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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:7.73E m/z Relative Abundance Precursor ion Figure S1A YVPVSLC(fgly)TPSRAAL (45-58)(C51 as fgly) Theoretical monoisotopic mass = (2+) y y6y6 1+ y7y7 y8y8 y y b b8b8 1+ b [b 8 -H 2 O] 1+ CID-MS2 (using ion trap) (m/z , 2+) Figure S1B Y V PVS L C(fgly) T PSRA A L (45-58) b8b8 b 13 y6y6 y 12 y7y7 y8y8 NL:1.30E m/z Relative Abundance

2 NL:3.19E m/z Relative Abundance Precursor ion Figure S2A YVPVSLCTPSRAAL (45-58)(C51 as free cys) Theoretical monoisotopic mass = (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 [b 12 -H 2 O] 2+ b4b4 1+ NL:4.67E m/z Relative Abundance CID-MS2 (using ion trap) (m/z , 2+) Figure S2B YVPV SLCT PSRAA L (45-58) b8b8 y6y6 y 12 b2b2

3 NL:2.92E m/z Relative Abundance Precursor ion Figure S3ARMSRGGCSGL ( )(C276 as free cys) Theoretical monoisotopic mass = (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 ( ) CID-MS2 (using ion trap) (m/z , 2+) [b 7 -H 2 O] 1+ [b 6 -H 2 O] 1+ [M-SCH 2 ] 2+ c9c9 y9y9 b3b3 NL:1.10E m/z Relative Abundance Figure S3B

4 NL:1.07E m/z Relative Abundance Theoretical monoisotopic mass = (3+) Monoisotopic mass Precursor ion Figure S4A NL:2.76E m/z Relative Abundance 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) [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) y 16 2+(P2) b 18 3+(P2) y 14 2+(P2) y 19 2+(P2) x5 CID-MS2 (using ion trap) (m/z , 3+) Figure S4B 3+ b9b9 LRCGKGTTYEGGVRE (P1) ( ) (C282) FTQGSAHSDTTADPACHASSSL(P2)( ) (C396) y9y9 b 18 y 19 b5b5 b 10 y5y5 y 13

5 NL:9.16E m/z Relative Abundance 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+ · · · [M-NH 3 ] 2+ · · · Z 14 3+(P2) Z 20 3+(P2) x2 Z 19 2+(P2) C 14 2+(P1) Z 20 2+(P2) LRCGKGTTYEGGVRE (P1) ( ) (C282) FTQGSAHSDTTADPACHASSSL(P2)( ) (C396) c2c2 z 10 c7c7 z 20 c7c7 c 14 z5z5 z8z8 Figure S4C ETD-MS 2 (using ion trap) (m/z , 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 DQGPCQ (p1) ( ) D L T CFPPATPC D GGC (p2) ( ) [M+3H] 2+ P1 1+ P2 1+ [M+3H] 3+ ETD-MS2 (using ion trap) (m/z , 3+) NL:7.67E Figure S8

10 y6y6 1+ y7y7 y b b b y9y9 1+ b CID-MS 2 (using Orbitrap) (m/z , 2+) Theoretical monoisotopic mass = (2+) DQGPCQDLTCF P PA T PCDGGC ( ) (C138-C143, C150-C154) Enzyme digestion: Lys-C + Trypsin + PNGase F + Asp-N at pH8 Figure S9A y6y6 y 10 b 11 b m/z Relative Abundance z= z= z= z= z= z= z= z= z=? z= z=? z=? z= z= z=? z= z= z= z=1 Precursor ion, 2+ A Scrambled Disulfide

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

12 HRFLGIPYSHDQGPCQNLTCFPPATPCDGGCDQGLVPIPLL ( ) (C138-C154, C143-C150) b b b b b y 3 1+ y 5 1+ b b 6 1+ b b b b b x20 x5 x20 CID-MS2 (using ion trap) (m/z , 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 = (4+) Figure S10 Precursor ion, 4+ Nested Disulfide (pepsin digestion)

13 NL:1.01E m/z Relative Abundance PALQICCHPGCTPRPACCHCPDPHA ( ) (C470-C482, C471-C484, and C475-C481) 3+ Precursor ion Figure S11 Theoretical monoisotopic mass = (3+)

14 y3y3 1+ b5b5 b b b y b 22 y3y3 CID-MS 2 (m/z , 3+) 14 PALQ ICCHPGCTPRPACCHC PD PH A ( ) y 21 b 20 Figure S12 NL:6.58E

15 y 5 1+ NL:3.12E m/z x50x20x50 z 5 1+ y 6 1+ b 8 1+ [b 8 -SH 2 ] 1+ b b [M-SH2] 2+ y b [Internal cleavage of y17 and y3] 1+ S·SH PA L Q I CC H PG CTPRPAC C H C PD PHA ( ) SH S· b 22 y 17 b8b8 y5y5 CID-MS 3 (using ion trap) m/z (ETD)→ (CID-MS3) b 7 1+ z 6 1+ y 7 1+ b b y c z b 4 1+ [Internal cleavage of y17 and y3-SH] 1+ z Figure S13 b 17 +S 1+ b 17


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