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...
15
NL: 7.73E8 729.0 729.5 730.0 730.5 731.0 731.5 732.0 732.5 733.0 733.5 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 y 6 1+ y 7 1+ y 8 1+ y 10 1+ y 12 1+ b 12 1+ 845.4 b 8 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) b 8 b 13 y 6 y 12 y 7 y 8 NL:1.30E7 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 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 482.30 235.2 1256.6 1065.6 360.2 1435.6
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Transcript of 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...
NL:7.73E8
729.0 729.5 730.0 730.5 731.0 731.5 732.0 732.5 733.0 733.5
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
729.9020
730.4028
730.9041
731.4056731.9074
2+Precursor ion
Figure S1A YVPVSLC(fgly)TPSRAAL (45-58)(C51 as fgly)Theoretical monoisotopic mass = 729.9012 (2+)
y122+
614.5y6
1+
y71+
y81+ y10
1+ y121+
b121+845.4
b81+
b132+
[b8-H2O]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)
b8 b13
y6y12 y7y8
NL:1.30E7
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400m/z
0
10
20
30
40
50
60
70
80
Rel
ativ
e A
bu
nd
ance
599.0
827.4
664.5
715.4
1196.61000.4800.5565.2
895.4743482.30235.2 1256.61065.6360.2 1435.6
NL:3.19E7
738.0 738.5 739.0 739.5 740.0 740.5 741.0 741.5 742.0 742.5 743.0
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lativ
e A
bund
ance
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+)
y61+
[M-H2O]2+
[y12-H2O]1+
[b8-2H2O]1+
[y10-H2O]1+
b21+
[M-SH2]2+
[M-SCH2]2+
[y12-H2O]2+
b132+
[b12-H2O]2+
b41+
NL:4.67E5
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400m/z0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bu
nd
ance
729.9
614.5
827.3
1196.61000.6893.9460.2 1309.7380.2263.3 1418.9
CID-MS2 (using ion trap)(m/z 738.90, 2+)
Figure S2B
YVPV SLCT PSRAA L (45-58)
b8
y6y12
b2
NL:2.92E7
511.8 512.0 512.2 512.4 512.6 512.8 513.0 513.2 513.4 513.6 513.8 514.0 514.2 514.4 514.6m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
512.2453
512.7463
513.2459513.7459
2+
Precursor ionFigure S3A RMSRGGCSGL (270-279)(C276 as free cys)
Theoretical monoisotopic mass = 512.2448 (2+)
[M-H2O] 2+
b92+
b42+b3
2+
y91+
b81+
[b8-H2O]1+
b41+
R MS R GG C S G L (270-279)
CID-MS2 (using ion trap) (m/z 512.22, 2+)
[b7-H2O]1+
[b6-H2O]1+
[M-SCH2] 2+
c92+
y9
b3
NL:1.10E6
200 300 400 500 600 700 800 900 1000m/z0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bu
nd
ance
456.0
503.7
447.0867.5
964.5488.3 818.2731.2 875.5396.1 531.2266.6 628.3358.6189.1 835.5800.3 947.3704.5 893.9323.2 600.1 1027.7
Figure S3B
NL: 1.07E8
1270.0 1271.0 1272.0 1273.0 1274.0 1275.0m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
1272.5826
1272.24951272.9169
1273.25071271.9153
1273.5851
1273.9186
Theoretical monoisotopic mass = 1271.9117 (3+)
Monoisotopic mass
Precursor ion
Figure S4A
NL: 2.76E5
400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
1248.8
1320.9
1443.0
829.9
931.7 1650.31184.1
1544.0
1142.0
787.9 1068.7 1375.2517.6 1501.3 1720.1 1980.7
y51+(P1)
[b11-2H2O] 4+(P1)
b9 1+(P2)
[y16-H2O] 3+(P2)
y133+(P1)
1239.8
[y9-H2O] 2+(P2)
y92+(P2)
b13 1+(P2)
y132+(P2)
b5 2+(P1) y15
2+(P2)
b10 2+(P1)
1612.8
y162+(P2)
b18 3+(P2) y14
2+(P2)
y192+(P2)
x5 x5
CID-MS2 (using ion trap)(m/z 1272.58, 3+)
Figure S4B
3+
b9
LR CGKGT TYEGGVRE (P1) (280-294) (C282)
FTQGSAHSDT TADPA CHASSSL(P2)(381-402) (C396)
y9
b18
y19 b5 b10
y5y13
NL: 9.16E5
200 400 600 800 1000 1200 1400 1600 1800 2000m/z0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
1909.0
813.8
1267.41097.0
1684.5451.3
1626.11843.91122.4
894.5 1298.51184.9746.5 1462.2 1711.8502.4 1594.8630.3 995.5394.3266.2 1341.8
C4 1+(P2)
C2 1+(P2) C3
1+(P2) Z51+(P1) Z6
1+(P1) C7 1+(P2)
[P1] 2+
[P2] 2+
Z81+(P1)
[M-H2O] 3+ · ·
Z102+(P2)
C7 2+(P1)
Z182+(P2)
[M] 2+ · · ·
1901.1[M-NH3]
2+ · · ·
Z143+(P2)
Z203+(P2)
x2
Z192+(P2)
C142+(P1)
1777.5
Z202+(P2)
LR CGKGT TYEGGVRE (P1) (280-294) (C282)
FTQGSAHSDT TADPA CHASSSL(P2)(381-402) (C396)
c2
z10
c7
z20c7 c14
z5z8Figure S4C
ETD-MS2 (using ion trap)(m/z 764.95, 5+)
FLGIPYSHDQGPCQNLTCFPPATPCDGGCDQGLVPIPLLANLSVEAQPPWLPGLEARC138 C143 C150 C154
Figure S5
Nested Disulfides with Correct and Scrambled Configurations in a Tryptic Peptide
A (correct)
FLGIPYSHDQGPCQNLTCFPPATPCDGGCDQGLVPIPLLANLSVEAQPPWLPGLEARC138 C143 C150 C154
B (scramble 1)
FLGIPYSHDQGPCQNLTCFPPATPCDGGCDQGLVPIPLLANLSVEAQPPWLPGLEARC138 C143 C150 C154
C (scramble 2)
FLGIPYSHDQGPCQNLTCFPPATPCDGGCDQGLVPIPLLANLSVEAQPPWLPGLEARC138 C143 C150 C154
Figure S6 Digestion Strategy for Nested Disulfides
A (Lys-C + trypsin)
C138 C143 C150 C154
B (Lys-C + trypsin + PNGase F)
FLGIPYSH DQGPCQ DLTCFPPATPCDGGC DQGLVPIPLLA DLSVEAQPPWLPGLEARC138 C143 C150 C154
C (Lys-C + trypsin + PNGase F + Asp-N)
FLGIPYSHDQGPCQDLTCFPPATPCDGGCDQGLVPIPLLADLSVEAQPPWLPGLEAR
Figure S7 Digestion Strategy for Nested Disulfides
FLGIPYSH DQGPCQ DLTCFPPATPCDGGC DQGLVPIPLLA DLSVEAQPPWLPGLEARC138 C143 C150 C154
Lys-C + trypsin + PNGase F + Asp-N
DQGPCQ DLTCFPPATPCDGGCC138 C143 C150 C154
DQGPCQ
DLTCFPPATPCDGGC
C138
C143 C150
C154
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
200 400 600 800 1000 1200 1400 1600 1800 2000m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bu
nd
ance
714.5
1070.6 1426.1647.4 1782.81368.8915.5 1225.5842.6 1881.5283.3 517.2429.4 1590.8202.2
Figure S8
y61+ y7
1+
y101+
b111+
b141+
b151+818.2810
y91+
b121+
CID-MS2 (using Orbitrap)(m/z 1061.40, 2+)
1061.0 1062.0 1063.0 1064.0m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
1061.39901060.8974
1061.8992
1062.3996
1062.905
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 S9Ay6y10
b11 b15
400 600 800 1000 1200 1400 1600 1800 2000m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bu
nd
ance
915.3339z=1
1043.3848z=2650.1906
z=1 1206.4578z=1
549.1428z=1
1471.6019z=1 1572.6499
z=1
376.7184z=8
503.0838z=?
1754.5939z=1777.9993
z=?1858.2398
z=?1622.5437z=1
1303.5108z=1
1130.8780z=?
699.3681z=1
1436.4742z=1
871.4703z=1
1974.7286z=1
Precursor ion, 2+
A Scrambled Disulfide
30 40 50 60 70Time (min)
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
48.7953.94
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
HRFLGIPYSHDQGPCQNLTCFPPATPCDGGCDQGLVPIPLL (124-164) (C138-C154, C143-C150)
b38 3+
NL:2.17E5
300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z0
20
40
60
80
100
Rel
ativ
e A
bund
ance
1275.31345.1
1415.4
1033.5342.3
1061.6980.9
1713.6552.3 662.4
1242.4 1903.8724.8 1769.5
b39 4+
b404+
1009.2
b38 4+
b37 4+
y31+
y51+ b11
2+
b6 1+ b35
3+
b36 3+ b40
3+
b32 2+ b35
2+
1863.1
x20 x5 x5 x20
CID-MS2 (using ion trap)(m/z 1094.52, 4+)
HRFLGIPYSHDQGPCQ NLTCFPPATPCDGGCDQGLVPIPLLb6 b40
y3y6
1092.0 1093.0 1094.0 1095.0 1096.0 1097.0m/z0
20
40
60
80
100
Rel
ativ
e A
bund
ance
1094.5201 1094.7703
1094.2699 1095.0205
1095.27121094.0192
1095.5216
1095.7723
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)
NL:1.01E8
873.5 874.0 874.5 875.0 875.5 876.0 876.5 877.0 877.5 878.0
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lativ
e A
bund
ance
874.6909875.0243
874.3569875.3581
875.6916
876.0251876.3585
PALQICCHPGCTPRPACCHCPDPHA (465-489) (C470-C482, C471-C484, and C475-C481)
3+
Precursor ion
Figure S11
Theoretical monoisotopic mass = 874.3564 (3+)
y31+
b51+ b20
2+
b222+
b243+
1106.8
y212+
b22
y3
CID-MS2
(m/z 874.69, 3+)
14
PALQ ICCHPGCTPRPACCHC PD PH A (465-489)
y21
b20
Figure S12
NL: 6.58E6
400 600 800 1000 1200 1400 1600 1800 2000m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bu
nd
ance
1149.7
324.2 845.11043.7 1227.6786.3 1775.5655.4523.4 1888.61637.51462.1
y5 1+
NL: 3.12E5
400 600 800 1000 1200 1400 1600 1800 2000
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
1150.8
1760.01044.8
1436.81985.2536.4 1295.1864.6 1485.8
1676.8947.9 1588.9 1864.0639.4521.4 1403.9830.6 1267.8727.5
x50 x20 x50
z5 1+ y6
1+
b8 1+
[b8-SH2] 1+
b20 2+
b22 2+
[M-SH2] 2+
y171+
b191+
[Internal cleavage of y17 and y3] 1+
S· SH
PA L Q I CC H PG CTPRPAC C H C PD PHA (465-489)
SHS· b22
y17
b8
y5
CID-MS3 (using ion trap)m/z 656.60 (ETD)→1312.6 (CID-MS3)
b7 1+
z6 1+
y7 1+
b24 2+
b181+y15
1+
c18 1+z15
1+
b4 1+
[Internal cleavage of y17 and y3-SH] 1+
z14 1+
Figure S13
b17+S1+
b17
