID-MS2 (using ion trap) (m/z 1094.52, 4+) HRFLGIPYSHDQGPCQNLTCFPPATPCDGGCDQGLVPIPLL b6b6 b 40 y3y3 y6y6 Enzyme digestion: pepsin at pH 2 (since the N-linked site is not fully glycosylated, a non-glycosylated counterpart was selected for interpretation) Theoretical monoisotopic mass = 1094.0161 (4+) Figure S10 Precursor ion, 4+ Nested Disulfide (pepsin digestion)

13 NL:1.01E8 873.5874.0874.5875.0875.5876.0876.5877.0877.5878.0 m/z 0 10 20 30 40 50 60 70 80 90 100 Relative Abundance 874.6909 875.0243 874.3569 875.3581 875.6916 876.0251 876.3585 PALQICCHPGCTPRPACCHCPDPHA (465-489) (C470-C482, C471-C484, and C475-C481) 3+ Precursor ion Figure S11 Theoretical monoisotopic mass = 874.3564 (3+)

14 y3y3 1+ b5b5 b 20 2+ b 22 2+ b 24 3+ 1106.8 y 21 2+ b 22 y3y3 CID-MS 2 (m/z 874.69, 3+) 14 PALQ ICCHPGCTPRPACCHC PD PH A (465-489) y 21 b 20 Figure S12 NL:6.58E6 400600800100012001400160018002000 1149.7 324.2 845.1 1043.7 1227.6786.31775.5 655.4523.41888.61637.51462.1

15 y 5 1+ NL:3.12E5 400600800100012001400160018002000 m/z 1150.8 1760.0 1044.8 1436.8 1985.2 536.4 1295.1 864.6 1485.8 1676.8 947.9 1588.9 1864.0 639.4521.4 1403.9 830.6 1267.8 727.5 x50x20x50 z 5 1+ y 6 1+ b 8 1+ [b 8 -SH 2 ] 1+ b 20 2+ b 22 2+ [M-SH2] 2+ y 17 1+ b 19 1+ [Internal cleavage of y17 and y3] 1+ S·SH PA L Q I CC H PG CTPRPAC C H C PD PHA (465-489) SH S· b 22 y 17 b8b8 y5y5 CID-MS 3 (using ion trap) m/z 656.60 (ETD)→1312.6 (CID-MS3) b 7 1+ z 6 1+ y 7 1+ b 24 2+ b 18 1+ y 15 1+ c 18 1+ z 15 1+ b 4 1+ [Internal cleavage of y17 and y3-SH] 1+ z 14 1+ Figure S13 b 17 +S 1+ b 17


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