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Supplementary Figures
Supplementary Figure 1. Comparative evaluation of different XL-MS acquisition strategies at the
redundant CSM level at 1% FDR. Significance was determined by a two-sided Z-test. (analysis at unique
CSM level is shown in Fig. 1).
Supplementary Figure 2. Quality assessment of cross-links from multiple XL-MS acquisition strategies
utilizing false search space (left panel) and precision (right panel). CSMs were identified using XlinkX at
1%FDR with additional filtering cut-offs for ‘XlinkX Score’.
Supplementary Figure 3. Dissection of CSMs from the ensemble i.e., CID-MS2-MS3-ETD-MS2
approach at 1% FDR in terms of quality of the two inherent approaches (i.e., CID-MS2-MS3 and CID-
MS2-ETD-MS2). Venn Diagrams show the overlap of CSMs at 1% FDR, along with further filtering at
different ‘XlinkX Score’ cut-offs. Left panel shows CSM overlap between the two approaches (fraction
of mis-identifications is shown in the parentheses) and the right panel shows overlap for the inter-protein
cross-links (precision is given in parentheses). XlinkX was used to process the raw files for six E. coli CID-
MS2-MS3-ETD-MS2 XL-MS fractions from Liu et al9.
Supplementary Figure 4. Venn diagrams showing the overlap of CSMs from MaXLinker at 1% FDR with
CSMs identified by XlinkX at 1% FDR with further filtering at different ‘XlinkX Score’ cut-offs for
XlinkX (using the same set of raw files for six E. coli MS2-MS3 XL-MS fractions from Liu et al9). Left
panel shows results for analysis using false search space (fraction of mis-identifications given in parentheses
below the number of CSMs) and the right panel represents precision-based analysis (precision given in
parentheses below the number of interprotein CSMs).
Supplementary Figure 5. Illustration of MaXLinker’s ‘MS2 Rescue’ module, in a case where the precursor
mass validation step fails along with uninformative MS3 spectra for either of the peptides
Supplementary Figure 6. Illustration of MaXLinker’s ‘MS2 Rescue’ module, in a case where the
precursor mass validation step fails along with all four MS3 spectra potentially representing different
charge states of one peptide.
P = 1.1 x 10-9
Fraction of m
is-identifications(%)
All
CSM
s
500
10
30
00
1000
1500
2000
20P = 7.9 x 10-9
P = 2.2 x 10-4
MS3
-Onl
y
CID-
MS2
-ETD
-MS2
CID-
MS2
-MS3
CID-
MS2
-MS3
-ETD
-MS2
Supplementary Figure 1
∆ XlinkX Score ≥ 10
∆ XlinkX Score ≥ 20
∆ XlinkX Score ≥ 40
∆ XlinkX Score ≥ 50
Fraction of m
is-identifications(%)
Fraction of m
is-identifications(%)
Fraction of m
is-identifications(%)
Fraction of m
is-identifications(%)
Fraction of m
is-identifications(%)
10
30
0
20
40
10
30
0
20
40
10
30
0
20
40
10
30
0
20
40
10
30
0
20
40
Uni
que
CSM
s
200
0
400
600
200
0
400
200
0
400
200
0
400
200
0
400
200
0
400
600
Precision (%)
25
75
0
50
100
200
0
400
Precision (%)
25
75
0
50
100
200
0
400
Precision (%)
25
75
0
50
100
200
0
400
Precision (%)
25
75
0
50
100
200
0
400
Precision (%)
25
75
0
50
100
MS3
-Onl
yCI
D-M
S2-E
TD-M
S2CI
D-M
S2-M
S3CI
D-M
S2-M
S3-E
TD-M
S2
∆ XlinkX Score ≥ 30
Uni
que
CSM
sU
niqu
e CS
Ms
Uni
que
CSM
sU
niqu
e CS
Ms
Uni
que
CSM
sU
niqu
e CS
Ms
Uni
que
CSM
sU
niqu
e CS
Ms
Uni
que
CSM
s
MS3
-Onl
yCI
D-M
S2-E
TD-M
S2
CID-
MS2
-MS3
CID-
MS2
-MS3
-ETD
-MS2
Supplementary Figure 2
∆ XlinkX Score ≥ 10
All
∆ XlinkX Score ≥ 20
∆ XlinkX Score ≥ 30
∆ XlinkX Score ≥ 40
∆ XlinkX Score ≥ 50
MS2-MS2 MS2-MS3
386(7.5%)
142(37.3%)
157(65.0%)
MS2-MS2 MS2-MS3
303(2.3%)
124(19.4%)
61(36.1%)
MS2-MS2 MS2-MS3
244(2.1%)
149(10.7%)
25(8.0%)
188(0.0%)
180(7.8%)
18(5.6%)
MS2-MS2 MS2-MS3
137(0.0%)
208(6.3%)
12(0.0%)
MS2-MS2 MS2-MS3
101(0.0%)
221(5.4%)
14(0.0%)
MS2-MS2 MS2-MS3 MS2-MS2 MS2-MS3
MS2-MS2 MS2-MS3
MS2-MS2 MS2-MS3
MS2-MS2 MS2-MS3
MS2-MS2 MS2-MS3
57(50.9%)
43(14.0%)
72(4.2%)
31(48.4%)
2(100%)
14(92.9%)
MS2-MS2 MS2-MS3
29(48.3%)
2(100%)
16(87.5%)
27(37.0%)
6(83.3%)
23(82.6%)
28(28.6%)
5(60.0%)
31(74.2%)
26(19.2%)
25(12.0%)
43(65.1%)
Supplementary Figure 3
∆ XlinkX Score ≥ 10
∆ XlinkX Score ≥ 20
∆ XlinkX Score ≥ 30
∆ XlinkX Score ≥ 40
∆ XlinkX Score ≥ 50
37(83.7%)
46(17.4%)
38(50.0%)
36(86.1%)
30(23.3%)
39(48.7%)
36(86.1%)
24(25.0%)
39(48.7%)
309(0.7%)
148(18.9%)
84(1.2%)
296(0.3%)
110(12.7%)
97(2.1%)
288(0.4%)
94(10.6%)
105(1.9%)
280(0.4%)
78(7.7%)
113(1.8%)
275(0.4%)
66(6.1%)
118(1.7%)
35(85.7%)
22(27.3%)
40(50.0%)
34(85.3%)
20(25.0%)
41(51.2%)
MaXLinker
MaXLinker
MaXLinker
MaXLinker
MaXLinker
MaXLinker
MaXLinker
MaXLinker
MaXLinker
MaXLinkerXlinkX v2.0
XlinkX v2.0
XlinkX v2.0
XlinkX v2.0
XlinkX v2.0 XlinkX v2.0
XlinkX v2.0
XlinkX v2.0
XlinkX v2.0
XlinkX v2.0
Supplementary Figure 4
Peptide A
Peptide B Uninformative MS3 spectrum Uninformative MS3 spectrum
AShortScan: 21166m/z: 574.31909z: 2+m: 1147.63091
ALongScan: 21167m/z: 590.30536z: 2+m: 1179.60344
BShortScan: 21170m/z: 565.81036z: 2+m: 1130.61345
BLongScan: 21171m/z: 581.79999z: 2+m: 1162.59270
precursor mass - (AShort + BLong + H2O - H+) = 479.12078precursor mass - (ALong+ BShort + H2O - H+) = 479.12750
Precursorm/z: 702.34265
z: 4+m: 2806.34713
QYLT[K]ELAK
EFADNLDSDF[K]VR
Peptide B
BShortm/z: 805.37466z: 2+m: 1609.74204
Derived BLongm/z: 821.36093z: 2+m: 1641.71458
MS2 rescue m
odule
Derived BShort = precursor mass - (ALong+ H2O - H+)Derived BLong = precursor mass - (AShort+ H2O - H+)
PSM search on MS2 spectrum oncewith each derived (‘Long’ and ‘Short’ )
mass as precursor
Validate derived masses in the MS2 spectrum
High con�dencePSMs
MaXLinker’s further validation �lters
Intraprotein cross-link in E. coli’s 30S ribosomal protein S3
(uniprot: P0A7V3)
Supplementary Figure 5
Peptide A
Peptide B
AShortScan: 12705 m/z: 639. 36523 z: 2+m: 1277. 72319
ALongScan: 12704m/z: 655.35364z: 2+m: 1309.70000
BLongScan: 12706m/z: 437. 23773 z: 3+m: 1309. 69864
BShortScan: 12707m/z: 426. 58060z: 3+m: 1277. 72724
precursor mass - (AShort + BLong + H2O - H+) = 351. 22760 precursor mass - (ALong+ BShort + H2O - H+) = 351.23301
Precursorm/z: 564.05511
z: 4+m: 2253.19697
VNAL[K]EQA
LTAH[K]NAVTLR
Peptide A
Derived AShortm/z: 926.49669 z: 1+m: 926. 49669
Derived ALongm/z: 958.46808z: 1+m: 958.46808
MS2 rescue m
odule
Derived AShort = precursor mass - (BLong+ H2O - H+)Derived ALong = precursor mass - (BShort+ H2O - H+)
PSM search on MS2 spectrum oncewith each derived (‘Long’ and ‘Short’ )
mass as precursor
High con�dencePSMs
MaXLinker’s further validation �lters
Intraprotein cross-link in E. coli’s Histidinol dehydrogenase
(uniprot: P06988)
Validate derived masses in the MS2 spectrum
Supplementary Figure 6