UV laser induced cross-linking in peptides.
Gabriella Leo1, Carlo Altucci2, Sandrine Bourgoin-Voillard3, Alfredo M. Gravagnuolo1,
Rosario Esposito2, Gennaro Marino1, Catherine E. Costello3, Raffaele Velotta2, Leila
Birolo1.
1Dipartimento
2Dipartimento
3Center
di Scienze Chimiche, Università di Napoli Federico II, Napoli, Italy ;
di Scienze Fisiche, Università di Napoli Federico II, Napoli, Italy;
for Biomedical Mass Spectrometry, Department of Biochemistry, Boston
University School of Medicine, Boston, Massachusetts 02118, USA.

Corresponding author: birolo@unina.it phone +39 (081) 679939; fax +39 (081) 674313
UV laser induced cross-linking in peptides
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Table S1. Assignments of the ESI-CID MS/MS spectrum (Fig. 2a) of xenopsin
homodimer.
Product ion assignment
a3
a4
b4
Calculated Experimental
Charge z error(ppm)
m/z
m/z*
269.1608
269.1601
1
-2.7
425.2619
425.2618
1
-0.3
453.2568
453.2569
1
0.0
b5
550.3095
550.3069
1
-4.8
a8αa6β-H2O
556.6476
556.6477
3
0.3
a8αa6β
562.6510
562.6511
3
0.3
a8αa7β
571.9826
571.9830
3
0.6
b8αb6β
600.3457
600.3459
3
0.4
b8αb7β
609.6773
609.6777
3
0.6
y4αy8β
753.4349
753.4354
2
0.7
y5αy8β
831.4855
831.4855
2
0.1
a8αa6β
843.4729
843.4734
2
0.6
b8αb6β
857.4703
857.4703
2
0.0
a8αa7β
900.0149
900.0154
2
0.5
b8αb7β
914.0124
914.0127
2
0.4
c5
567.3362
567.33625
1
0.2
[(2Mx -2H) + 3H]3+
653.3756
653.3759
3
0.4
[Mx+H]+
980.5676
980.5676
1
0.0
[(2Mx -2H-H2O) + 3H]3+
647.3721
647.3723
3
0.4
* product ion values calculated for z = 1
UV laser induced cross-linking in peptides
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Table S2. Assignments of the ESI-ECD MS/MS spectrum (Fig. 2b) of xenopsin
homodimer.
Product ion
assignment
c3
c5
Calculated Experimental
m/z
m/z*
314.1823
314.1823
567.3362
567.3361
Charge z
error(ppm)
1
1
0.0
-0.1
z5αz8β
x5αx8β
823.4762
844.4750
823.4767
844.4804
2
2
0.7
6.4
c8αc6β
865.9836
865.9836
2
0.0
z6αz8β
887.5236
887.5237
2
0.1
y6αy8β
895.5329
895.5326
2
-0.4
z7αz8β
916.0344
916.0342
2
-0.2
c8αc7β
922.5256
922.5254
2
-0.2
y7αy8β
924.0437
924.0455
2
1.9
z3αz8β
1392.7938
1392.7920
1
-1.3
z5αz8β
1645.9450
1645.9454
1
0.2
y5αy8β
1661.9340
1661.9637
1
17.8
c8αc6β
1730.9591
1730.9599
1
0.5
c8αc7β
1844.0440
1844.0441
1
0.0
[(2Mx -2H )+ 3H]3+
653.3756
653.37569
3
0.1
[(2Mx -2H)+ 3H]2+.
980.0637
980.06348
2
-0.2
* product ion values calculated for z = 1
UV laser induced cross-linking in peptides
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Table S3. Assignments of the ESI-CID MS/MS spectrum (Fig.3a) of angiotensin Ixenopsin dimer.
y4α
y2α
a6β
a6α
Calculated
m/z
257.1446
269.1608
354.7006
378.7112
b6α
392.7086
392.7085
2
-0.3
a7β
411.2427
411.2428
2
0.4
y3α
416.2292
416.2293
1
0.1
b4β
453.2568
453.2570
1
0.3
a8α
500.7718
500.7719
2
0.1
y4α
513.2820
513.2819
1
-0.3
b8α
514.7692
514.7692
2
-0.2
b4α
534.2671
534.2666
1
-0.9
c5β
a6αa8β
567.3362
578.6556
567.3377
578.6580
1
2
2.7
4.2
b9α
583.2987
583.2985
2
-0.3
b6αb8β
587.9873
587.9897
3
4.1
y9α
y10αy4β
b7αb8β
591.3325
607.9990
620.3382
591.3326
607.9977
620.3404
2
3
3
0.1
-2.1
3.5
b5α
a8αa8β
b8αb8β
647.3511
660.0294
669.3610
647.3511
660.0317
669.3633
1
3
3
0.0
3.5
3.4
b10αb6β
677.3526
677.3549
3
3.5
a10αa7β
705.7157
705.7178
3
3.0
b9αb8β
715.0473
715.0493
3
2.8
b6α
b5αb8β
784.4101
812.9478
784.4095
812.9519
1
2
-0.7
5.0
b6αb8β
881.4773
881.4810
2
4.2
b8αb8β
1003.5379
1003.5410
2
3.1
[(Mx + Ma -2H)+ 4H]4+
569.3128
569.3127
4
-0.2
Product ions assignment
* product ion values calculated for z = 1
Experimental
Charge z error(ppm)
m/z*
257.1446
2
-0.1
269.1608
1
0.0
354.7006
1
-0.1
378.7111
2
-0.2
UV laser induced cross-linking in peptides
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Table S4. Assignments of the ESI-ECD MS/MS spectrum (Fig. 3b) of angiotensin Ixenopsin dimer.
c2α
c3β
Calculated
m/z
289.1619
314.1823
c3α
z3α
388.2303
400.2105
388.2304
400.2105
1
1
0.3
0.0
y4α
513.2820
513.2820
1
0.0
z5α
634.3222
634.3225
1
0.5
c10αc6β
683.0306
683.0308
3
0.3
z10αz6β
697.3906
697.3891
3
-2.1
y10αy6β
702.7310
702.7301
3
-1.3
y9αy8β
715.0670
715.0670
3
0.0
z10αz7β
716.3986
716.3980
2
-0.9
c10αc7β/c9αc8β
720.7253
720.7255
3
0.3
z6α
747.4063
747.4066
1
0.5
y6α
763.4250
763.4251
1
0.2
c4αc8β
764.9228
764.9229
2
0.1
c5αc8β
821.4648
821.4649
2
0.1
z10αz3β
854.9605
854.9579
2
-3.0
c7α
898.4894
898.4892
1
-0.2
c7αc8β
938.5207
938.5207
2
0.1
z7αz8β
944.5121
944.5111
2
-1.1
z10αz5β
981.5348
981.5353
2
0.5
z8αz8β
994.0450
994.0451
2
0.1
y8αy8β
1002.0556
1002.0553
2
-0.3
c8αc8β
1012.0549
1012.0553
2
0.5
c10αc7β
1080.5806
1080.5801
2
-0.5
[(Mx + Ma -2H)+ 4H]4+
569.3128
569.3131
4
0.4
[(Mx + Ma -2H-NH3)+ 3H]3+
753.4084
753.40852
3
0.1
[(Mx + Ma -2H)+ 3H]3+
759.0839
759.0840
3
0.0
Product ion assignment
* product ion values calculated for z = 1
Experimental
Charge z error(ppm)
m/z*
289.1620
2
0.2
314.1823
1
0.2
UV laser induced cross-linking in peptides
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Figure S1. MALDI-TOF mass spectra of angiotensin I (Ma) not irradiated (panel A)
and irradiated for 10 sec (panel B).
UV laser induced cross-linking in peptides
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Figure S2. MALDI-TOF mass spectra of interleukin (Mi) not irradiated (panel A) and
irradiated for 10 sec (panel B).
UV laser induced cross-linking in peptides
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A
B
Figure S3. ESI-FT-ICR mass spectra of the [(2Mx -2H) + 3H]3+ ions that have been
selected for fragmentation in the ESI-CID (A) and ESI-ECD (B) MS/MS, respectively.
The error associated to this measurement and the resolution are also indicated.
UV laser induced cross-linking in peptides
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A
B
Figure S4. ESI-FT-ICR mass spectra of the [(Mx + Ma - 2H) + 4H]4+ ions that have
been selected for fragmentation in the ESI-CID (A) and ESI-ECD (B) MS/MS,
respectively. The error associated to this measurement and the resolution are also
indicated.
UV laser induced cross-linking in peptides
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Figure S5. ESI-ECD mass spectrum of xenopsin exposed to UV-laser in the
presence of DMPO. The [M + 2H]2+ peak at m/z 546.32 was selected as the
precursor ion. The asterisk indicates the residue at the adduct site. The ions
detected in the CID spectrum are indicated in red.
UV laser induced cross-linking in peptides
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m/z
Figure S6. ESI-ETD mass spectrum of xenopsin exposed to UV-laser in the
presence of MNP. The [M + 2H]2+ peak at m/z 534.32 was selected as the precursor
ion. The asterisk indicates the residue at the adduct site. The ion series detected in
the CID spectrum are indicated in red.
ESI-MS analysis showed the presence of a doubly charged ion at m/z 534.3219, a
value corresponding to an isotopic mass of 1067.6339 Da which is shifted by +
87.0684 Da with respect to the theoretical mass of the unmodified peptide. These
data suggest that a single MNP molecule was trapped on the xenopsin peptide.
MS/MS analyses (Fig. S1) on the SolariX FTMS of this ion were used to investigate
which amino acid of xenopsin was the target of the spin trap after UV laser crosslinking. As an example, the ETD spectrum of the [Mx + MMNP + 2H]2+ ion yielded the
charge-reduced ion [Mx + MMNP + 2H]2+ at m/z 1068.6437 (calc. m/z 1068.6438) and,
more interestingly, product ions of the c-, z- and y- series bearing the MNP molecule.
For example, the z5*, z6*, z7*, c6* and c7*, and y6* ions (calc. m/z 755.4688, m/z
883.5638, m/z 940.5853, m/z 840.4839, m/z 953.5679 and m/z 899.5825,
respectively) allowed us to localize the MNP on the Arg, Pro or Trp amino acids.
Interestingly, the c5 ion at m/z 567.3363 (calc. m/z 567.3362) corresponding to
cleavage of amino acid from the N-terminus of the peptide backbone devoid of MNP
was observed. Hence this ion and the product ions preserving the MNP moiety
suggested that Trp-6 is the amino acid involved in the bond with the spin trap MNP.
UV laser induced cross-linking in peptides
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Figure S7: ESI-ECD mass spectrum of angiotensin exposed to UV-laser in the
presence of DMPO. The [M + 3H]3+ peak at m/z 469.9235 was selected as the
precursor ion. The asterisk indicates the residue at the adduct site. The ions
detected in the CID spectrum are indicated in red.
UV laser induced cross-linking in peptides
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m/z
Figure S8: ESI-ECD mass spectrum of angiotensin exposed to UV-laser in the
presence of MNP. The [M + 3H]3+ peak at m/z 461.9227 was selected as the
precursor ion. The asterisk indicates the residue at the adduct site. The ions
detected in the CID spectrum are indicated in red.
UV laser induced cross-linking in peptides
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Figure S9. MALDI-TOF mass spectrum of xenopsin after exposure to the high
intensity UV laser for 1.56 s (1.3x106 µW cm-2, 160 µJ/pulse, 2 kHz repetition rate,
1.56 s), so that an energy of 0.5 J was released to the solution, which was then left
in the open air for 58 min. In the insets, zoom of selected m/z ranges.
UV laser induced cross-linking in peptides
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Fig. S10. Zoom of selected m/z ranges of the MALDI-TOF mass spectra of xenopsin
(A) and xenopsin after exposure to the high intensity (B) and to the low intensity UV
laser (C).