Abbreviation. Peptide Sequence. Parent z Light (m/z) Heavy (m/z). M1(ox). GGMoxQIFVK. 2. 448.2389. 451.2458. K6. MQIFVKGGTLTGK. 2. 690.3894. 693.898.
Supplemental Data 1.1
pS57
Ub (pSer57)
M1
50 40 30 20 10
TLS
0 10
D 90 Intensity (10^6)
80 70 60
20 30 40 Retention Time (min)
50
M1
50 40 pS65
30 20 10
EST
0 0
10
20 30 40 Retention Time (min)
50
60
K6 3
K4 8
K3 3
K2 9
K2 7
K1 1
K6 3
60 40 20 0
60
M1 - GGMQIFVK pS65 - EpSTLHLVLR EST - ESTLHLVLR
Ub (pSer65)
100
80
M
0
100 94%
0
K4 8
60
K6 K6 3 3_ ps 57
60
M1 - GGMQIFVK pS57 - TLpSDYNIQK TLS - TLSDYNIQK
50
80 79% 60 40 20
10%
0
K6 K6 3 3_ pS 65
70
20 30 40 Retention Time (min)
K3 3
Intensity (10^6)
80
10
9%
K4 8
C 90
0
20
K4 8
0
TITLE
K2 9
10
K3 3
20
40
K3 3
30
K2 7
40
60
K2 9
50
K2 9
M1
84%
K2 7
Ub (pSer20)
60
80
M
Intensity (10^6)
70
K2 7
pS20
K K1 11 1pS 20
80
100
0
K1 1
M1 - GGMQIFVK pS20 - TITLEVEPpSDTIENVK TITLE - TITLEVEPSDTIENVK
60
K1 1
90
50
K6
B
20 30 40 Retention Time (min)
K6
10
1
0
1
0
20
K6
100
40
K6
200
60
M 1
300
80
1
M1
96%
M
Ub
Distribution of ubiquitin chains (%)
400
100
Distribution of ubiquitin chains (%)
M1 - GGMQIFVK
Distribution of ubiquitin chains (%)
500
Distribution of ubiquitin chains (%)
Intensity (10^6)
A
Supplemental Data 1.2
150
Ub (pSer57) pS57 K6ox
50 TLS
0 0
D
Intensity (10^6)
150
10
20 30 40 Retention Time (min)
K6 - MQIFVKggTLTGK K6ox - MoxQIFVKggTLTGK pS65 - EpSTLHLVLR EST - ESTLHLVLR
100
Ub (pSer65) K6ox pS65
50
EST
0 0
10
20 30 40 Retention Time (min)
100
50
K6 3
K4 8
K3 3
K2 9
K2 7
K1 1
K6 3
96%
80 60 40 20 0
50
K6
K4 8
Intensity (10^6)
K6
100
0
K6 K6 3 3_ ps 57
200 K6 - MQIFVKggTLTGK K6ox - MoxQIFVKggTLTGK pS57 - TLpSDYNIQK TLS - TLSDYNIQK
60
77%
80 60 40 20
11%
0
K6 K6 3 3_ pS 65
50
K3 3
C
20 30 40 Retention Time (min)
K4 8
10
M
0
7%
K4 8
0
20
K2 9
K48
K3 3
20
40
K3 3
40
60
K2 7
K6ox
60
80
K2 9
pS20
89%
K2 9
Ub (pSer20)
80
100
M
K6
K2 7
100
100
0
K2 7
Intensity (10^6)
120
60
1
K6 - MQIFVKggTLTGK K6ox - MoxQIFVKggTLTGK pS20 - TITLEVEPpSDTIENVK
50
1
140
20 30 40 Retention Time (min)
M
B
10
1
0
K K1 11 1pS 20
0
20
K1 1
50
K1 1
100
40
K6
K6ox
M
150
60
K6
200
80
K6
250
96%
K6
Ub
300
100
1
K6
Distribution of ubiquitin chains (%)
Intensity (10^6)
350
Distribution of ubiquitin chains (%)
K6 - MQIFVKggTLTGK K6ox - MoxQIFVKggTLTGK
Distribution of ubiquitin chains (%)
400
Distribution of ubiquitin chains (%)
A
Supplemental Data 1.3
Ub (pSer57)
500
K48
400
pS57
300 200 100
K6ox
0 0
D
10
20 30 40 Retention Time (min)
50
Intensity (10^9)
K6
Ub (pSer65)
K48
1 pS65
0.5
K6ox
0 0
10
20 30 40 Retention Time (min)
50
60
K6 3
K4 8
K3 3
K2 9
K2 7
K1 1
K6
K6 3
K4 8
56%
60
40%
40 20 0
60
2
1.5
100
80
M
Intensity (10^6)
K6
K6 K6 3 3_ ps 57
700 600
100
0
80 60
55% 39%
40 20 0
K6 K6 3 3_ pS 65
60
K3 3
50
K4 8
20 30 40 Retention Time (min)
K4 8
10
K2 9
C
0
M
0
20
K3 3
0.2
40
K3 3
pS20
K6ox
47%
K2 7
0.4
49%
K2 9
0.6
60
K2 9
K6
K2 7
0.8
80
K2 7
Intensity (10^9)
1
K K1 11 1pS 20
1.2
100
K1 1
K48
K1 1
1.4
Ub (pSer20)
0
K6
60
K6
50
M
20 30 40 Retention Time (min)
1
10
20
1
B
0
40
1
0
50%
46%
K6
K6ox
60
1
0.5
80
M
Intensity (10^9)
K6
1
Distribution of ubiquitin chains (%)
K48
1.5
100
Distribution of ubiquitin chains (%)
Ub
Distribution of ubiquitin chains (%)
2
Distribution of ubiquitin chains (%)
A
A
Ub (pSer57)
120 100 80
pS57
60 40 20
TLS
0 0
10
D 100 90
20 30 40 Retention Time (min)
50
Intensity (10^6)
80 70 60 50 40 30
Ub (pSer65)
K11
pS65
20 10 0
EST
0
10
K63-pS65
K63
20 30 40 Retention Time (min)
100
50
60
K6 3
K4 8
K3 3
K2 9
K2 7
1 M
K6 3
95%
80 60 40 20 0
60
K11 - TLTGKggTITLEVEPSDTIENVK pS65 - EpSTLHLVLR EST - ESTLHLVLR
K4 8
K11
K6 K6 3 3_ ps 57
Intensity (10^6)
140
100
80
62%
60 40
33%
20 0
K6 K6 3 3_ pS 65
160
K3 3
K11 - TLTGKggTITLEVEPSDTIENVK pS57 - TLpSDYNIQK TLS - TLSDYNIQK
0
60
K4 8
180
50
K4 8
C
20 30 40 Retention Time (min)
K2 9
10
M
0
K3 3
0
20
K3 3
TITLE
K48 K11 K6
K6ox
1
5
K2 7
10
40
K2 9
15
60
K2 9
20
K2 7
pS20
K2 7
Ub (pSer20)
25
81%
80
1
30
100
0
60
K11 - TLTGKggTITLEVEPSDTIENVK K11-pS20 - TLTGKggTITLEVEPpSDTIENVK pS20 - TITLEVEPpSDTIENVK K11-pS20 TITLE - TITLEVEPSDTIENVK
35 Intensity (10^6)
50
M
40
20 30 40 Retention Time (min)
1
B
10
M
0
K1 1
0
20
K K1 11 1pS 20
100
K1 1
200
40
K1 1
300
60
K6
400
80
K6
500
95%
K6
Ub
600
100
K6
K11
Distribution of ubiquitin chains (%)
Intensity (10^6)
700
Distribution of ubiquitin chains (%)
K11 - TLTGKggTITLEVEPSDTIENVK
Distribution of ubiquitin chains (%)
800
Distribution of ubiquitin chains (%)
Supplemental Data 1.4
Supplemental Data 1.5
K6
0 10
20 30 40 Retention Time (min)
50
60
100
100
0 M
0
20
K6 3
K6ox
2
40
K4 8
K11
K3 3
4
K2 9
6
60
K2 7
K48
80
K1 1
8
91%
K6
Intensity (10^6)
10
100
1
K29
Distribution of ubiquitin chains (%)
Ub
K29 - AKggIQDK
Distribution of ubiquitin chains (%)
12
Distribution of ubiquitin chains (%)
A
B
30 pS65
20 10 EST
K48
K11
0 0
10
20 30 40 Retention Time (min)
50
60
K6 K6 3 3_ ps 57
K29
K6 K6 3 3_ pS 65
40 Intensity (10^6)
Ub (pSer65)
K29 - AKggIQDK pS65 - EpSTLHLVLR EST - ESTLHLVLR
0
60
K4 8
50
50
K4 8
C
20 30 40 Retention Time (min)
K3 3
10
M
0
91%
80 60 40 20 0
K3 3
0
17%
20
K2 9
K11
K2 9
TLS
K2 7
5
K2 7
10
40
K1 1
K29
60
1
15
75%
K1 1
20
80
K6
K48
25
K6
pS57
1
30 Intensity (10^6)
Ub (pSer57)
K29 - AKggIQDK pS57 - TLpSDYNIQK TLS - TLSDYNIQK
M
35
Supplemental Data 1.6
3
14
K48
Ub (pSer57)
12 Intensity (10^6)
2.5 Intensity (10^9)
K29
2 1.5 1
10 8 6 pS57
4 2 0
0.5
K6
K11 K63-pS57
0 10
D 25 20
1.5
Intensity (10^6)
Intensity (10^9)
2
20 30 40 Retention Time (min)
1
K48
Ub (pSer65)
15 10
pS65 K6
K6ox
0
K11 K63-pS65
0 0
10
20 30 40 Retention Time (min)
100
50
60
K6 3
K4 8
K3 3
K2 9
K2 7
K1 1
60
K6 3
55% 37%
40 20 0
60
K29
5
0.5
50
80
M
0
100
0
K4 8
60
K6 K6 3 3_ ps 57
C
50
80 60
45% 40
36%
20
9%
7%
0
K6 K6 3 3_ pS 65
20 40 30 Retention Time (min)
K3 3
10
K4 8
0
K4 8
0
6% K2 9
K6
20
K3 3
K11
K3 3
pS20
0
40
K2 7
K29
K2 9
0.5
5
60
K2 9
1
10
K2 7
1.5
79%
80
M
Intensity (10^6)
Intensity (10^9)
K48
2
100
K2 7
Ub (pSer20)
K K1 11 1pS 20
2.5 15
0
K1 1
60
K1 1
50
K6
B
20 30 40 Retention Time (min)
M
10
1
0
K6
K11
K6
0
20
K6
0
0.5
40
K6
2
48%
47%
1
4
60
1
1
6
80
1
1.5
8
100
M
Intensity (10^6)
2
K48
10
Distribution of ubiquitin chains (%)
12
2.5 Intensity (10^9)
Ub
K29
Distribution of ubiquitin chains (%)
14
Distribution of ubiquitin chains (%)
3
Distribution of ubiquitin chains (%)
A
Supplemental Data 1.7
pS57
200 100 0
D 25
10
50
K33
pS65
15 10 5 EST
0 0
10
20 30 40 Retention Time (min)
50
60
K6 3
K4 8
K3 3
K2 9
K2 7
K1 1
1 M
100
K6 3
K4 8
80 60 40 20 0
60
Ub (pSer65)
K33 - IQDKggEGIPPDQQR pS65 - EpSTLHLVLR EST - ESTLHLVLR
20 Intensity (10^6)
20 30 40 Retention Time (min)
99%
K6 K6 3 3_ ps 57
300
100
87% 80 60 40 20
10%
0
K6 K6 3 3_ pS 65
Ub (pSer57)
K33
0
0
60
K3 3
50
K4 8
K33 - IQDKggEGIPPDQQR pS57 - TLpSDYNIQK
400 Intensity (10^6)
30 40 Retention Time (min)
K4 8
500
20
K2 9
C
10
M
0
7%
K3 3
0
20
1
K6ox
K48 K6
K3 3
TITLE
10
K2 7
20
40
K2 9
30
60
K2 9
pS20
40
K2 7
50
86% 80
K2 7
K33
K K1 11 1pS 20
Ub (pSer20)
1
Intensity (10^6)
60
100
0
60
M
K33 - IQDKggEGIPPDQQR pS20 - TITLEVEPpSDTIENVK TITLE - TITLEVEPSDTIENVK
50
1
70
20 30 40 Retention Time (min)
M
B
10
Distribution of ubiquitin chains (%)
0
20
K1 1
0
40
K1 1
100
60
K6
200
80
K6
300
99%
K6
400
100
K6
500
Distribution of ubiquitin chains (%)
K33
600 Intensity (10^6)
Ub
K33 - IQDKggEGIPPDQQR
Distribution of ubiquitin chains (%)
700
Distribution of ubiquitin chains (%)
A
Supplemental Data 1.8
Intensity (10^6)
200
K48
Ub (pSer65) with UBE2R1
150 100
pS65
50 0 10
E 300
20 30 40 Retention Time (min)
K48
Ub (pSer65) with UBE2K
200 150 100
pS65
50 0 0
10
20 30 40 Retention Time (min)
100
50
60
K6 3
K4 8
K3 3
K2 9
K2 7
K1 1
K6
1
K6 3
K4 8
K3 3
K2 9
K2 7
K6 K6 3 3_ ps 57
60 40 20
12%
0
60
K48 UBE2K - LIFAGKggQLEDGR pS65 - EpSTLHLVLR
250 Intensity (10^6)
50
80%
80
M
0
100
0
60
K6 K6 3 3_ pS 65
K48 UBE2R1 - LIFAGKggQLEDGR pS65 - EpSTLHLVLR
50
86% 80 60 40 20
6% 0
K6 K6 3 3_ pS 65
250
20 30 40 Retention Time (min)
K4 8
D
10
M
0
K4 8
0
20
K4 8
K11
40
K3 3
pS57
60
K3 3
0.4 0.3 0.2 0.1
80
K3 3
Intensity (10^9)
Ub (pSer57)
K2 9
K48
0.8 0.7 0.6 0.5
97%
K2 9
K48 - LIFAGKggQLEDGR pS57 - TLpSDYNIQK
K2 9
1 0.9
0
60
K2 7
C
50
K2 7
20 30 40 Retention Time (min)
K2 7
10
K1 1pS 20
0
8% K1 1
0
20
K1 1
K6 K11
40
K1 1
TITLE
K6ox
60
K1 1
pS20
80%
80
K6
40 30 20 10
100
M
Ub (pSer20)
70 60 50
100
M
Intensity (10^6)
K48
1
K48 - LIFAGKggQLEDGR pS20 - TITLEVEPpSDTIENVK TITLE - TITLEVEPSDTIENVK
90 80
0
60
1
100
50
1
B
20 30 40 Retention Time (min)
20
1
10
40
M
0
60
K6
0
80
K6
100
95%
K6
200
Distribution of ubiquitin chains (%)
300
100
Distribution of ubiquitin chains (%)
Ub
400 Intensity (10^6)
K48
Distribution of ubiquitin chains (%)
K48 - LIFAGKggQLEDGR
Distribution of ubiquitin chains (%)
500
Distribution of ubiquitin chains (%)
A
Supplemental Data 1.9 100
K33 - IQDKggEGIPPDQQR pS20 - TITLEVEPpSDTIENVK TITLE - TITLEVEPSDTIENVK
M
100
140
20 30 40 Retention Time (min)
K63 - TLSDYNIQKggESTLHLVLR K63-pS65 - TLSDYNIQKggEpSTLHLVLR pS65 - EpSTLHLVLR
50
60
100
K63-pS65
Ub (pSer65)
120 100 80 pS65
60 40 20
K48
K63
0 0
10
5.5% 0
K4 8
10
20
K3 3
0
40
K2 9
K63
60
20 30 40 Retention Time (min)
50
60
94%
80 60 40 20 0
K4 8
Intensity (10^6)
160
pS57
80
K3 3
180
Ub (pSer57)
91%
K2 9
D
100
K63-pS57
K2 7
0
K63 - TLSDYNIQKggESTLHLVLR K63-pS57 - TLpSDYNIQKggESTLHLVLR pS57 - TLpSDYNIQK
0
60
K2 7
110 100 90 80 70 60 50 40 30 20 10
50
Distribution of ubiquitin chains (%)
Intensity (10^6)
C
20 30 40 Retention Time (min)
K1 1
10
Distribution of ubiquitin chains (%)
0
K4 8
0
20
K6 K6 3 3_ ps 57
TITLE
K1 1
K63
10
K6 K6 3 3_ pS 65
20
40
K3 3
30
K2 9
40
60
K2 7
50
K6
60
80
K K1 11 1pS 20
Ub (pSer20)
93%
K6
pS20
K6
Intensity (10^6)
70
60
1
80
50
M
90
20 30 40 Retention Time (min)
1
10
Distribution of ubiquitin chains (%)
0
B
6% 0 K6 3
0
20
K4 8
K6
K3 3
1
K2 9
K48
40
K2 7
K6ox
2
K1 1
K11
3
60
M
Intensity (10^6)
4
80
K6
Ub
5
91%
1
6
1
K63
K6 3
K63 - TLSDYNIQKggESTLHLVLR
M
7
Distribution of ubiquitin chains (%)
A
Supplemental Data 1.10
K48
300
pS57
250 K6
200
K11
150 100
K63-pS57
50
K33
K63
K6ox
0
D 400
20 30 40 Retention Time (min)
Ub (pSer65)
350
Intensity (10^6)
10
50
K48
250 K6 pS65
150 100
K11
K63-pS65
50
K6ox
K33
0 0
10
20 30 40 Retention Time (min)
50
60
K6 3
K4 8
K3 3
K2 9
K2 7
60
K6 3
K4 8
K3 3
51%
40
17%
20
14%
10%
8%
0
60
300
200
100
80
M
0
100
0
K6 K6 3 3_ ps 57
Ub (pSer57)
350
Intensity (10^6)
60
80 60
48% 40
19%
20
17% 9%
7%
0
K6 K6 3 3_ pS 65
400
50
K4 8
C
20 30 40 Retention Time (min)
K4 8
10
14%
9%
K2 9
0
12%
M
0
K11-pS20
20%
20
K3 3
K6ox
K3 3
pS20
100
45% 40
K2 7
K6
60
K2 9
300
80
K2 9
Intensity (10^6)
400
200
100
K2 7
K48
K2 7
Ub (pSer20)
14%
0 K1 1
60
K K1 11 1pS 20
50
K1 1
500
20 30 40 Retention Time (min)
K1 1
10
M
B
0
17% 8%
1
0
K6ox
K33
21%
20
1
0.2
K63
K6
0.4
40%
40
K6
K11
1
K6
M
0.6
60
K6
0.8
80
K6
1
1
K48
Distribution of ubiquitin chains (%)
Intensity (10^9)
1.2
100
Distribution of ubiquitin chains (%)
Ub
Distribution of ubiquitin chains (%)
1.4
Distribution of ubiquitin chains (%)
A
Supplemental Data 1.11 Abbreviation
Parent z
Light (m/z)
Heavy (m/z)
GGMoxQIFVK
2
448.2389
451.2458
K6
MQIFVKGGTLTGK
2
690.3894
693.898
K6
MQIFVKGGTLTGK
3
460.5954
462.9344
K6(ox)
MoxQIFVKGGTLTGK
2
698.3869
701.8954
K6(ox)
MoxQIFVKGGTLTGK
3
465.927
468.2661
K11
TLTGKGGTITLEVEPSDTIENVK
2
1201.6367
1204.6436
K11
TLTGKGGTITLEVEPSDTIENVK
3
801.4269
803.4315
K27
TITLEVEPSDTIENVKGGAK
2
1051.6698
1054.6478
K27
TITLEVEPSDTIENVKGGAK
3
701.4489
703.4343
K29
AKGGIQDK
1
816.4574
822.4775
K29
AKGGIQDK
2
408.7323
411.7424
K33
IQDKGGEGIPPDQQR
3
546.6129
548.6175
K48
LIFAGKGGQLEDGR
2
730.8964
734.405
K48
LIFAGKGGQLEDGR
3
487.6001
489.9391
K63
TLSDYNIQKGGESTLHLVLR
3
748.7376
751.0767
K11_pS20
TLTGKGGTITLEVEPpSDTIENVK
2
1241.6198
1244.6267
K11_pS20
TLTGKGGTITLEVEPpSDTIENVK
3
828.0823
830.0869
K27_pS20
TITLEVEPpSDTIENVKGGAK
2
1091.038
1094.0449
K27_pS20
TITLEVEPpSDTIENVKGGAK
3
727.6944
729.699
TITLEVEPSDTIENVK TITLEVEPSDTIENVK TITLEVEPpSDTIENVK TITLEVEPpSDTIENVK
2 3 2 3
894.4673 596.6473 934.4505 623.3027
897.4742 598.6519 937.4574 625.3073
K63_pS57
TLpSDYNIQKGGESTLHLVLR
3
775.3931
777.7321
K63_pS57
TLpSDYNIQKGGESTLHLVLR
4
581.7966
583.5509
TLpSDYNIQKESTLHLVLR TLpSDYNIQKESTLHLVLR TLSDYNIQKESTLHLVLR TLSDYNIQKESTLHLVLR TLpSDYNIQK TLpSDYNIQK TLSDYNIQK TLSDYNIQK
2 3 2 3 1 2 1 2
1105.5645 737.3787 1065.5813 710.7233 1161.5187 581.263 1081.5524 541.2798
1109.0731 739.7178 1070.5854 714.0594 1168.5359 584.7716 1088.5696 544.7884
K63_pS65
TLSDYNIQKGGEpSTLHLVLR
3
775.3931
777.7321
K63_pS65
TLSDYNIQKGGEpSTLHLVLR
4
581.7966
5835509
TLS-EpS65 TLS-EpS65 TLS-EST TLS-EST EST_pS65 EST_pS65 EST EST
TLSDYNIQKEpSTLHLVLR TLSDYNIQKEpSTLHLVLR TLSDYNIQKESTLHLVLR TLSDYNIQKESTLHLVLR EpSTLHLVLR EpSTLHLVLR ESTLHLVLR ESTLHLVLR
2 3 2 3 2 3 2 3
1105.5645 737.3787 1065.5813 710.7233 574.2972 383.2006 534.314 356.5451
1109.0731 739.7178 1070.5854 714.0594 577.8058 385.5396 539.3182 359.8812
M1(ox)
TITLE TITLE TITLE_pS20 TITLE_pS20
TL_pS57-EST TL_pS57-EST TLS-EST TLS-EST TL_pS57 TL_pS57 TLS TLS
Peptide Sequence
3. Supplemental Data 1 Supplemental Data 1, related to Figure 3 and 5. Targeted mass spectrometry data and peptide sequences. Supplemental Data 1.1 related to Figure 3 and 5. Targeted mass spectrometry using AQUA peptides on purified trimers. M1 (assembled with Ube1, UBE2L3 and HOIP) on either Ub (A), Ub (pSer20) (B), Ub (pSer57) (C) or Ub (pSer65) (D). Supplemental Data 1.2 related to Figure 3 and 5. Targeted mass spectrometry using AQUA peptides on purified trimers. K6 chains (assembled with Ube1, UBE2L3, NleL) on either UbK48R (A), UbK48R (pSer20) (B), UbK48R (pSer57) (C) or UbK48R (pSer65) (D). Supplemental Data 1.3 related to Figure 3 and 5. Targeted mass spectrometry using AQUA peptides on total reactions. Reactions with Ube1, UBE2L3 and NleL with either Ub (A), Ub (pSer20) (B), Ub (pSer57) (C) or Ub (pSer65) (D). Supplemental Data 1.4 related to Figure 3 and 5. Targeted mass spectrometry using AQUA peptides on purified trimers. K11 chains (assembled with Ube1, UBE2S-UBP and AMSH) on either Ub (A), Ub (pSer20) (B), Ub (pSer57) (C) or Ub (pSer65) (D). Supplemental Data 1.5 related to Figure 3 and 5. Targeted mass spectrometry using AQUA peptides on purified trimers. K29 chains (assembled with Ube1, UBE2D3, UBE3C and vOTU) on either Ub (A), Ub (pSer57) (B) or Ub (pSer65) (C). Supplemental Data 1.6 related to Figure 3 and 5. Targeted mass spectrometry using AQUA peptides on total reactions. Reactions with Ube1, UBE2D3 and UBE3C with either Ub (A), Ub (pSer20) (B), Ub (pSer57) (C) or Ub (pSer65) (D). Supplemental Data 1.7 related to Figure 3 and 5. Targeted mass spectrometry using AQUA peptides on purified trimers. K33 chains (assembled with Ube1, UBE2D1, AREL1, OTUB1 and Cezanne-EK) on either Ub (A), Ub (pSer20) (B), Ub (pSer57) (C) or Ub (pSer65) (D). Supplemental Data 1.8 related to Figure 3 and 5. Targeted mass spectrometry using AQUA peptides on purified trimers. K48 chains (assembled with Ube1, UBE2R1 or UBE2K) on either Ub (A), Ub (pSer20) (B), Ub (pSer57) (C), Ub (pSer65) with UBE2R1 (D) or with UBE2K (E). Supplemental Data 1.9 related to Figure 3 and 5. Targeted mass spectrometry using AQUA peptides on purified trimers.
K63 chains (assembled with Ube1, UBE2N and UBE2V1) on either Ub (A), Ub (pSer20) (B), Ub (pSer57) (C) or Ub (pSer65) (D). Supplemental Data 1.10 related to Figure 3 and 5. Targeted mass spectrometry using AQUA peptides on total reactions. Reaction with Ube1, UBE2L3 and HHARI with either Ub (A), Ub (pSer20) (B), Ub (pSer57) (C) or Ub (pSer65) (D). Supplemental Data 1.11 related to Figure 3 and 5. List of isotopically labelled peptides used to monitor polyUb and phosphorylated polyUb linkages. Signature peptides representing isopeptide-linked polyUb linkages are denoted by a subscript “GG” adjacent to the modified lysine. Signature peptides representing phosphorylated peptides are denotes by a “p” adjacent to modified serine. Isotopically labelled amino acids are denoted in blue and the oxidation state of Met-containing peptides is denoted by “ox”. For each peptide, the optimal precursor ions to the HCD collision energy of 30% were selected for the PRM analysis.
