Supporting Information
LC-MS/MS Analysis and Comparison of Oxidative Damages on Peptides
Induced by Pathogen Reduction Technologies for Platelets
Michel Prudent§, Giona Sonego§, Mélanie Abonnenc, Jean-Daniel Tissot, Niels Lion*
Service Régional Vaudois de Transfusion Sanguine, Unité de Recherche et Développement,
Lausanne, Switzerland
§ MP and GS contributed equally to this work.
Address reprint request to : Niels Lion, Service Régional Vaudois de Transfusion Sanguine,
1066 Epalinges, Switzerland, Tel : +41 21 314 65 68, Fax: +41 21 314 65 78 Email:
niels.lion@mavietonsang.ch
S1
Setup of homemade Mirasol system
Figure S1. UV spectral chart used for the irradiation of Riboflavin
(http://www.lighting.philips.com/pwc_li/main/application_areas/assets/phototheraphy/philips_photothera
py_lamps_catalogue.pdf)
Riboflavin (%)
Photodegradation of Riboflavin
100
90
80
70
60
50
40
30
20
10
0
Mirasol (12.5min - 78.9%)
0
10 20 30 40 50 60 70 80 90
Illumination time (min)
Figure S2. Calibration of Mirasol setup: Riboflavin degradation in presence of UVB with the homemade
system. Mirasol conditions were reached after 12.5 min of irradiation.
S2
UV Chromatograms (214 nm)
1
*
Figure S3. UV chromatogram of peptide M + UV. (1) 11.6 min peptide M (m/z 636.2). *: contaminant
coming from InterSol.
2
*
1
Figure S4. UV chromatogram of peptide M Intercept. (1) 10.8 min oxidation (m/z 668.1), (2) 11.6 min
peptide M (m/z 636.2). Other main peaks correspond to degradation products of amotosalen (see Figure
1). *: contaminant coming from InterSol.
S3
*
2
1
Figure S5. UV chromatogram of peptide M Mirasol. (1)10.6 min oxidation (m/z 652.2 / 668.1), (2) 11.6
min peptide M (m/z 636.2). Other main peaks correspond to degradation products of riboflavin (see
Figure 1). *: contaminant coming from InterSol.
3
1
*
2
Figure S6. UV chromatogram of peptide Fib seq + UV. (1) 22.2 min Fib seq peptide z=2 (m/z 625.7), (2)
22.4 min methasulfonic acid z=2 (m/z 649.2), (3) 23.9 min dimer z=3 (m/z 832.6) *: contaminant coming
from InterSol.
S4
4
3
12
*
Figure S7. UV chromatogram of peptide Fib seq Intercept. (1) 21.2 min sulfonic acid Z=2 (m/z 649.2),
(2) 21.8 min oxidation + 32 Da Z=3 (m/z 843.3), (3) 22.0 min oxidation + 30 Da z=3 (m/z 842.6), (4) 22.8
min dimer (m/z 1248.9). Other main peaks correspond to degradation products of amotosalen (see Figure
1). *: contaminant coming from InterSol.
*
1 23
Figure S8. UV chromatogram of peptide Fib seq Mirasol. (1) 21.3 min sulfonic acid z=2 (m/z 649.2), (2)
22.2 min oxidation + 32 Da z=3 (m/z 843.3), (3) 22.8 min dimer (m/z 1248.9). Other main peaks
correspond to degradation products of riboflavin (see Figure 1). *: contaminant coming from InterSol.
S5
*
1
2
Figure S9. UV chromatogram of peptide cC + UV. (1) 15.2 min cC peptide (m/z 548.2), (2) 16.4 min
dimer z=2 (m/z 547.2). *: contaminant coming from InterSol.
*
3
1 2 45
Figure S10. UV chromatogram of peptide cC Intercept. (1) 12.9 min sulfonic acid (m/z 596.2), (2) 15.2
min (m/z 548.2) monomer, (3) 16.4 dimer z=2 (m/z 547.2), (4) 16.9 min unknown (m/z 555.2), (5) 18.3
min +30 Da oxidation unknown (m/z 578.2). Other main peaks correspond to degradation products of
amotosalen (see Figure 1). *: contaminant coming from InterSol.
S6
*
12
3 4
Figure S11. UV chromatogram of peptide cC Mirasol. (1) 13.0 min sulfonic acid (m/z 596.2), (2)
13.8/14.8 min (m/z 548.2)monomer, (3) 16.5 dimer Z=2 (m/z 547.2), (4) 18.3 min +30 Da oxidation
unknown (m/z 578.2). Other main peaks correspond to degradation products of riboflavin (see Figure 1).
*: contaminant coming from InterSol.
1
*
Figure S12. UV chromatogram of peptide angII + UV. (1) 13.3 min angII z=2 (m/z 523.7). This
chromatogram was obtained with gradient used for M peptide. *: contaminant coming from InterSol.
S7
4
3
2 5
1
*
Figure S13. UV chromatogram of peptide angII Intercept. (1) 22.0 min double ox. (m/z 538.7), (2) 22.4
min single ox. (m/z 531.7), (3) 22.7 min double ox. (m/z 539.7), (4) 23.2 min AngII peptide (m/z 523.7),
(5) 23.8 min single ox. (m/z 530.7). All these MS peaks are z = 2. Other main peaks correspond to
degradation products of amotosalen (see Figure 1). *: contaminant coming from InterSol.
2
3
4
6
1 5
*
Figure S14. UV chromatogram of peptide angII Mirasol. (1) 22.0 min double ox. (m/z 538.7), (2) 22.7
min double ox. (m/z 539.7), (3) 23.2 min single ox. (m/z 530.7), (4) 23.9 min unknown ox. + 46 Da (m/z
546.7), (5) 24.6 min unknown ox. (m/z 555.8), (6) 22.5 min single ox. (m/z 531.7). All these MS peaks are
z=2. Other main peaks correspond to degradation products of riboflavin (see Figure 1). *: contaminant
S8
coming from InterSol.
1
*
Figure S15. UV chromatogram of peptide cH + UV. (1) 13.1 min monomer (m/z 582.3). *: contaminant
coming from InterSol.
1
*
22
Figure S16. UV chromatogram of peptide cH Intercept. (1) 13.0 min monomer (m/z 582.3), (2) 14.1/15.0
min single oxidation (m/z 596.2). Other main peaks correspond to degradation products of amotosalen
S9
(see Figure 1). *: contaminant coming from InterSol.
*
122
Figure S17. UV chromatogram of peptide cH Mirasol. (1) 13.4 min monomer (m/z 582.3), (2) 14.3/15.2
min single oxidation (m/z 596.2). Other main peaks correspond to degradation products of riboflavin (see
Figure 1). *: contaminant coming from InterSol.
S10
1
*
Figure S18. UV chromatogram of peptide YY + UV. (1) 18.5 min monomer (m/z 345.2). *: contaminant
coming from InterSol.
3
*
12
Figure S19. UV chromatogram of peptide YY Intercept. (1) 17.2 min double ox. (375.1 m/z), (2) 17.7
min double ox. (377.1 m/z), (3) 18.5 min monomer (345.2 m/z). Other main peaks correspond to
S11
degradation products of amotosalen (see Figure 1). *: contaminant coming from InterSol.
3
*
1 2
Figure S20. UV chromatogram of peptide YY Mirasol. (1) 16.0/17.5 min double oxidation (m/z 377.1),
(2) 18.7 min monomer (m/z 345.2). Other main peaks correspond to degradation products of riboflavin
(see Figure 1). *: contaminant coming from InterSol.
S12
Tandem mass spectra of the oxidized peptides reported in Table 2.
cC (parent ion m/z 596.2)
Figure S21. Tandem mass spectrum of oxidized cysteine (+ 48 Da). *: modified amino-acids.
cC (parent ion m/z 547.7 dimer)
Figure S22. Tandem mass spectrum of oxidized cysteine (-2 Da). *:modified amino-acids.
cC (parent ion m/z 578.2)
Figure S23. Tandem mass spectrum of oxidized cysteine (+30 Da). *: modified amino-acids.
S13
cC (parent ion m/z 555.7 dimer)
Figure S24. Tandem mass spectrum of oxidized cysteine (+16 Da) not identified. *: modified aminoacids.
Fib seq (parent ion m/z 1248.9 dimer)
Figure S25. Tandem mass spectrum of oxidized cysteine (-2 Da). *: modified amino-acids.
S14
Fib seq (parent ion m/z 649.7)
Figure S26. Tandem mass spectrum of oxidized cysteine (+ 48 Da). In blue are 2+ ions peaks. *:
modified amino-acids.
YY (parent ion m/z 375.1)
Figure S27. Tandem mass spectrum of oxidized tyrosine (+ 30 Da). *: modified amino-acids.
S15
YY (parent ion m/z 377.1)
Figure S28. Tandem mass spectrum of oxidized tyrosine (+ 32 Da). *: modified amino-acids.
M (parent ion m/z 668.2)
Figure S29. Tandem mass spectrum of oxidized tyrosine (+ 16 Da). *: modified amino-acids.
S16
AngII (parent ion m/z 538.7)
Figure S30 . Tandem mass spectrum of oxidized tyrosine (+ 30 Da). In blue are 2+ ions peaks. *:
modified amino-acids.
AngII (parent ion m/z 531.7)
Figure S31 . Tandem mass spectrum of oxidized histidine (+ 16 Da). In blue are 2+ ions peaks. *:
modified amino-acids.
S17
AngII (parent ion m/z 539.7)
Figure S32 . Tandem mass spectrum of oxidized histidine (+ 32 Da). In blue are 2+ ions peaks. *:
modified amino-acids.
Ang II (parent ion m/z 546.7 dimer)
Figure S33. Tandem mass spectrum of oxidized histidine and tyrosine(+ 46 Da). +32 Da confirmed on
histidine (see y3, y3 and b6 ions). Probable+14 Da on tyrosine. *: modified amino-acids.
S18
cH (parent ion m/z 596.2)
Figure S34. Tandem mass spectrum of oxidized histidine (+ 14 Da). *: modified amino-acids.
S19
t-test analyses
t-test total ox
R Console Page 1
R version 3.0.1 (2013-05-16) -- "Good Sport"
Copyright (C) 2013 The R Foundation for Statistical Computing
Platform: x86_64-w64-mingw32/x64 (64-bit)
R is free software and comes with ABSOLUTELY NO WARRANTY.
You are welcome to redistribute it under certain conditions.
Type 'license()' or 'licence()' for distribution details.
R is a collaborative project with many contributors.
Type 'contributors()' for more information and
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'help.start()' for an HTML browser interface to help.
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> cC
Error: object 'cC' not found
>
> cC.intercept=c(86.30,81.84,88.41)
> cC.mirasol=c(94.30,88.40,93.72)
>
> (cC=t.test(cC.intercept,cC.mirasol,paired=T))
Paired t-test
data: cC.intercept and cC.mirasol
t = -8.5222, df = 2, p-value = 0.01349
alternative hypothesis: true difference in means is not equal to 0
95 percent confidence interval:
-9.967276 -3.279391
sample estimates:
mean of the differences
-6.623333
>
> cH
Error: object 'cH' not found
>
> cH.intercept=c(9.92,10.31,11.33)
> cH.mirasol=c(91.46,83.19,70.49)
>
> (cH=t.test(cH.intercept,cH.mirasol,paired=T))
Paired t-test
data: cH.intercept and cH.mirasol
t = -10.927, df = 2, p-value = 0.008271
alternative hypothesis: true difference in means is not equal to 0
95 percent confidence interval:
-99.22666 -43.16000
sample estimates:
mean of the differences
-71.19333
>
> cW
Error: object 'cW' not found
>
> cW.intercept=c(19.14,17.75,21.92)
> cW.mirasol=c(100.0,100.0,100.0)
S20
>
> (cW=t.test(cW.intercept,cW.mirasol,paired=T))
Paired t-test
data: cW.intercept and cW.mirasol
t = -65.5836, df = 2, p-value = 0.0002324
alternative hypothesis: true difference in means is not equal
95 percent confidence interval:
-85.67114 -75.12220
sample estimates:
mean of the differences
R Console Page 2
-80.39667
>
> YY
Error: object 'YY' not found
>
> YY.intercept=c(3.86,2.74,4.37)
> YY.mirasol=c(44.73,15.20,16.97)
>
> (YY=t.test(YY.intercept,YY.mirasol,paired=T))
Paired t-test
data: YY.intercept and YY.mirasol
t = -2.3264, df = 2, p-value = 0.1455
alternative hypothesis: true difference in means is not equal
95 percent confidence interval:
-62.62276 18.66943
sample estimates:
mean of the differences
-21.97667
>
> M
Error: object 'M' not found
>
> M.intercept=c(0.98,1.19,1.21)
> M.mirasol=c(14.55,6.92,11.19)
>
> (M=t.test(M.intercept,M.mirasol,paired=T))
Paired t-test
data: M.intercept and M.mirasol
t = -4.3074, df = 2, p-value = 0.0499
alternative hypothesis: true difference in means is not equal
95 percent confidence interval:
-19.50931486 -0.01068514
sample estimates:
mean of the differences
-9.76
>
> angII
Error: object 'angII' not found
>
> angII.intercept=c(49.62,44.91,17.27)
> angII.mirasol=c(51.73,38.10,18.85)
>
> (angII=t.test(angII.intercept,angII.mirasol,paired=T))
Paired t-test
data: angII.intercept and angII.mirasol
t = 0.36, df = 2, p-value = 0.7533
alternative hypothesis: true difference in means is not equal
S21
to 0
to 0
to 0
to 0
95 percent confidence interval:
-11.3906 13.4706
sample estimates:
mean of the differences
1.04
>
> Fib
Error: object 'Fib' not found
>
> Fib.intercept=c(100.0,100.0,100.0)
> Fib.mirasol=c(100.0,100.0,100.0)
>
> (Fib=t.test(Fib.intercept,Fib.mirasol,paired=T))
Paired t-test
data: Fib.intercept and Fib.mirasol
R Console Page 3
t = NaN, df = 2, p-value = NA
alternative hypothesis: true difference in means is not equal to 0
95 percent confidence interval:
NaN NaN
sample estimates:
mean of the differences
0
>
>
t-test remaining peptides
R Console Page 1
R version 3.0.1 (2013-05-16) -- "Good Sport"
Copyright (C) 2013 The R Foundation for Statistical Computing
Platform: x86_64-w64-mingw32/x64 (64-bit)
R is free software and comes with ABSOLUTELY NO WARRANTY.
You are welcome to redistribute it under certain conditions.
Type 'license()' or 'licence()' for distribution details.
R is a collaborative project with many contributors.
Type 'contributors()' for more information and
'citation()' on how to cite R or R packages in publications.
Type 'demo()' for some demos, 'help()' for on-line help, or
'help.start()' for an HTML browser interface to help.
Type 'q()' to quit R.
>
> cC
Error: object 'cC' not found
>
> cC.intercept=c(13.70,18.16,11.59)
> cC.mirasol=c(5.70,11.60,6.28)
>
> (cC=t.test(cC.intercept,cC.mirasol,paired=T))
Paired t-test
data: cC.intercept and cC.mirasol
t = 8.5222, df = 2, p-value = 0.01349
alternative hypothesis: true difference in means is not equal to 0
95 percent confidence interval:
3.279391 9.967276
sample estimates:
mean of the differences
S22
6.623333
>
> cH
Error: object 'cH' not found
>
> cH.intercept=c(90.08,89.69,88.67)
> cH.mirasol=c(8.54,16.81,29.51)
>
> (cH=t.test(cH.intercept,cH.mirasol,paired=T))
Paired t-test
data: cH.intercept and cH.mirasol
t = 10.927, df = 2, p-value = 0.008271
alternative hypothesis: true difference in means is not equal to 0
95 percent confidence interval:
43.16000 99.22666
sample estimates:
mean of the differences
71.19333
>
> cW
Error: object 'cW' not found
>
> cW.intercept=c(80.86,82.25,78.08)
> cW.mirasol=c(0.0,0.0,0.0)
>
> (cW=t.test(cW.intercept,cW.mirasol,paired=T))
Paired t-test
data: cW.intercept and cW.mirasol
t = 65.5836, df = 2, p-value = 0.0002324
alternative hypothesis: true difference in means is not equal to 0
95 percent confidence interval:
75.12220 85.67114
sample estimates:
R Console Page 2
mean of the differences
80.39667
>
> YY
Error: object 'YY' not found
>
> YY.intercept=c(96.14,97.26,95.63)
> YY.mirasol=c(55.27,84.80,83.03)
>
> (YY=t.test(YY.intercept,YY.mirasol,paired=T))
Paired t-test
data: YY.intercept and YY.mirasol
t = 2.3264, df = 2, p-value = 0.1455
alternative hypothesis: true difference in means is not equal to 0
95 percent confidence interval:
-18.66943 62.62276
sample estimates:
mean of the differences
21.97667
>
> M
Error: object 'M' not found
>
> M.intercept=c(99.02,98.79,98.81)
S23
> M.mirasol=c(85.45,93.08,88.81)
>
> (M=t.test(M.intercept,M.mirasol,paired=T))
Paired t-test
data: M.intercept and M.mirasol
t = 4.2955, df = 2, p-value = 0.05015
alternative hypothesis: true difference in means is not equal to 0
95 percent confidence interval:
-0.01630495 19.53630495
sample estimates:
mean of the differences
9.76
>
> angII
Error: object 'angII' not found
>
> angII.intercept=c(50.38,55.09,82.73)
> angII.mirasol=c(48.27,61.90,81.15)
>
> (angII=t.test(angII.intercept,angII.mirasol,paired=T))
Paired t-test
data: angII.intercept and angII.mirasol
t = -0.36, df = 2, p-value = 0.7533
alternative hypothesis: true difference in means is not equal to 0
95 percent confidence interval:
-13.4706 11.3906
sample estimates:
mean of the differences
-1.04
>
> Fib
Error: object 'Fib' not found
>
> Fib.intercept=c(0.0,0.0,0.0)
> Fib.mirasol=c(0.0,0.0,0.0)
>
> (Fib=t.test(Fib.intercept,Fib.mirasol,paired=T))
Paired t-test
R Console Page 3
data: Fib.intercept and Fib.mirasol
t = NaN, df = 2, p-value = NA
alternative hypothesis: true difference in means is not equal to 0
95 percent confidence interval:
NaN NaN
sample estimates:
mean of the differences
0
>
>
t-test cysteine oxidation
R Console
R version
Copyright
Platform:
R is free
Page 1
3.0.1 (2013-05-16) -- "Good Sport"
(C) 2013 The R Foundation for Statistical Computing
x86_64-w64-mingw32/x64 (64-bit)
software and comes with ABSOLUTELY NO WARRANTY.
S24
You are welcome to redistribute it under certain conditions.
Type 'license()' or 'licence()' for distribution details.
R is a collaborative project with many contributors.
Type 'contributors()' for more information and
'citation()' on how to cite R or R packages in publications.
Type 'demo()' for some demos, 'help()' for on-line help, or
'help.start()' for an HTML browser interface to help.
Type 'q()' to quit R.
> cC_dimer
Error: object 'cC_dimer' not found
>
> cC_dimer.intercept=c(71.85,72.48,63.10)
> cC_dimer.mirasol=c(35.04,27.69,25.37)
>
> (cC_dimer=t.test(cC_dimer.intercept,cC_dimer.mirasol,paired=T))
Paired t-test
data: cC_dimer.intercept and cC_dimer.mirasol
t = 15.78, df = 2, p-value = 0.003992
alternative hypothesis: true difference in means is not equal to 0
95 percent confidence interval:
28.93098 50.62235
sample estimates:
mean of the differences
39.77667
>
> Fib_dimer
Error: object 'Fib_dimer' not found
>
> Fib_dimer.intercept=c(74.94,76.95,63.99)
> Fib_dimer.mirasol=c(18.14,40.08,46.54)
>
> (Fib_dimer=t.test(Fib_dimer.intercept,Fib_dimer.mirasol,paired=T))
Paired t-test
data: Fib_dimer.intercept and Fib_dimer.mirasol
t = 3.2607, df = 2, p-value = 0.08257
alternative hypothesis: true difference in means is not equal to 0
95 percent confidence interval:
-11.83678 85.91678
sample estimates:
mean of the differences
37.04
>
> cC_ox
Error: object 'cC_ox' not found
>
> cC_ox.intercept=c(14.45,9.36,25.30)
> cC_ox.mirasol=c(59.26,60.71,68.35)
>
> (cC_ox=t.test(cC_ox.intercept,cC_ox.mirasol,paired=T))
Paired t-test
data: cC_ox.intercept and cC_ox.mirasol
t = -18.3777, df = 2, p-value = 0.002948
alternative hypothesis: true difference in means is not equal to 0
95 percent confidence interval:
-57.26743 -35.53923
sample estimates:
mean of the differences
R Console Page 2
S25
-46.40333
>
> Fib_ox
Error: object 'Fib_ox' not found
>
> Fib_ox.intercept=c(25.06,23.05,36.01)
> Fib_ox.mirasol=c(81.86,59.92,53.46)
>
> (Fib_ox=t.test(Fib_ox.intercept,Fib_ox.mirasol,paired=T))
Paired t-test
data: Fib_ox.intercept and Fib_ox.mirasol
t = -3.2607, df = 2, p-value = 0.08257
alternative hypothesis: true difference in means is not equal to 0
95 percent confidence interval:
-85.91678 11.83678
sample estimates:
mean of the differences
-37.04
>
S26