Supporting information Oxidation of Tyrosine-Phosphopeptides by ...
Supporting information
Oxidation of Tyrosine-Phosphopeptides by Titanium Dioxide Photocatalysis Miina Ruokolainen†, Elisa Ollikainen†, Tiina Sikanen†, Tapio Kotiaho†,‡, Risto Kostiainen*† †
Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, P.O. Box 56 (Viikinkaari 5E), FI-00014, FINLAND, ‡ Department of Chemistry, University of Helsinki, P.O. BOX 55 (A.I. Virtasen aukio 1), FI00014, FINLAND [email protected], [email protected], [email protected], [email protected], [email protected] Corresponding author: (email) [email protected], (tel.) + 358 2941 59134
S1
Chemicals
The insulin receptor peptides TRDIYETDYYRK (IR0), TRDIpYETDYYRK (IR1A), TRDIYETDpYYRK (IR1B), TRDIYETDYpYRK (IR1C), and TRDIpYETDpYpYRK (IR3) were from Anaspec (Fremont, CA, USA) (Table S1). TiO2 Degussa P-25, formic acid (98–100%) and LC-MS grade methanol were from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Ammonium hydroxide was from Mallinckrodt Baker B.V. (Denventer, the Netherlands). Water was purified with a Milli-Q Plus (Millipore, Molsheim, France) purification system. Table S1. Abbreviations, sequences, formulae and monoisotopic masses of the peptides used. Abbreviation Sequence Formula Mmonoisotope IR0 TRDIYETDYYRK C72H107N19O24 1621.77 IR1A TRDIpYETDYYRK C72H108N19O27P 1701.74 IR1B TRDIYETDpYYRK C72H108N19O27P 1701.74 IR1C TRDIYETDYpYRK C72H108N19O27P 1701.74 IR3 TRDIpYETDpYpYRK C72H110N19O33P3 1861.67
Ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS)
The samples were analyzed using ACQUITY UPLC™ (Waters, Milford, MA, USA) and Xevo™ Q-TOF-MS (Waters, Manchester, UK) in the positive electrospray ionization mode. A Vanguard BEH C18 precolumn (5 mm x 2.1 mm i.d., 1.7 µm, Waters) was used prior to the analytical ACQUITY UPLC BEH C-18 column (100 mm x 2.1 mm i.d., 1.7 µm, Waters). The chromatographic parameters, solvent gradients used for analysis of reaction products and mass spectrometric parameters are in presented Tables S2-S5. Figure S1 presents extracted ion chromatograms of the oxidation products of the peptides. The chromatograms have been smoothed by Savitsky-Golay method (window size: 3 scans, number of smooths: 2). Figure S2 presents kinetic plots of the formation and degradation of the oxidation products. Table S6 presents accurate masses of the diagnostic product ions of the oxidized peptides. The measured masses of the oxidation products have been corrected using the relative deviation of the measured mass of the corresponding non-oxidized peptide in the same run.
S2
Table S2. Chromatographic parameters used for analysis of the photocatalytic reaction products of the phosphopeptides. Parameter Eluent A Eluent B Flow rate Column temperature Sample tray temperature Injection mode Injection volume
0.1% formic acid 0.1% formic acid in MeOH/H2O 98:2 450 µl min-1 40 °C 8 °C partial loop 5 µL
Table S3. Solvent gradients used for analysis of the photocatalytic reaction products of IR0 and IR3. IR0 Time (min) 0.00-4.50 4.50-8.50 8.50-9.00 9.00-9.10 9.10-12.50
B (%) 14 14 → 30 30 30 → 14 14
IR3 Time (min) 0.00-5.00 5.00-10.00 10.00-12.00 12.00-12.10 12.10-15.00
B (%) 10 10 → 30 30 30 → 10 10
Table S4. Solvent gradients used for analysis of the photocatalytic reaction products of IR1A, IR1B and IR1C. IR1A Time (min) 0.00-1.00 1.00-5.00 5.00-8.00 8.00-8.50 8.50-8.60 8.60-12.00
B (%) 10 → 14 14 14 → 30 30 30 →10 10
IR1B Time (min) 0.0-4.00 4.00-8.00 8.00-11.00 11.00-11.50 11.50-11.60 11.60-15.00
B (%) 10 → 12 12 12 → 30 30 30 → 10 10
IR1C Time (min) 0.00-0.50 0.50-6.00 6.00-9.00 9.00-9.50 9.50-9.60 9.60-13.00
B (%) 10 → 14 14 14 → 30 30 30 → 10 10
S3
Table S5. MS-parameters used for detection of the photocatalytic reaction products. Parameter Capillary voltage (kV) Extraction cone voltage (V) Sampling cone voltage (V) MS scan range (m/z) Product ion scan range (m/z) Scan time (s) Desolvation gas Collision gas Ion source temperature External reference (Lockspray)
IR0 IR1A, IR1B, IR1C IR3 3 3 4 0 0 0 20 20 45 400–1650 400–1750 400–1900 70–1650 70–1750 0.15 0.15 0.15 -1 Nitrogen (1000 L h , 500 C) Argon 150 C leucine enkephalin (concentration 2 ng µL-1, flow rate 20 µL min-1) at m/z 556.2771
S4
Figure S1. Extracted ion [M+3H]+3 chromatograms (EIC) of the oxidation products of the peptides from the UV exposure time which yielded highest abundance of the oxidation products (see Fig. S2).
S5
a) Degradation of the peptides (loss of the non-oxidized form) 100% IR0 IR1A Relative abundance
80%
IR1B IR1C
60%
40%
20%
0% 0
5
10
15
UV exposure time (min)
b) Formation of the initial reaction products 100 sum of M+O products of IR0 sum of M+O products of IR1A
Abundance
80
sum of M+2O products of IR1A sum of M+O products of IR1B
60
sum of M+O products of IR1C
40
20
0 0
5
10
15
UV exposure time (min)
Figure S2. a) Degradation of the peptides IR0, IR1A, IR1B and IR1C and b) formation of the initial reaction products under TiO2 photocatalysis calculated using the triply protonated ion.
S6
Table S6. The measured accurate masses of the oxidation products and their diagnostic product ions, and the deviations from the calculated masses. Single oxidized product ions are bolded and double oxidized product ions are bolded and underlined.
peptide/ oxidation tR product site (min) IR0 5.98 M+O Y9 1.90 M+O Y10 2.42 M+O Y5 4.31
precursor [M+3H]+3 m/z ΔM 541.5880 -0.0110 546.9300 -0.0006 546.9303 -0.0003 546.9284 -0.0022
b4 b5 m/z ΔM m/z 486.2705 0.0029 649.3353 486.2678 0.0002 649.3318 486.2664 -0.0012 649.3326 486.2692 0.0016 665.3261
ΔM 0.0044 0.0009 0.0017 0.0003
b6 m/z ΔM 778.3777 0.0042 778.3781 0.0046 778.3747 0.0012 794.3679 -0.0005
y3 m/z 466.2802 466.2787 482.2715 466.2791 466.2833 482.2759 466.2786 466.2683 466.2697 482.2766 466.2777 482.2757 466.2791 482.2758 466.2828 466.2807 466.2735 498.2608
ΔM 0.0024 0.0009 -0.0012 0.0013 0.0055 0.0032 0.0008 -0.0095 -0.0081 0.0039 -0.0001 0.0030 0.0013 0.0031 0.0050 0.0029 -0.0043 -0.0068
M+O
Y9 and Y10 a
4.57
546.9297
-0.0009 486.2672 -0.0004 649.3322
0.0013
778.3705 -0.0030
IR1A M+O
Y9
5.43 2.09
568.2451 573.5847
-0.0093 486.2681 -0.0014 486.2681
0.0005 0.0005
729.2985 729.2994
0.0013 0.0022
858.3416 858.3528
M+O
Y9 and Y10 a
2.29
573.5857
-0.0004 486.2702
0.0026
729.2919 -0.0053
858.3350 -0.0048
M+O
Y9 and Y10 a
2.57
573.5847
-0.0014 486.2687
0.0011
729.3034
0.0062
858.3338 -0.0060
M+O
Y9 and Y10 a
4.00
573.5880
0.0019
486.2699
0.0023
729.3001
0.0029
858.3419
M+O M+2O M+2O c M+2O d M+2O e M+2O f IR1B M+O M+O IR1C M+O M+O M+O M+O M+O
pY5 Y9 I4 Y10 Y9 b I4 Y5 Y10 Y9 Y9 Y5 Y9 pY10
7.00 2.62 2.94 3.13 3.30 3.67 9.64 7.42 7.57 5.20 1.94 2.14 3.61 3.80 8.29
573.5876 578.9168 578.9179 578.9175 578.9156 578.9163 568.2471 573.5871 573.5871 568.2452 573.5843 573.5843 573.5843 573.5859 573.5861
0.0015 -0.0009 0.0002 -0.0002 -0.0021 -0.0014 -0.0073 0.0010 0.0010 -0.0092 -0.0018 -0.0018 -0.0018 -0.0002 0.0000
486.2668 486.2682 518.2555 486.2684 486.2660 518.2527 486.2697 486.2640 486.2678 486.2697 486.2651 486.2656 486.2698 486.2723 486.2669
-0.0008 0.0006 -0.0019 0.0008 -0.0016 -0.0047 0.0021 -0.0036 0.0002 0.0021 -0.0025 -0.0020 0.0022 0.0047 -0.0007
745.2896 -0.0025 729.2986 0.0014 761.2942 0.0072 729.2996 0.0024 729.2967 -0.0005
874.3416 0.0069 858.3373 -0.0025 890.3182 -0.0114 858.3480 0.0082 858.3567 0.0169
649.3341 665.3259 649.3336 649.3344 649.3337 649.3317 665.3306 649.3328 649.3340
466.2786 0.0008 778.3761 0.0026 466.2807 0.0029 794.3617 -0.0067 466.2861 0.0083 778.3770 0.0035 482.2652 -0.0075 778.3770 0.0035 546.2476 0.0035 778.3776 0.0041 546.2446 0.0005 778.3693 -0.0042 546.2481 0.0040 794.3718 0.0034 546.2481 0.0040 778.3787 0.0052 546.2379 -0.0062 778.3809 0.0074 562.2235 -0.0155
0.0032 0.0001 0.0027 0.0035 0.0028 0.0008 0.0048 0.0019 0.0031
0.0018 0.0130
0.0021
y4 m/z 629.3459 645.3387 645.3387 629.3466
ΔM 0.0048 0.0027 0.0027 0.0055
y5 y6 m/z ΔM m/z ΔM 744.3729 0.0049 845.4203 0.0046 760.3688 0.0059 861.4236 0.0130 760.3576 -0.0053 861.4058 -0.0048 744.3713 0.0033 845.4113 -0.0044
645.3351 -0.0009
760.3674
629.3433 645.3416
0.0022 0.0056
744.3699 0.0019 845.4206 0.0049 760.3570 -0.0059 861.4099 -0.0007
645.3362
0.0002
760.3448 -0.0181 861.3829 -0.0277
645.3183 -0.0177
861.4063 -0.0043
645.3373
0.0013
760.3665
0.0045
0.0036
861.4125
861.4115
0.0019
0.0009
629.3419 0.0008 661.3193 -0.0116 629.3332 -0.0079 661.3388 0.0079
744.3670 -0.0010 845.4262 0.0105 776.3694 0.0116 877.3884 -0.0171 744.3679 -0.0001 845.4075 -0.0082 776.3466 -0.0112
629.3448 709.3119 709.3125
744.3541 824.3389 824.3418 840.3364 824.3397 840.3281 840.3273 824.3371 840.3309 840.3250
0.0037 0.0045 0.0051
709.3129 0.0055 725.3021 -0.0002 725.3077 0.0054 709.3181 0.0107 725.3036 0.0013 725.2978 -0.0045
-0.0139 0.0046 0.0075 0.0072 0.0054 -0.0012 -0.0019 0.0028 0.0017 -0.0042
845.4189 925.3837 925.3938 941.3820 925.3867 941.3782 941.3787 925.3826 941.3779 941.3832
0.0032 0.0017 0.0118 0.0051 0.0047 0.0013 0.0018 0.0006 0.0010 0.0063
a
Two oxidation products that could not be resolved chromatographically. b No oxidized product ions were observed, but the non-oxidized product ions rule out other oxidation sites. Other diagnostic product ions (Δ M): c b3, m/z 373.1776 (-0.0059); d x2, m/z 329.1960 (-0.0023), e z3, m/z 449.2387 (-0.0123) and b8, m/z 1074.4222 (0.0078), and f b3, m/z 373.1876 (0.0041).
S7
Oxidation of Tyrosine-Phosphopeptides by Titanium Dioxide Photocatalysis Miina Ruokolainen†, Elisa Ollikainen†, Tiina Sikanen†, Tapio Kotiaho†,‡, Risto Kostiainen*† †
Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, P.O. Box 56 (Viikinkaari 5E), FI-00014, FINLAND, ‡ Department of Chemistry, University of Helsinki, P.O. BOX 55 (A.I. Virtasen aukio 1), FI00014, FINLAND [email protected], [email protected], [email protected], [email protected], [email protected] Corresponding author: (email) [email protected], (tel.) + 358 2941 59134
S1
Chemicals
The insulin receptor peptides TRDIYETDYYRK (IR0), TRDIpYETDYYRK (IR1A), TRDIYETDpYYRK (IR1B), TRDIYETDYpYRK (IR1C), and TRDIpYETDpYpYRK (IR3) were from Anaspec (Fremont, CA, USA) (Table S1). TiO2 Degussa P-25, formic acid (98–100%) and LC-MS grade methanol were from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Ammonium hydroxide was from Mallinckrodt Baker B.V. (Denventer, the Netherlands). Water was purified with a Milli-Q Plus (Millipore, Molsheim, France) purification system. Table S1. Abbreviations, sequences, formulae and monoisotopic masses of the peptides used. Abbreviation Sequence Formula Mmonoisotope IR0 TRDIYETDYYRK C72H107N19O24 1621.77 IR1A TRDIpYETDYYRK C72H108N19O27P 1701.74 IR1B TRDIYETDpYYRK C72H108N19O27P 1701.74 IR1C TRDIYETDYpYRK C72H108N19O27P 1701.74 IR3 TRDIpYETDpYpYRK C72H110N19O33P3 1861.67
Ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS)
The samples were analyzed using ACQUITY UPLC™ (Waters, Milford, MA, USA) and Xevo™ Q-TOF-MS (Waters, Manchester, UK) in the positive electrospray ionization mode. A Vanguard BEH C18 precolumn (5 mm x 2.1 mm i.d., 1.7 µm, Waters) was used prior to the analytical ACQUITY UPLC BEH C-18 column (100 mm x 2.1 mm i.d., 1.7 µm, Waters). The chromatographic parameters, solvent gradients used for analysis of reaction products and mass spectrometric parameters are in presented Tables S2-S5. Figure S1 presents extracted ion chromatograms of the oxidation products of the peptides. The chromatograms have been smoothed by Savitsky-Golay method (window size: 3 scans, number of smooths: 2). Figure S2 presents kinetic plots of the formation and degradation of the oxidation products. Table S6 presents accurate masses of the diagnostic product ions of the oxidized peptides. The measured masses of the oxidation products have been corrected using the relative deviation of the measured mass of the corresponding non-oxidized peptide in the same run.
S2
Table S2. Chromatographic parameters used for analysis of the photocatalytic reaction products of the phosphopeptides. Parameter Eluent A Eluent B Flow rate Column temperature Sample tray temperature Injection mode Injection volume
0.1% formic acid 0.1% formic acid in MeOH/H2O 98:2 450 µl min-1 40 °C 8 °C partial loop 5 µL
Table S3. Solvent gradients used for analysis of the photocatalytic reaction products of IR0 and IR3. IR0 Time (min) 0.00-4.50 4.50-8.50 8.50-9.00 9.00-9.10 9.10-12.50
B (%) 14 14 → 30 30 30 → 14 14
IR3 Time (min) 0.00-5.00 5.00-10.00 10.00-12.00 12.00-12.10 12.10-15.00
B (%) 10 10 → 30 30 30 → 10 10
Table S4. Solvent gradients used for analysis of the photocatalytic reaction products of IR1A, IR1B and IR1C. IR1A Time (min) 0.00-1.00 1.00-5.00 5.00-8.00 8.00-8.50 8.50-8.60 8.60-12.00
B (%) 10 → 14 14 14 → 30 30 30 →10 10
IR1B Time (min) 0.0-4.00 4.00-8.00 8.00-11.00 11.00-11.50 11.50-11.60 11.60-15.00
B (%) 10 → 12 12 12 → 30 30 30 → 10 10
IR1C Time (min) 0.00-0.50 0.50-6.00 6.00-9.00 9.00-9.50 9.50-9.60 9.60-13.00
B (%) 10 → 14 14 14 → 30 30 30 → 10 10
S3
Table S5. MS-parameters used for detection of the photocatalytic reaction products. Parameter Capillary voltage (kV) Extraction cone voltage (V) Sampling cone voltage (V) MS scan range (m/z) Product ion scan range (m/z) Scan time (s) Desolvation gas Collision gas Ion source temperature External reference (Lockspray)
IR0 IR1A, IR1B, IR1C IR3 3 3 4 0 0 0 20 20 45 400–1650 400–1750 400–1900 70–1650 70–1750 0.15 0.15 0.15 -1 Nitrogen (1000 L h , 500 C) Argon 150 C leucine enkephalin (concentration 2 ng µL-1, flow rate 20 µL min-1) at m/z 556.2771
S4
Figure S1. Extracted ion [M+3H]+3 chromatograms (EIC) of the oxidation products of the peptides from the UV exposure time which yielded highest abundance of the oxidation products (see Fig. S2).
S5
a) Degradation of the peptides (loss of the non-oxidized form) 100% IR0 IR1A Relative abundance
80%
IR1B IR1C
60%
40%
20%
0% 0
5
10
15
UV exposure time (min)
b) Formation of the initial reaction products 100 sum of M+O products of IR0 sum of M+O products of IR1A
Abundance
80
sum of M+2O products of IR1A sum of M+O products of IR1B
60
sum of M+O products of IR1C
40
20
0 0
5
10
15
UV exposure time (min)
Figure S2. a) Degradation of the peptides IR0, IR1A, IR1B and IR1C and b) formation of the initial reaction products under TiO2 photocatalysis calculated using the triply protonated ion.
S6
Table S6. The measured accurate masses of the oxidation products and their diagnostic product ions, and the deviations from the calculated masses. Single oxidized product ions are bolded and double oxidized product ions are bolded and underlined.
peptide/ oxidation tR product site (min) IR0 5.98 M+O Y9 1.90 M+O Y10 2.42 M+O Y5 4.31
precursor [M+3H]+3 m/z ΔM 541.5880 -0.0110 546.9300 -0.0006 546.9303 -0.0003 546.9284 -0.0022
b4 b5 m/z ΔM m/z 486.2705 0.0029 649.3353 486.2678 0.0002 649.3318 486.2664 -0.0012 649.3326 486.2692 0.0016 665.3261
ΔM 0.0044 0.0009 0.0017 0.0003
b6 m/z ΔM 778.3777 0.0042 778.3781 0.0046 778.3747 0.0012 794.3679 -0.0005
y3 m/z 466.2802 466.2787 482.2715 466.2791 466.2833 482.2759 466.2786 466.2683 466.2697 482.2766 466.2777 482.2757 466.2791 482.2758 466.2828 466.2807 466.2735 498.2608
ΔM 0.0024 0.0009 -0.0012 0.0013 0.0055 0.0032 0.0008 -0.0095 -0.0081 0.0039 -0.0001 0.0030 0.0013 0.0031 0.0050 0.0029 -0.0043 -0.0068
M+O
Y9 and Y10 a
4.57
546.9297
-0.0009 486.2672 -0.0004 649.3322
0.0013
778.3705 -0.0030
IR1A M+O
Y9
5.43 2.09
568.2451 573.5847
-0.0093 486.2681 -0.0014 486.2681
0.0005 0.0005
729.2985 729.2994
0.0013 0.0022
858.3416 858.3528
M+O
Y9 and Y10 a
2.29
573.5857
-0.0004 486.2702
0.0026
729.2919 -0.0053
858.3350 -0.0048
M+O
Y9 and Y10 a
2.57
573.5847
-0.0014 486.2687
0.0011
729.3034
0.0062
858.3338 -0.0060
M+O
Y9 and Y10 a
4.00
573.5880
0.0019
486.2699
0.0023
729.3001
0.0029
858.3419
M+O M+2O M+2O c M+2O d M+2O e M+2O f IR1B M+O M+O IR1C M+O M+O M+O M+O M+O
pY5 Y9 I4 Y10 Y9 b I4 Y5 Y10 Y9 Y9 Y5 Y9 pY10
7.00 2.62 2.94 3.13 3.30 3.67 9.64 7.42 7.57 5.20 1.94 2.14 3.61 3.80 8.29
573.5876 578.9168 578.9179 578.9175 578.9156 578.9163 568.2471 573.5871 573.5871 568.2452 573.5843 573.5843 573.5843 573.5859 573.5861
0.0015 -0.0009 0.0002 -0.0002 -0.0021 -0.0014 -0.0073 0.0010 0.0010 -0.0092 -0.0018 -0.0018 -0.0018 -0.0002 0.0000
486.2668 486.2682 518.2555 486.2684 486.2660 518.2527 486.2697 486.2640 486.2678 486.2697 486.2651 486.2656 486.2698 486.2723 486.2669
-0.0008 0.0006 -0.0019 0.0008 -0.0016 -0.0047 0.0021 -0.0036 0.0002 0.0021 -0.0025 -0.0020 0.0022 0.0047 -0.0007
745.2896 -0.0025 729.2986 0.0014 761.2942 0.0072 729.2996 0.0024 729.2967 -0.0005
874.3416 0.0069 858.3373 -0.0025 890.3182 -0.0114 858.3480 0.0082 858.3567 0.0169
649.3341 665.3259 649.3336 649.3344 649.3337 649.3317 665.3306 649.3328 649.3340
466.2786 0.0008 778.3761 0.0026 466.2807 0.0029 794.3617 -0.0067 466.2861 0.0083 778.3770 0.0035 482.2652 -0.0075 778.3770 0.0035 546.2476 0.0035 778.3776 0.0041 546.2446 0.0005 778.3693 -0.0042 546.2481 0.0040 794.3718 0.0034 546.2481 0.0040 778.3787 0.0052 546.2379 -0.0062 778.3809 0.0074 562.2235 -0.0155
0.0032 0.0001 0.0027 0.0035 0.0028 0.0008 0.0048 0.0019 0.0031
0.0018 0.0130
0.0021
y4 m/z 629.3459 645.3387 645.3387 629.3466
ΔM 0.0048 0.0027 0.0027 0.0055
y5 y6 m/z ΔM m/z ΔM 744.3729 0.0049 845.4203 0.0046 760.3688 0.0059 861.4236 0.0130 760.3576 -0.0053 861.4058 -0.0048 744.3713 0.0033 845.4113 -0.0044
645.3351 -0.0009
760.3674
629.3433 645.3416
0.0022 0.0056
744.3699 0.0019 845.4206 0.0049 760.3570 -0.0059 861.4099 -0.0007
645.3362
0.0002
760.3448 -0.0181 861.3829 -0.0277
645.3183 -0.0177
861.4063 -0.0043
645.3373
0.0013
760.3665
0.0045
0.0036
861.4125
861.4115
0.0019
0.0009
629.3419 0.0008 661.3193 -0.0116 629.3332 -0.0079 661.3388 0.0079
744.3670 -0.0010 845.4262 0.0105 776.3694 0.0116 877.3884 -0.0171 744.3679 -0.0001 845.4075 -0.0082 776.3466 -0.0112
629.3448 709.3119 709.3125
744.3541 824.3389 824.3418 840.3364 824.3397 840.3281 840.3273 824.3371 840.3309 840.3250
0.0037 0.0045 0.0051
709.3129 0.0055 725.3021 -0.0002 725.3077 0.0054 709.3181 0.0107 725.3036 0.0013 725.2978 -0.0045
-0.0139 0.0046 0.0075 0.0072 0.0054 -0.0012 -0.0019 0.0028 0.0017 -0.0042
845.4189 925.3837 925.3938 941.3820 925.3867 941.3782 941.3787 925.3826 941.3779 941.3832
0.0032 0.0017 0.0118 0.0051 0.0047 0.0013 0.0018 0.0006 0.0010 0.0063
a
Two oxidation products that could not be resolved chromatographically. b No oxidized product ions were observed, but the non-oxidized product ions rule out other oxidation sites. Other diagnostic product ions (Δ M): c b3, m/z 373.1776 (-0.0059); d x2, m/z 329.1960 (-0.0023), e z3, m/z 449.2387 (-0.0123) and b8, m/z 1074.4222 (0.0078), and f b3, m/z 373.1876 (0.0041).
S7
