Electronic Supplementary Material (ESI) for Chemical Science.
This journal is © The Royal Society of Chemistry 2014
Monrad et al.
page S1
SUPPLEMENTARY INFORMATION
Dissecting the Reaction of Phase II Metabolites of Ibuprofen
and Other NSAIDS with Human Plasma Protein
Rune Nygaard Monrad,a James C. Errey,a Conor S. Barry,a Mazhar Iqbal,b Xiaoli Meng,b Lisa
Iddon,b Jennifer A. Perrie,b John R. Harding,c Ian D. Wilson,c Andrew V. Stachulski,b
and Benjamin G. Davis*a
Table of Contents
Page
Additional experimental methods. ....................................................................................................... 2 Raw and processed protein mass spectra ............................................................................................. 5 Mass spectra of tryptic peptides ........................................................................................................ 18 Reactive Accessibility of HSA .......................................................................................................... 29 Control Reaction of HSA with D-glucuronic acid ............................................................................ 31 General Synthetic Methods for AGs.................................................................................................. 33 (4-Isobutyl)phenylacetyl 1β-acyl glucuronide (ibufenac acyl glucuronide) 1a ................................ 34 [(2R)-2-Methyl-(4-isobutyl)]phenyl]acetyl 1β-acyl [(R)-ibuprofen acyl glucuronide) 1b ............... 34 [(2S)-2-Methyl-(4-isobutyl)]phenyl]acetyl 1β-acyl [(S)-ibuprofen acyl glucuronide) 1c ................ 35 [2,2-Dimethyl-(4-isobutyl)]phenyl]acetic acid .................................................................................. 35 [2,2-Dimethyl-(4-isobutyl)]phenyl]acetyl 1β-acyl glucuronide 1d ................................................... 36 4-Bromobenzoyl 1β-acyl glucuronide 2 ............................................................................................ 36 [3-(4-bromo-2-fluorobenzyl)-4-oxo-3H-phthalazin-1-yl]acetyl 1β-acyl glucuronide ...................... 36 (Ponolrestat 1β-acyl glucuronide) 3 .................................................................................................. 36 References.......................................................................................................................................... 37 Monrad et al.
page S2
Additional experimental methods.
Size-exclusion chromatography. Desalting and removal of excess reagents were carried out by
PD10 size-exclusion chromatography (10 kDa molecular weight cutoff). The PD10 column was
washed with buffer or water (25 mL), the protein sample (2.5 mL) was loaded and eluted with
buffer or water (3.5 mL).
Water. Water (H2O) was purified using a Milli-Q purification system.
Determination of protein concentration. Protein concentrations were measured by UV
absorbance. Either based on the extinction coefficient at 280 nm (ε280nm = 28730 M-1 cm-1 for
unmodified HSA) calculated from the amino acid sequence using the following protein
concentration calculater: http://www.mrc-lmb.cam.ac.uk/ms/methods/proteincalculator.html. The
concentration of protein was also determined using Bradford assay with BSA as a standard, which
agreed favourably with the concentration obtained by A280.
Protein mass spectrometry. The protein solutions were analysed under denaturing conditions by
liquid chromatography (Waters, Milford, MA) coupled to positive electrospray ionization time of
flight mass spectrometry (ESI+ TOF MS, Micromass, UK) using a Phenomenex Jupiter 5u C4
300Å 250 x 4.6 mm column. The protein solution was injected and eluted at 1 mL/min using a 35
min linear gradient method from solvent A (water/5% acetonitrile/0.1% formic acid) and solvent B
(acetonitrile/0.1% formic acid), see Table 1 below. All solvents were degassed by sonication for 15
min prior to use. The output of the liquid chromatography was split 1:4 (mass spectrometer:waste)
and injected into the mass spectrometer with a scan range of 300–1800 m/z, capillary voltage
Monrad et al.
page S3
2500 V, cone voltage of 30 V, source temperature of 80°C, and desolvation temperature of 200°C.
Albumin elutes at 13.5 min and is visualized as a charge envelope from 950 to 1750 m/z
representing +69 to +41 charges. The spectrum was then deconvolved to the uncharged parent mass
using MaxEnt 1 (Micromass). Myoglobin (horse heart) was used as a calibration standard and to
test the sensitivity of the system. The following section shows the raw protein spectra followed by
their deconvoluted spectra. Both the full spectrum and a zoom of the most interesting peaks are
included.
Table 1.
Time
(min)
0.0
5.0
20.0
25.0
30.0
35.0
Chromatographic conditions for protein mass
spectrometry.
%A
%B
Flowrate
Curve
mL/min
95
5
1
1
95
5
1
1
5
95
1
6
5
95
1
1
95
5
1
6
95
5
1
1
Tryptic digestion and MS/MS analysis. 50 µL of a solution of modified HSA (approx. 4 g/L) in
NH4HCO3 (50 mM, pH 8.0) was added trypsin (10 µg, 1 g/L in 50 mM NH4HCO3) and incubated at
37 ºC for 16 h. The resultant solution was added 1 % aq. formic acid (5 µL) and analysed by liquid
chromatography (Agilent) coupled to ESI+ TOF MS (Q-Tof microTM, Micromass, UK) using a
Phenomenex Jupiter 5u C18 300Å 150 x 0.5 mm column. The tryptic peptides were injected and
eluted at 15 µL/min using a 90 min linear gradient method from solvent A (water/0.1% formic acid)
and solvent B (acetonitrile/0.1% formic acid), see Table 2 below. The output of the liquid
chromatography was injected into the mass spectrometer with a scan range of 100–2800 m/z,
Monrad et al.
page S4
capillary voltage 3000 V, cone voltage of 35 V, source temperature of 80°C, and desolvation
temperature of 200°C.
Table 2.
Time
(min)
0.0
5.0
30.0
60.0
70.0
90.0
Chromatographic conditions for
analysis of tryptic peptides.
%A
%B
Flowrate
µL/min
95
5
15
95
5
15
60
40
15
5
95
15
95
5
15
95
5
15
Monrad et al.
page S5
Raw and processed protein mass spectra
Human serum albumin
1127 1147
100
1187
1108
1090
TOF MS ES+
272
1209
1232
1255
1279 1304
1072
1055
1330
1331
1304
1039
1023
%
1305
1255
1008
978 993
1256
978
950
1358
1386 1416
14161447
1480
1479
1305 1331
1514
1359 1387 1417
1280 1305
1480
1333 1362
1414 1420 1451
1387
1550
1587
1626
0
m/z
950
1000
1050
1100
1150
1200
1250
1300
1350
1400
1450
1500
1550
1600
1650
Human serum albumin
TOF MS ES+
3.07e3
66437
100
HSA, found
66437
HSA, expected 66438
%
66487
66598
0
64000
mass
65000
66000
67000
68000
69000
70000
71000
Human serum albumin
TOF MS ES+
3.07e3
66437
100
%
66487
66598
0
mass
65600
65800
66000
66200
66400
66600
66800
67000
67200
67400
67600
The two most significant impurities (66487 and 66598 Da) in the purified HSA correspond to small
amounts of post-translationally modified protein. Both acetylation (∆MW = 42 Da) and
glycosylation with glucose or another hexose (∆MW = 162 Da, ~9% here) are found in HSA isolated
from humans.1-4
Monrad et al.
page S6
p-Bromobenzoic acid AG 5.0 mM
1153 1173
100
1133
TOF MS ES+
272
1216
1238
1262 1286
1078 1096
12621262
1312
1045 1061
1262
1045
%
1013
998
983
0
950
1338 1366
1366
1338
1395 1425
1339
1395 14251456
1425
1489
1456
1523
1559
m/z
1000
1050
1100
1150
1200
1250
1300
1350
1400
1450
1500
1550
1600
1650
p-Bromobenzoic acid AG 5.0 mM
TOF MS ES+
1.80e3
66801
100
HSA, found
HSA, expected
Glycation, found
Glycation, expected
66441
66441
66438
66801
66797
%
0
64000
mass
66000
65000
68000
67000
69000
70000
71000
p-Bromobenzoic acid AG 5.0 mM
TOF MS ES+
1.80e3
66801
100
66441
%
66850
66610
66744
66971
67171
0
mass
65750
66000
66250
66500
66750
67000
67250
67500
67750
68000
68250
68500
Monrad et al.
page S7
p-Bromobenzoic acid AG 0.5 mM
100
1114
1153 1173
TOF MS ES+
370
1216
12381262
1286
1096
1078
1312
1262 1311
1338
1061
1366
1394
1045
%
1424
1029
1013
983 998
1456
1489
1523
1559
1524
1481
969
0
950
m/z
1000
1050
1100
1150
1200
1250
1300
1350
1400
1450
1500
1550
1600
1650
p-Bromobenzoic acid AG 0.5 mM
TOF MS ES+
3.13e3
66798
100
HSA, found
HSA, expected
Glycation, found
Glycation, expected
66439
66439
66438
66798
66797
%
0
64000
mass
66000
65000
68000
67000
69000
70000
71000
p-Bromobenzoic acid AG 0.5 mM
TOF MS ES+
3.13e3
66798
100
66439
%
66848
66486
66603
66539
66742
66898 66960
0
66200
66400
66600
66800
67000
67200
67400
67600
mass
67800
Monrad et al.
page S8
Statil AG 5.0 mM
TOF MS ES+
154
1167
100
1090
1127
1187 1209
1232
1304
1330
1055 1073
1358
1358
1331
%
1386 1416
1023
1331
1008
993
978
964
1447
1480
1514
1416
1417 1479
1387
1359
0
m/z
950
1000
1050
1100
1150
1200
1250
Statil AG 5.0 mM
1350
1400
1450
1500
HSA, found
HSA, expected
Transacylation (TA), found
TA, expected
Glycation (Glyc.), found
Glyc., expected
Both TA and Glyc., found
Both TA and Glyc., expected
66440
100
1300
1550
1600
1650
66440
66438
66812
66811
66984
66987
67366
67360
1700
TOF MS ES+
1.18e3
%
66812
67366
66984
0
64000
mass
65000
66000
68000
67000
69000
70000
71000
Statil AG 5.0 mM
TOF MS ES+
712
66440
100
%
66491
66812
66602
66760
0
65600
66984
66864
67366
mass
65800
66000
66200
66400
66600
66800
67000
67200
67400
67600
Monrad et al.
page S9
Statil AG 0.5 mM
TOF MS ES+
181
1127 1147
100
1187 1209 1255
1108
1090
1232
1073
1279
1056
%
1304
1330 1358
1023 1039
1305
1255
1008
1386
1305
1280
978 992
1305
1332
964
1416
1358
1360
1387
950
1447 1480
1514
1447
1550
1513
1417
1515
1587
1627
0
m/z
950
1000
1050
1100
1150
1200
1250
1300
1350
1400
1450
HSA, found
HSA, expected
Transacylation (TA), found
TA, expected
Glycation (Glyc.), found
Glyc., expected
66641
66438
66820
66811
66995
66987
1500
1550
1600
1650
Statil AG 0.5 mM
66441
100
TOF MS ES+
1.42e3
%
66820 66995
0
64000
mass
65000
66000
67000
68000
69000
70000
71000
Statil AG 0.5 mM
TOF MS ES+
548
66441
100
66488
%
66603
66540
66820
66995
0
mass
65800
66000
66200
66400
66600
66800
67000
67200
67400
Monrad et al.
page S10
Ibufenac AG 5.0 mM
100
1108
1255 1279
1304
1330
1209
1090
%
TOF MS ES+
213
1231
1127 1146 1187
1357
1073
1056
1023 1055
1385 1415
1389
1445
1478
1023
1449
1008
0
950
1511
1546
1486
m/z
1000
1050
1100
1150
1200
1250
1300
1400
1450
1500
1550
HSA, found
HSA, expected
Transacylation (TA), found
TA, expected
Glycation (Glyc.), or double TA, found
Glyc., expected
Double TA, expected
Both Glyc. and TA, or triple TA, found
Both Glyc. and TA, expected
Triple TA, expected
Both Glyc. and double TA, or
Glyc., or quadruple TA, found
Both Glyc. and double TA, expected
Double Glyc., expected
Quadruple TA, expected
Ibufenac AG 5.0 mM
66440
100
1350
%
66610
66787
1600
1650
66440
66438
66610
66612
66787
66788
66786
66957
66962
66960
double
67133
67136
67138
67134
1700
TOF MS ES+
2.52e3
66957 67133
0
64000
mass
65000
66000
68000
67000
69000
70000
71000
Ibufenac AG 5.0 mM
TOF MS ES+
1.65e3
66440
100
66610
%
66787
66484
66653
66957
67133
0
mass
65600
65800
66000
66200
66400
66600
66800
67000
67200
67400
67600
67800
68000
Monrad et al.
page S11
Ibufenac AG 0.5 mM
1147 1167 1231
1209
100
TOF MS ES+
153
1255
1279 1304
1090
1330
1385
1357
1415
1056 1073
1446
1386
1415 1478
1056
%
1023 1039
1386
9931008
1446
1511 1546
1622
1479 1546
1584
1622
0
m/z
950
1000
1050
1100
1150
1200
1250
1300
1350
1400
1450
1500
1550
1600
1650
1700
1750
Ibufenac AG 0.5 mM
TOF MS ES+
1.91e3
66444
100
HSA,
found
HSA, expected
Transacylation (TA), found
TA, expected
Glycation (Glyc.), or double TA, found
Glyc., expected
Double TA, expected
%
66444
66438
66610
66612
66792
66788
66786
66610
66792
0
64000
mass
65000
66000
67000
68000
69000
70000
71000
Ibufenac AG 0.5 mM
TOF MS ES+
1.12e3
66444
100
%
66486
66610 66792
0
mass
65000
65250
65500
65750
66000
66250
66500
66750
67000
67250
67500
67750
68000
Monrad et al.
page S12
(R)-Ibuprofen AG 5.0 mM
100
1090.2
TOF MS ES+
122
1146.6
1187.5
1127.2 1166.6 1209.0 1254.7
1278.8
1303.8 1329.9
1072.7
1039.1
1357.0 1385.2
1023.3
%
1414.7 1445.5
1007.7
1477.6
1511.2
978.1
964.0
1546.3
1583.1
1621.8
1662.4
0
m/z
900
950
1000
1050
1100
1150
1200
1250
1300
1350
1400
1450
1500
1550
1600
1650
1700
1750
(R)-Ibuprofen AG 5.0 mM
TOF MS ES+
2.44e3
66442
100
HSA, found
HSA, expected
Transacylation (TA), found
TA, expected
Glycation (Glyc.), found
Glyc., expected
Both TA and Glyc., found
Both TA and Glyc., expected
%
66442
66438
66627
66626
66802
66802
66990
66990
66990
66627
66802
0
64000
mass
65000
66000
68000
67000
69000
70000
71000
(R)-Ibuprofen AG 5.0 mM
TOF MS ES+
1.32e3
66442
100
%
66627
66484
66802
66540
66990
0
mass
65800
66000
66200
66400
66600
66800
67000
67200
67400
Monrad et al.
page S13
(R)-Ibuprofen AG 0.5 mM
100
1127.1
TOF MS ES+
216
1166.6 1187.4 1209.0
1254.6
1108.3
1090.2
1072.6
1303.8
1329.8 1356.9
1385.2
1055.6
%
1414.6
1445.4
1039.2
1023.2
1477.5
1511.0
1546.3
1583.1
1007.7
978.1
1621.7
1662.3
1705.0
0
950
1000
1050
1100
1150
1200
1250
1300
1350
1400
1450
1500
1550
1600
1650
1700
m/z
1750
(R)-Ibuprofen AG 0.5 mM
TOF MS ES+
5.39e3
66438
100
HSA, found
HSA, expected
Transacylation (TA), found
TA, expected
Glycation (Glyc.), found
Glyc., expected
%
66438
66438
66637
66626
66797
66802
66637
66797
0
64000
mass
65000
66000
67000
68000
69000
70000
71000
(R)-Ibuprofen AG 0.5 mM
TOF MS ES+
1.93e3
66438
100
%
66482
66637
66537
66601
0
66000
66100
66200
66300
66400
66500
66600
66797
66700
66800
66900
mass
67000
Monrad et al.
page S14
(S)-Ibuprofen AG 5.0 mM
100
1127
1090 1108
1167
TOF MS ES+
148
1231
1209
1254 1279
1090
1072
1039 1056
1304
1357
1330 1357
1357
%
1008 1023
993
1385
1415
1477
1446 1478
1386
1511
1419
1449
1511 15111547
1583
1400
1600
0
m/z
950
1000
1050
1100
1150
1200
1250
1300
1350
1450
1500
1550
1650
1700
1750
(S)-Ibuprofen AG 5.0 mM
TOF MS ES+
2.40e3
66439
100
HSA, found
HSA, expected
Transacylation (TA), found
TA, expected
Glycation (Glyc.), found
Glyc., expected
%
66439
66438
66625
66626
66801
66802
6662566801
0
64000
mass
65000
66000
68000
67000
69000
70000
71000
(S)-Ibuprofen AG 5.0 mM
TOF MS ES+
938
66439
100
%
66625
66801
66482
66536
66672
66972
67147
0
66000
66200
66400
66600
66800
67000
67200
67400
67600
mass
67800
Monrad et al.
page S15
(S)-Ibuprofen AG 0.5 mM
TOF MS ES+
228
1146.4
1187.5
1127.2
1254.5 1278.7
1209.0
100
1108.3
1303.8 1329.8
1356.9
1072.51090.2
1055.6
1385.4 1414.5
%
1445.4
1477.7
1007.8
1511.2
1546.2 1583.2
978.2992.7
0
m/z
1000
1050
1100
1200
1150
1250
1300
1350
1400
1450
1500
1550
1600
1650
1700
(S)-Ibuprofen AG 0.5 mM
TOF MS ES+
3.99e3
66442
100
HSA, found
HSA, expected
Transacylation (TA), found
TA, expected
Glycation (Glyc.), found
Glyc., expected
%
66442
66438
66631
66626
66802
66802
66631
66802
0
64000
mass
65000
66000
68000
67000
69000
70000
71000
(S)-Ibuprofen AG 0.5 mM
TOF MS ES+
1.50e3
66442
100
%
66481
66802
66540 66607 66631
0
mass
66100
66200
66300
66400
66500
66600
66700
66800
66900
67000
Monrad et al.
page S16
Dimethyl Ibu-analogue AG 5.0 mM
TOF MS ES+
103
1146.6 1166.6
1209.0
1127.1
1254.6
1090.21108.4
100
1278.7 1303.7
1072.6
1329.8 1356.9
1385.2
1414.7 1445.4
1055.7
%
1023.2
1477.5
1511.0
1546.2
1583.0
978.1992.7
1621.8
950.2
1662.3
0
m/z
950
1000
1050
1100
1150
1200
1250
1300
1350
1400
1450
1550
1500
1600
1650
1700
Dimethyl Ibu-analogue AG 5.0 mM
TOF MS ES+
2.10e3
66439
100
HSA, found
HSA, expected
Transacylation (TA), found
TA, expected
Glycation (Glyc.), found
Glyc., expected
66439
66438
66635
66640
66813
66816
%
66635
66813
0
64000
mass
65000
66000
68000
67000
69000
70000
71000
Dimethyl Ibu-analogue AG 5.0 mM
TOF MS ES+
805
66439
100
%
66485
66540
66597
66813
66635
0
mass
65800
66000
66200
66400
66600
66800
67000
67200
Monrad et al.
page S17
Dimethyl Ibu-analogue AG 0.5 mM
TOF MS ES+
193
1147
100
1167
1255
1187
1127
1231
1279 1304
1090 1108
1330 1357
1073
%
1385
1415
13041330
1445
1386
1056
1056
1039
993 1008
1358
1331
978
1478
1546
1511
1546
1415 1446
1386
15111512
1583
1446
1387 1418
1547
1512
1583
1622
0
1000
1050
1100
1150
1200
1250
1300
1350
1400
1450
1500
1550
1600
1650
1700
m/z
1750
Dimethyl Ibu-analogue AG 0.5 mM
TOF MS ES+
2.58e3
66442
100
HSA, found
HSA, expected
66442
66438
%
0
64000
mass
66000
65000
68000
67000
69000
70000
71000
Dimethyl Ibu-analogue AG 0.5 mM
TOF MS ES+
646
66442
100
%
66485
66602
66262
66330 66384
66544
0
65900
66000
66100
66200
66300
66400
66500
66600
66700
66800
66900
mass
67000
Monrad et al.
page S18
Mass spectra of tryptic peptides
K-137, K(+)YLYEIAR, Glycosylation, retention time 33.49 min
Y-ions
m/z predicted
m/z found
Y1
175.1
175.2
Y2
246.2
246.3
Y4
488.3
488.6
IA
185.1
185.2
EI
243.1
243.3
YE
293.1
293.3
Y5
651.3
651.8
Y6
764.4
764.9
Y7
927.5
928.1
Internal ions
m/z predicted
m/z found
Mass spectra K-137, K(+)YLYEIAR, Glycosylation, retention time 33.49 min
52Bw4-5-2
52Bw4-5-2
RNM_52Bw4-5 688 (28.365) Cm (666:1030)
TOF MSMS 704.10ES+ RNM_52Bw4-5 688 (28.365) Cm (666:1030)
1.27e3 100
380.4
100
TOF MSMS 704.10ES+
201.2
266
183.3
120.2
175.2
261.3
129.2
235.3
233.3
183.3
%
%
158.2
147.2 226.3
211.3
453.5
341.4
165.2
454.5
0
100
527.6
662.7 712.9
538.6
713.8
582.6
300
400
500
600
700
199.2
197.3
171.2
181.2
181.2
198.2
190.2
176.2
171.3
169.2
170.2 172.2
165.2
160.2
814.9
179.2
177.2
174.3
195.2
200.3
191.2
189.2
195.2
188.3
192.2 193.2 196.2
203.2 204.3
202.2
195.3
182.2
178.2
m/z
200
173.2
184.3
409.5
197.3
186.2
167.2
166.2 169.2
165.2
408.5
318.4
201.2
187.2
187.2
169.2
159.2
162.2
159.2
340.4
185.2
169.2
800
900
1000
0
1100
m/z
158
52Bw4-5-2
160
162
164
166
168
170
172
174
176
178
180
182
184
186
188
190
192
194
196
198
200
202
204
52Bw4-5-2
RNM_52Bw4-5 688 (28.365) Cm (666:1030)
TOF MSMS 704.10ES+ RNM_52Bw4-5 688 (28.365) Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (666:1030)
651.8
90 100
487.6
100
TOF MSMS 704.10ES+
28.8
487.6
488.6
487.7
487.5
652.8
483.5
645.9
489.5
489.6
487.4
485.6
485.6
484.6
486.4
483.4
483.4
484.3
484.0
485.3
485.9
651.7
488.9
492.6
489.8
489
490
492.7
492.4
491.4
491.7
492.4
491.9
491.4
491.2
%
492.7
646.9
485
486
487
488
491
492
650.6
649.5
647.5
651.8
653.5
650.5
652.3
100
647
648
649
650
651
652
%
774.9
767.9
769.9
768.9
770.9
653
654
655
657.6
656
658.5
657
658
659
765.9
757.9
759.9
760.7
760.9 761.9
761.0 762.0
922.0
775.9
766.9
766.9
775.8
759
760
761
762
767.9
763.8
763
764
765
931.1
932.0
933.1
934.0
935.1
937.1
936.1
769.9
769.8
771.0
771.9 772.9
775.0 775.8
772.8 773.0
774.0
778.0
920.0
766
767
768
769
770
771
772
773
774
775
776
777
922.3
928.1
928.0
923.9
925.1
925.0
927.9
927.2
926.1
929.1
930.1
931.1
936.2
936.0
937.1
935.0
938.0
935.9
931.9
933.1 933.9
932.4
939.0
939.0
939.2
0
778
m/z
920
52Bw4-5-2
921.1 921.8
939.1
938.1
TOF MSMS 704.10ES+
39
923.1
924.1
922.2 923.1
%
776.9
m/z
758
930.1
927.1
926.1
0
TOF MSMS 704.10ES+ RNM_52Bw4-5 688 (28.365) Cm (666:1030)
922.2
44 100
762.8 763.9
759.0
921.0
766.0
764.8
756.9
%
756.7
929.1
925.1
777.9
920.1
765.0
100
TOF MSMS 704.10ES+
24.1
924.1
776.9
771.9 772.9 773.9
0
RNM_52Bw4-5 688 (28.365) Cm (666:1030)
921
922
923
924
925
926
927
928
929
930
931
932
933
934
935
936
937
938
939
52Bw4-5-2
RNM_52Bw4-5 688 (28.365) Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (666:1030)
TOF MSMS 704.10ES+ RNM_52Bw4-5 688 (28.365) Cm (666:1030)
245.2
210 100
284.3
TOF MSMS 704.10ES+
204
243.3
249.3
245.3
283.3
293.3
244.3
243.3
294.3
295.3
299.4
298.3
286.3
291.3
288.3
287.3
245.2
244.3
290.3
285.3
301.3
300.3
304.4
302.3
296.3 297.3
292.3
244.3
243.3
306.3
305.3
303.3
289.3
244.2
249.2
244.3
249.3
307.3 308.3 309.3
246.3
0
RNM_52Bw4-5 688 (28.365) Cm (666:1030)
TOF MSMS 704.10ES+
279
284.3
247.3
%
249.4
251.3
247.3
283.3
246.2
243.4
251.3
293.3
286.3
287.3
298.3 299.3
304.4
302.3
305.3
290
292
294
296
298
300
302
304
306
245.1
243.4
248.2
307.3 308.3 309.3
308
310
250.4
249.2
251.4
248.5
m/z
288
252.2
246.4
242.4
306.3
303.3
0
286
250.3
250.4
301.3
296.3 297.3
292.3
252.3
252.3
248.3
301.3
299.4
295.3
291.3
288.3
288.4
247.3
294.3
290.3
290.4
285.3
284
656.6
658.7
659.7
658.8
658.8
659.5
928.1
775.9
766.9
756.8
761.9 762.9 763.9
756.0
757.9 758.9 759.9 760.9
%
657.7 657.8
656.8
657.9
656.9
923.1
765.9
100
656.7
m/z
646
%
%
655.8
TOF MSMS 704.10ES+ RNM_52Bw4-5 688 (28.365) Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (666:1030)
922.1
30.9 100
764.9
757
655.7
653.8 654.6 654.8
653.9
655.6
653.9
653.6
652.6
651.0
657.7
655.7
653.8
652.6
52Bw4-5-2
RNM_52Bw4-5 688 (28.365) Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (666:1030)
0
756
652.8
652.7
0
493
52Bw4-5-2
100
648.6
649.3
m/z
484
652.7
648.8
650.8
649.8
647.8 648.7 648.8
650.7
646.8 647.7
647.9
649.6
649.9
650.9
646.6
646.8
645.9
0
483
652.7
650.8
490.7
490.4
TOF MSMS 704.10ES+
50
651.8
651.8
100
492.6
491.4
489.4
488.3
487.1
491.6
491.5
490.6491.4
488.7
487.8
487.8
487.4
486.4
486.3
485.4
490.4
487.7
486.6
484.7 485.4
483.7
483.7
490.5
489.6
484.4
483.6
482.7
0
RNM_52Bw4-5 688 (28.365) Cm (666:1030)
491.6
486.5 486.6
656.7
491.5
484.6
483.6
483.5
657.7
655.7
654.7
488.5
484.5
483.6
658.7
646.7
653.7
484.5
%
650.8
649.7
648.7
647.7
%
488.6
0
m/z
243
244
245
246
247
248
249
250
251
252
Monrad et al.
page S19
K-195, ASSAK(+)QR, Transacylation, retention time 24.36 min
B- and Y-ions
B2
m/z predicted
m/z found
159.1
159.3
B6 (includes
modification)
747.4
748.2
SA
159.1
159.3
SS
175.1
175.3
Y1
Y2
175.1
175.3
303.2
303.5
Internal ions
m/z predicted
m/z found
K(+)Q
431.3
431.7
AK(+)Q
502.3
502.8
Mass spectra K-195, ASSAK(+)QR, Transacylation, retention time 24.36 min
52Bw4-2
52Bw4-2
RNM_52Bw4-2 541 (25.515) Cm (465:542)
TOF MSMS 921.80ES+ RNM_52Bw4-2 539 (25.476) Cm (464:540)
157.3
170 100
244.4
100
TOF MSMS 921.80ES+
18
927.5
159.3
928.6
922.5
157.3 157.3
%
136.2
%
922.4
147.3
226.4
928.6
928.7
226.4
928.7
158.3
156.3
156.2
164.3
155.3
325.6
373.6
290.4
373.6
226.3
921.5
0
100
155.3
776.3 810.4
1612.6
928.9 1004.6
1119.7
1249.0
1584.3
155.8
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
158.3
157.7
156.2
156.9
159.3
161.3
158.4
158.0
157.2
158.8 159.2
159.8
160.2
160.4
160.0
161.2
162.3
162.3
164.3
163.3
163.2 163.3
161.8 162.2
162.4 163.1
162
163
165.3
164.2 164.3
163.7
165.3
164.9
165.2
0
m/z
1600
156
52Bw4-2
165.3
158.2 158.3
156.3
155.4
826.3
159.3
156.2
928.8
905.4
m/z
200
157.2 157.3
921.4
444.7496.8
506.8
159.2
158.3
158.2
157
158
159
160
161
164
165
52Bw4-2
RNM_52Bw4-2 539 (25.476) Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (464:540)
TOF MSMS 921.80ES+ RNM_52Bw4-2 539 (25.476) Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (464:540)
5.42 100
748.2
100
TOF MSMS 921.80ES+
17.4
175.3
757.2
%
%
753.2
749.1
747.2
743.2
745.2
744.1
758.1
752.2
750.2
751.1
746.1
754.1
181.3
173.3
171.3
176.3
172.3
170.2
168.3
0
RNM_52Bw4-2 539 (25.476) Cm (464:540)
0
TOF MSMS 921.80ES+ RNM_52Bw4-2 539 (25.476) Cm (464:540)
12 100
748.2
100
169.3
755.3 756.0
0
743
175.3
753.1
749.1
749.2 750.2
744.1 744.3
745.3
747.1
743.2
745.1
746.2
747.1
747.9
744.1
745.0
746.1
746.0
748.5
750.1
750.0
757.2
757.2
757.1
753.3
751.3
752.0 752.2
753.0
750.9
756.0
755.1
754.0 754.2
755.3
754.9
%
745
746
747
748
749
750
751
752
753
754
755
756
173.3
175.4
758.1 758.3
756.9
169.3
168.3 169.3
169.2
m/z
744
TOF MSMS 921.80ES+
34
175.3
748.1
748.0
179.3
175.3
748.4
%
177.3
757
173.3 173.3
172.3
172.4
175.3
174.1 174.2
175.2
174
175
176.3
177.3
177.4 178.2
176.2 176.4
181.3
181.2 181.4
179.3
180.3
182.3
179.2 179.4
180.8
182.2
182.3
0
758
m/z
168
52Bw4-2
171.3
171.2 171.3
170.3
169
170
171
172
173
176
177
178
179
180
181
182
52Bw4-2
RNM_52Bw4-2 539 (25.476) Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (464:540)
TOF MSMS 921.80ES+ RNM_52Bw4-2 539 (25.476) Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (464:540)
422.6
412.6
6.04 100
302.4
100
306.4
411.6
303.5
413.6
307.5
%
308.5
% 409.6
305.5
427.6
424.7
304.4
301.4
TOF MSMS 921.80ES+
11.3
421.6
416.6 417.6
309.5
429.7
425.7
430.7 431.7 433.7
423.6
415.6
428.6
419.6
418.6
0
RNM_52Bw4-2 539 (25.476) Cm (464:540)
0
TOF MSMS 921.80ES+ RNM_52Bw4-2 539 (25.476) Cm (464:540)
15 100
302.4
302.4
100
302.5
301.4
303.6
305.5
305.4
305.5
305.4
304.5
303.4
306.4306.5
306.4
304.4
410.6 411.6
308.5
308.4
306.4
308.6
307.6 308.3
306.3
307.4
303.1
308.8
308.0
307.1
302
303
304
305
306
307
308
309
RNM_52Bw4-2 539 (25.476) Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (464:540)
496.8
100
TOF MSMS 921.80ES+
11.0
495.8
476.7 477.7
478.7
471.7
460.8 461.7 464.7 465.7
% 458.7
463.7
459.7
466.7
468.7
497.8
482.8 483.8
472.7
475.7
489.7
479.7 480.7
474.7
469.8
485.8
487.7
491.7
490.7
494.8
502.8
500.8
501.8
504.7
499.7
0
RNM_52Bw4-2 539 (25.476) Cm (464:540)
496.8
100
503.8
TOF MSMS 921.80ES+
22
495.8
457.7
%
460.8
460.7
459.8
477.7 478.7
462.7
480.8
471.7 472.8
482.8 483.8
464.8 465.7
475.7
479.7
463.6
484.8
467.7 468.7 470.7
480.8
474.7
470.7
481.7
495.8
489.7
487.8
495.8
491.8
497.8
495.7
493.8
501.7 502.7
504.7
503.8 505.8
499.7
0
m/z
458
460
462
464
466
468
470
472
474
476
478
480
482
484
486
488
490
492
494
496
498
500
502
418.7
504
421.6
421.6
420.6
424.6
427.6
427.6
429.7 430.7
433.8
431.7
427.6
428.6
432.8
439.7
435.7
438.7
439.6
0
410
52Bw4-2
424.7
416.6
415.6
417.6 419.6
309.5
309.4
309.6
309.3
309.7
m/z
422.6 422.6
413.7
%
307.5
307.5
0
301
TOF MSMS 921.80ES+
22
422.6
412.7
304.5
304.4
302.1
441.7
435.6 438.7 439.6
437.7
440.7
436.7
412.6
303.5
303.5
301.3
434.7
422.6
412.6
301.4
301.5
301.4
%
432.7
412
414
416
418
420
422
424
426
428
430
432
434
436
438
440
441.6
441.6
m/z
442
Monrad et al.
page S20
K-199, LK(+)CASLQK, Glycosylation, retention time 27.80 min
B- and Y-ions
m/z predicted
m/z found
B1
114.1
114.2
Y1
147.1
147.3
Y2
275.2
275.5
Y3
388.3
388.6
Y5
546.3
546.8
CA
175.1
175.3
SL
201.1
201.3
ASL
272.2
272.4
K(+)CASL
427.7 (doubly charged)
427.6, 428.2
Internal ions
m/z predicted
m/z found
K(+)CASLQ
491.8 (doubly charged)
491.7, 492.3
K-199, LK(+)CASLQK, Glycosylation, retention time 27.80 min
52Bw4-3
52Bw4-3
RNM_52Bw4-3 621 (27.076) Cm (617:691)
TOF MSMS 621.60ES+ RNM_52Bw4-3 621 (27.076) Cm (617:691)
109 100
120.2
100
TOF MSMS 621.60ES+
21
115.2
115.2 115.2
110.2
129.3
116.2
130.2
%
%
147.3
116.2
221.3
231.4
258.4 269.4
115.2
258.4
175.3
211.4
285.4
175.3
324.5 340.5
114.2
286.4
158.3
460.8
340.5
306.4
114.2
460.9
0
100
114.2
597.9
m/z
125
150
175
200
225
250
275
300
325
350
375
400
425
450
475
500
525
550
575
600
115.2
114.3
115.3
116.2
116.2
115.0
117.2
115.8
117.2
117.2
0
m/z
625
114
52Bw4-3
116.2
115.3
114.2
460.7
373.6 389.6
115
116
117
118
52Bw4-3
RNM_52Bw4-3 621 (27.076) Cm (617:691)
386.5
100
TOF MSMS 621.60ES+ RNM_52Bw4-3 621 (27.076) Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (617:691)
546.8
13 100
389.6
TOF MSMS 621.60ES+
2.26
389.6
542.8
540.8
386.5 386.6
384.5
0
RNM_52Bw4-3 621 (27.076) Cm (617:691)
387.6
390.7
387.5
384.7
386.7
385.5
384.7
386.5
385.3
386.1
388.7
387.7
386.8
389.5
389.1
387.5
386.4
390.5
388.1 388.4
387.4
391.5
390.7
391.4
390.4
388.0
389.1
544.8
391.5
390.5
389.3
540.8
540.8
391.8
391.4
%
391.8 392.4
390.2
387
388
389
390
391
543.8
543.7
542.7
544.8 544.9
542.9
544.7
543.7
m/z
386
545.8
546.9
545.9 546.8
542.7 542.8
541.9
541.7
541.3
392.3
0
385
TOF MSMS 621.60ES+
8
542.8
100
390.5
388.5
387.5
385.7
385.3
552.9
552.0
549.9
385.6
384.5
550.8
548.8
543.8
541.9
386.6
385.6
%
544.8
391.6
388.6
387.6
547.8
545.8
%
389.5
388.6
545.0
544.7
547.8
547.9
547.0
546.7
547.8
545.7
548.8
548.8
548.6
550.7 550.8
550.9
550.6
549.8 549.9
549.7
550.6
548.4
551.2
552.9
552.8 552.9
552.0
551.9 552.1
553.1
552.7
m/z
541
542
543
544
545
546
547
548
549
550
551
552
553
52Bw4-3
RNM_52Bw4-3 621 (27.076) Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (617:691)
TOF MSMS 621.60ES+ RNM_52Bw4-3 621 (27.076) Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (617:691)
271.4
13.7 100
201.3
100
TOF MSMS 621.60ES+
9.10
276.4
199.3
282.4
272.4
273.4
270.4
195.3
197.3
%
280.4
200.3
192.3
197.3
198.3
192.3
197.3
197.3
195.3 195.4
197.3
196.3
193.3
194.4
196.3
193.3
193.4
195.2
196.2
197.1
193.0
271.4
201.3
195.3
195.4
195.3
%
0
TOF MSMS 621.60ES+ RNM_52Bw4-3 621 (27.076) Cm (617:691)
271.4
23 100
201.3
201.3
199.3
198.2
197.4
200.3
198.3
199.2
200.2
203.3
204.4
203.3
204.3
207.3
205.3
203.4
208.3
207.3
206.3
203.3
208.3
203.4
205.3
207.3
205.3
202.3
207.4
202.2
206.2 206.4
204.9
204.2
208.3
200.4
201.2
0
m/z
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
RNM_52Bw4-3 621 (27.076) Sb (2,40.00 ); Cm (617:691)
TOF MSMS 621.60ES+
6
494.8
100
491.7
491.6
490.7
%
489.9
490.7
493.7
491.8
491.6
491.0
492.7
493.7
492.7
491.9
491.6
492.3
494.6
492.8
494.2
493.6
492.5
493.8
495.7
495.0
495.9
495.4 495.6
494.6
0
RNM_52Bw4-3 621 (27.076) Cm (617:691)
491.7
491.6
490.7
489.9
490.7
491.6
491.0
490.2 490.5
492.7
493.7
492.7
491.6
491.3 491.5
493.7
491.8
491.9
492.3
492.2
496.2
TOF MSMS 621.60ES+
7
494.8
100
489.8
492.8
494.6
494.2
493.6
492.5
493.1 493.3 493.6
493.8
494.1
495.7
495.0
494.6
495.9
495.4 495.6
494.6
496.2
496.3
495.3
0
m/z
490
491
492
493
494
TOF MSMS 621.60ES+
17
276.4
273.3
273.4
277.4
495
496
270.4
271.4 271.5
270.5
270.5
276.4
272.5
273.5
272.3
272.1
272.5
271.3
282.4
282.4
276.4
279.4
280.4
281.4
280.4 281.4
272.3
%
274.4
274.5
274.3
275.5
275.4
275.3
274.2
276.4
275.5
277.4 277.5
276.5
279.4
278.4
278.4 278.5
277.5
279.3
276.2
277.3
278.2
279.2
276
277
278
279
280.5281.4
279.5
282.4
281.5
281.3
282.5
283.4
282.5
281.8
279.9
280.9
283.5
283.3
0
m/z
271
52Bw4-3
489.8
272.4
273.4
202.3
201.4
201.3
199.4
199.2
271.5
271.5
201.4
199.3 199.4
283.4
278.4
275.4
274.4
206.3
0
RNM_52Bw4-3 621 (27.076) Cm (617:691)
100
281.4
279.4
208.3
205.3
196.3
194.3
%
207.3
204.3
193.3
277.4
203.3
198.3
202.3
%
553.7
0
392
52Bw4-3
545.9
272
273
274
275
280
281
282
283
Monrad et al.
page S21
K-205, LKCASLQK(+), Glycosylation, retention time 27.80 min
B- and Y-ions
m/z predicted
m/z found
B1
B4
B5
B6
114.1
114.2
416.2
416.7
503.3
503.7
616.3
617.1
Y1 (includes
modification)
497.2
497.7
CA
175.1
175.3
SL
201.1
201.3
CAS
262.1
262.4
ASL
272.2
272.4
KCA
303.1
303.5
Y3 (includes
modification)
738.4
738.2
Internal ions
m/z predicted
m/z found
CASLQ
503.2
503.7
KCASL
503.3
503.7
Mass spectra K-205, LKCASLQK(+), Glycosylation, retention time 27.80 min
52Bw4-3
52Bw4-3
RNM_52Bw4-3 621 (27.076) Cm (617:691)
TOF MSMS 621.60ES+ RNM_52Bw4-3 621 (27.076) Cm (617:691)
109 100
120.2
100
TOF MSMS 621.60ES+
21
115.2
115.2 115.2
110.2
129.3
116.2
130.2
%
%
147.3
116.2
221.3
231.4
258.4 269.4
115.2
258.4
175.3
211.4
285.4
175.3
324.5
340.5
114.2
286.4
158.3
460.8
340.5
306.4
373.6
114.2
460.9
0
100
114.2
597.9
m/z
125
150
175
200
225
250
275
300
325
350
375
115.3
114.2
460.7
389.6
400
425
450
475
500
525
550
575
600
115.2
114.3
116.2
116.2
117.2
115.8
117.2
117.2
0
m/z
625
114
52Bw4-3
116.2
115.3
115.0
115
116
117
118
52Bw4-3
RNM_52Bw4-3 621 (27.076) Sm (Mn, 2x5.00); Sb (2,40.00 ); Sb (2,40.00 ); Cm (617:691)
TOF MSMS 621.60ES+ RNM_52Bw4-3 621 (27.076) Sm (Mn, 2x5.00); Sb (2,40.00 ); Sb (2,40.00 ); Cm (617:691)
423.6
1.99 100
417.6
100
TOF MSMS 621.60ES+
1
506.8
416.7
415.6
413.6
414.6
503.8
421.6
418.6
%
%
419.6
422.6
502.7
504.7
505.7
501.7
420.6
0
RNM_52Bw4-3 621 (27.076) Cm (617:691)
0
TOF MSMS 621.60ES+ RNM_52Bw4-3 621 (27.076) Cm (617:691)
9 100
416.7
100
413.6
% 413.6
416.6
415.6
415.6 415.6
414.6 414.7
413.7
413.5
417.6
417.6 417.7
416.7
417.7
414.7 415.5
414.4
417.5
415.5
415.1
417.4
423.6
419.6
419.7
418.6
421.6 421.7
420.6
419.6
417.7
421.6
419.7 420.6
417.7
416.1
416.5
503.7
421.6
418.6
415.7
413.8
418.5
418.8
418.5
420.5
419.4
422.5
421.2
420.5
423.6
422.4
423.8
423.5
424.4
m/z
415
416
417
418
419
420
421
422
423
504.8
503.4
503.6
505.7
505.8
507.6
506.6
504.9
504.6
504.0
506.8
505.9
505.4
505.6
506.2
507.2
506.4
507.1
507.6
0
m/z
424
502
52Bw4-3
504.7
503.9
503.1
502.4 502.6
506.7
504.7
503.8
503.6
502.1
423.5
503.8
502.8
502.6
501.8
%
0
414
502.7
501.7
423.6 423.7
422.7
421.8 422.5
422.8
421.5
TOF MSMS 621.60ES+
6
506.8
423.7
413.6
413.7
503
504
505
506
507
52Bw4-3
RNM_52Bw4-3 621 (27.076) Sm (Mn, 2x5.00); Sb (2,40.00 ); Sb (2,40.00 ); Cm (617:691)
TOF MSMS 621.60ES+ RNM_52Bw4-3 621 (27.076) Sm (Mn, 2x5.00); Sb (2,40.00 ); Sb (2,40.00 ); Cm (617:691)
494.8
1 100
100
TOF MSMS 621.60ES+
1.56
623.0
618.0
617.1
500.7
%
615.0
620.0
0
TOF MSMS 621.60ES+ RNM_52Bw4-3 621 (27.076) Cm (617:691)
494.8
6 100
618.0
617.0
615.0
616.9
615.9 616.0
615.1
614.2
493.7
623.0
614.9
618.0
618.9
617.1 617.9
615.7
615.6
616.4 616.8
619.1
620.0
618.8
619.9
617.6
619.7
621.0
620.5620.8
622.9
621.8 622.0
621.2
496.7
620.4
621.8
622.8
622.7
623.6
616
617
618
619
620
621
622
493.8
494.6
493.6
495.4
TOF MSMS 621.60ES+
7
496.9 497.6
498.6
498.3
500.7
499.6
497.9 498.6
497.3
499.8
498.7 498.8
497.8
496.6
500.8
499.7
497.7
495.9
495.6
494.6
498.8
499.2
498.4
501.6 501.7
500.6
500.9
499.9 500.6
499.4
501.8
500.9 501.6
500.4
501.3
502.0
501.6
0
623
494
52Bw4-3
497.7
496.8
495.7
495.0
494.2
493.6
m/z
615
494.6
623.1
%
622.2
0
614
500.7
623.0
617.9
493.7
613.9
501.7
495.8
621.0
617.1
100
498.7
499.8
616.0
613.9
497.7
493.7
0
RNM_52Bw4-3 621 (27.076) Cm (617:691)
%
496.7
%
621.9
619.0
495
496
497
498
499
500
m/z
502
501
52Bw4-3
RNM_52Bw4-3 621 (27.076) Sm (Mn, 2x5.00); Sb (2,40.00 ); Sb (2,40.00 ); Cm (617:691)
TOF MSMS 621.60ES+ RNM_52Bw4-3 621 (27.076) Sm (Mn, 2x5.00); Sb (2,40.00 ); Sb (2,40.00 ); Cm (617:691)
175.3
1.10 100
738.2
100
TOF MSMS 621.60ES+
17.7
211.4
183.3
%
199.3
%
727.4
732.2
730.3
734.0
739.1
740.1
731.1
738.3
735.2
748.2
731.2
724.2
725.1
727.0
728.0 729.2
730.3
740.1
732.3
734.0
737.2
738.1
742.2
741.3
746.8 748.1
745.7
734.0
745.6
753.3
749.2
750.9
754.2
m/z
726
728
730
732
734
736
738
740
742
744
746
748
750
155.3
752
187.3
183.3
176.3 181.3
181.3
173.3
165.2
159.3 164.3
162.3
201.3
201.3
197.3
201.3
195.3
204.4
195.3
187.3
191.3
207.3
189.3
181.2
195.3
0
m/z
754
155
52Bw4-3
166.2
167.2169.3
158.3
153.2
183.3
183.3
175.3
158.3
156.2
%
752.1
750.8
0
724
211.4
209.3
175.3
156.3
748.0
TOF MSMS 621.60ES+
32
175.3
751.3
748.3
743.1 744.3
207.3
189.3
153.2
0
TOF MSMS 621.60ES+ RNM_52Bw4-3 621 (27.076) Cm (617:691)
5 100
738.1
100
177.3
165.3
203.3
195.3
191.3
171.3
159.3
753.3
754.2
750.9
746.1 746.8
736.2
181.3
176.3
167.3
751.3
743.1 744.3
0
RNM_52Bw4-3 621 (27.076) Cm (617:691)
%
173.3
155.3
735.2
729.1
185.3
166.3
731.1
724.0
209.3
201.3
187.3
158.3
156.3
748.2
160
165
170
175
180
185
190
195
200
205
210
52Bw4-3
RNM_52Bw4-3 621 (27.076) Sm (Mn, 2x5.00); Sb (2,40.00 ); Sb (2,40.00 ); Cm (617:691)
TOF MSMS 621.60ES+ RNM_52Bw4-3 621 (27.076) Sm (Mn, 2x5.00); Sb (2,40.00 ); Sb (2,40.00 ); Cm (617:691)
19.2 100
269.4
100
TOF MSMS 621.60ES+
14.2
324.5
332.5
306.4
268.4
%
262.4
267.4
264.4
261.4
265.4
266.4
300.5
273.4
302.4
301.4
323.5
310.5
303.5
272.4
270.4
328.5
314.5
%
271.4
263.4
260.4
315.5
316.5
312.5
305.5
304.5
308.5
326.5
325.5
320.5 321.5
333.5
334.5
329.5
330.5
318.5
336.5
327.5
331.5
0
RNM_52Bw4-3 621 (27.076) Cm (617:691)
0
TOF MSMS 621.60ES+ RNM_52Bw4-3 621 (27.076) Cm (617:691)
39 100
269.4
100
TOF MSMS 621.60ES+
28
324.5
324.5
269.4
269.4
324.4
269.4
306.4
303.5
263.4
%
260.4
260.4
260.5 261.4
260.3
261.2
269.3 269.5
271.4
268.4
265.4
273.3
267.4
270.4 271.4
271.5 272.4
268.5
265.3 265.5 266.4
268.3
269.5
270.4
273.4
264.4
266.5
272.3
268.5
272.5
263.3 263.4
271.4
269.5
264.4
267.5
273.5
266.3
272.3
267.3
263.5
268.8
270.5
262.5
264.5
268.0
272.5
270.2
273.6
262.4
262.4
262.3
262.1
0
260
261
262
263
%
300.5
263.4
264
265
266
267
268
269
270
271
272
273
m/z
274
302.5
302.4
303.5
305.5
306.5
310.5
312.4
328.5
324.4
314.5
315.5
317.5
316.5
310.4
320.5
332.5
329.4
326.5
325.5
326.6
321.5 323.5
321.5
332.5
332.4
320.4
333.5
333.5
333.5
334.5 336.5
331.5
337.5
335.5
0
m/z
300
302
304
306
308
310
312
314
316
318
320
322
324
326
328
330
332
334
336
Monrad et al.
page S22
The obtained LC–MS/MS data (Table 2 in the manuscript and the K-199 and K-205 MS/MS spectra
above) with m/z 621.6 and a retention time of 27.80 min could in theory be ascribed glycosylation
at both lysines 199 and 205. Due to the typical inability of trypsin to tolerate a modification at
lysines at the P1 position we believe that the ion with m/z 621.6 and a retention time of 27.80 min
corresponds to a modification at K-199, but it could in principle be the K-205 modification - or both
simultaneously.
Monrad et al.
page S23
K-436, VGSK(+)CCK, Transacylation, retention time 24.16 min
B- and Y-ions
m/z predicted
m/z found
B1
B2
B3
B4 (includes
modification)
546.3
546.8
100.1
100.2
157.1
157.3
244.1
244.4
GS
145.1
145.3
CC
207.0
207.3
SK(+)
390.2
390.5
Y1
Y2
147.1
147.3
250.1
250.4
Internal ions
m/z predicted
m/z found
K(+)C
406.2
407.6
Mass spectra K-436, VGSK(+)CCK, Transacylation, retention time 24.16 min
52Bw4-1-2
52Bw4-1-2
RNM_52Bw4-1 497 (24.660) Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (454:535)
100
112.2
120.2
TOF MSMS 451.20ES+ RNM_52Bw4-1 497 (24.660) Cm (454:535)
55.3 100
TOF MSMS 451.20ES+
46
102.2
198.3
237.4
181.3
201.3
%
209.4
173.3
157.3
136.2
252.4
235.3
459.7
312.5 320.4
269.4
340.5
270.4
348.5
546.8
476.7
417.6
563.9
0
RNM_52Bw4-1 497 (24.660) Cm (454:535)
100
112.2 120.2
120.2
TOF MSMS 451.20ES+
89
102.2
237.4
101.2
181.2 181.3
%
252.4
173.3
129.2
100.2
209.4
130.2
252.4
269.4 280.5
312.4
546.8546.9
476.8
417.7
160
180
200
220
240
260
280
300
320
340
360
380
400
420
440
460
504.7
480
500
101.3
100.3
101.3
547.0 563.9
m/z
140
102.1
101.2
546.8
459.7
320.4 340.5
357.5
120
100.2
100.2
198.3
201.3
201.3
181.3
0
100
%
198.3
520
540
560
580
52Bw4-1-2
0
100
103.2
103.2
102.3
104.2
102
103
104
TOF MSMS 451.20ES+ RNM_52Bw4-1 497 (24.660) Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (454:535)
38 100
157.3
100
157.3
%
211.4
207.3
206.3
210.3
208.4
205.3
157.3
153.3
204.3
0
RNM_52Bw4-1 497 (24.660) Cm (454:535)
158.3
153.2
%
153.2
149.2
149.2
150.3
151.2
150.2
151.3
155.2
153.3 154.3
152.3
151.3 152.3
154.3
209.4
159.3
155.3
156.2
156.3
156.2
154.2
153.2
151.3
158.2
155.2 155.3
155.2
158.2
157.2
156.2
209.3
159.2 159.3
158.3
158.2
%
160.3
162.3
160.3
162.2
161.2
160.2
161.3
159.2
159.1
163.3 164.3
164.2 164.3
165.3
165.3
205.3
204.4
m/z
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
206.3
206.2
205.3
207.3
209.4
209.3
208.4
208.4
209.2
211.4
211.4
211.4
210.3
210.3
210.4
209.3
207.4
206.4
209.5
211.3
211.2
0
165
m/z
204
52Bw4-1-2
207.3
207.3 207.4
206.3
204.3
0
149
TOF MSMS 451.20ES+
46
209.4
100
155.3
TOF MSMS 451.20ES+
23.0
209.4
157.3
148
105
52Bw4-1-2
RNM_52Bw4-1 497 (24.660) Cm (454:535)
148.2
105.2
m/z
101
205
206
207
208
209
210
211
52Bw4-1-2
RNM_52Bw4-1 497 (24.660) Cm (454:535)
TOF MSMS 451.20ES+ RNM_52Bw4-1 497 (24.660) Cm (454:535)
66 100
237.4
100
TOF MSMS 451.20ES+
31
546.8
237.4
252.4
546.9
546.8
%
%
235.3
235.4
252.4
237.3
547.0
237.5
252.4
245.4
238.4
239.4
235.3
241.4
235.4
234.4
236.4
236.3 237.2
234.4
239.3
239.4
241.4 242.4
240.4
237.5
245.4
239.3
239.4
239
240
244.4
242.4 242.4
246.3
243.4 245.3
246.4
248.4
246.4 247.4
546.7
253.4
251.3
249.4 250.4
251.4
250.4
248.4
251.5
250.3 250.4
547.8
254.4
251.4
251.4
252.5 253.4
253.3
254.5 255.4
0
236
237
238
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
147.3
147.2
144.3
151.3
148.2
149.2
148.3
150.3
151.3
150.2
152.2
%
147.2 147.3
146.3
144.2
145.3
144.3
143.9
144.2
144.9
145.2
145.2
146.2 146.3
145.9 146.2
146.9
147.2
147.9
149.3
151.2 151.3
148.2 148.3
149.2
148.2
149.2 149.3
150.2
148.2
149.2
150.2
148
149
150
148.3
151.2
151.3
150.7 151.1
152.0
152.2
0
m/z
144
145
146
147
151
152
539.8
555.8 556.9
548.8 549.8
541.9
573.8
564.9
574.8
561.8
558.0
573.8
565.9 568.0
0
m/z
530
TOF MSMS 451.20ES+
10
147.3
100
563.9
563.8 564.0
545.8
545.8
531.9
533.9 535.9
m/z
235
530.8
254.3 254.5
52Bw4-1-2
RNM_52Bw4-1 497 (24.660) Cm (454:535)
563.9
547.9
254.5
532
534
536
538
540
542
544
546
548
550
552
554
556
558
560
562
564
566
568
570
572
574
Monrad et al.
page S24
K-525, K(+)QTALVELVK, Glycosylation, retention time 18.86 min
B- and Y-ions
B1 (includes
modification)
479.2
479.7
m/z predicted
m/z found
Y1
Y3
Y4
Y8
147.1
147.2
359.3
359.5
488.3
488.6
872.5
873.2
Internal ions
m/z
predicted
m/z
found
TA
173.1
AL
185.1
LV
213.2
ALV
284.2
TAL
286.2
ELV
342.2
VEL
342.2
LVE
342.2
TALV
385.2
LVEL
455.3
QTALV
513.3
TALVE
514.3
173.3
185.3
213.3
284.4
286.4
342.5
342.5
342.5
385.5
455.6
513.7
514.7
Mass spectra K-525, K(+)QTALVELVK, Glycation, retention time 18.86 min
52Bw4-6-2
RNM_52Bw4-6 198 (18.852) Cm (176:231)
100
129.2
TOF MSMS 740.70ES+
89
129.2
136.2
136.2
147.2
226.3
249.4
%
110.2
321.4
226.3
147.3
218.4
321.5
249.4
186.3
187.3
325.5
318.4
284.4
330.5
744.2
742.0
423.5
424.5
394.5
315.4
744.3
314.4
175.3
744.3
0
100 125
52Bw4-6-2
m/z
150
175
200
225
250
275
300
325
350
375
400
425
450
475
RNM_52Bw4-6 198 (18.852) Cm (176:231)
100
147.2
%
500
525
550
575
600
625
650
675
700
725
750
775
800
TOF MSMS 740.70ES+
59
147.2
147.3
147.2
144.2
145.3
152.3
152.3
148.3
147.2
146.2
148.2
155.3
155.3
153.2
153.3
152.2
153.2
152
153
156.2
156.2
155.3
154.3
0
m/z
145
146
147
148
149
150
151
154
155
156
52Bw4-6-2
52Bw4-6-2
RNM_52Bw4-6 198 (18.852) Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (176:231)
TOF MSMS 740.70ES+ RNM_52Bw4-6 198 (18.852) Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (176:231)
6.28 100
359.5
100
TOF MSMS 740.70ES+
2.81
488.6
358.5
486.6
490.6
485.7
%
362.5
361.5
%
363.5
487.7
489.6
492.6
357.6
360.5
491.7
493.2
0
RNM_52Bw4-6 198 (18.852) Cm (176:231)
0
TOF MSMS 740.70ES+ RNM_52Bw4-6 198 (18.852) Cm (176:231)
486.6
11 100
359.5
100
359.5
358.5
488.6
358.4
359.4
360.5
357.6
359.6
358.3
357.7
358.0
358.7
358.1
359.0 359.3
359.0
360.4
360.4
359.7
485.7
363.5
486.6 486.7
%
359.7
360.2
360.1
360.2
360.5
361.4
360.6
362.3
361.6
360.7
361.3
362.5
362.0
361.9
362.3
362.3
363.3
363.1
362.9
363.1
0
363.3
359
360
361
362
363
486.5
487.6
485.9
489.7
493.6
488.5
487.7
486.7
490.6
487.8
487.8
489.5
488.5
489.7
490.7
488.8
491.8
491.7
490.4
493.5
492.5
492.7
363.6
485.2
485.6
487.0
487.5
488.1
488.3
489.2
489.8
490.9
491.6
491.9
492.8
493.2
363.7
486.2 486.5
487.3
489.1
363.9
m/z
358
485.8
363.4
362.6
361.6
361.2
488.7
361.5
360.5
359.4
359.3
358.2
363.6
362.4
361.4
358.6
358.4
490.6
362.5
362.5
%
TOF MSMS 740.70ES+
9
490.1
490.3
491.3
492.2
492.5
493.1
0
493.5
m/z
485
486
487
488
489
490
491
492
493
Monrad et al.
page S25
52Bw4-6-2
52Bw4-6-2
RNM_52Bw4-6 198 (18.852) Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (176:231)
TOF MSMS 740.70ES+ RNM_52Bw4-6 198 (18.852) Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (176:231)
1 100
100
TOF MSMS 740.70ES+
496.7
5.92
495.7
477.7
880.3
478.6
479.6
497.7
470.6
879.2
%
467.6
%
873.2
883.3
882.5
486.6
494.6
482.6
483.6
472.6
490.6
485.7
484.7
493.6
487.7
474.6
878.1
489.6
492.6
886.3
875.1
872.3
480.6
475.6
471.6
465.6 466.6
885.2
884.2
873.4
481.6
476.6
468.7
874.2
871.3
488.6
469.7
491.7
877.2
0
RNM_52Bw4-6 198 (18.852) Cm (176:231)
873.2
100
879.3
0
TOF MSMS 740.70ES+ RNM_52Bw4-6 198 (18.852) Cm (176:231)
4 100
880.3
TOF MSMS 740.70ES+
496.7
12
477.7
495.7
497.7
496.6
873.1
879.2
882.5
880.6
883.3 883.4
884.2
885.2
477.6
469.7
478.7 479.7
497.7
486.6
470.6
469.6
467.6
%
871.3 871.6
874.0
872.3
874.3
875.2
877.2 878.1 878.3
879.1
880.2
881.6 882.3
884.4
885.3
886.4
%
488.6
471.6
467.6
465.6
468.6
875.0
876.7 877.2 877.8
879.0
884.1
885.1
886.2
887.6
m/z
872
873
874
875
876
877
878
879
880
881
882
883
884
885
886
480.7
490.6
482.6
498.7
481.6
477.5
485.7
482.6
474.6 475.6
489.7
483.7
490.6
487.6
484.7
495.6
493.6
488.5
483.7
495.6
492.5 493.5 494.7
888.4 888.6
0
871
477.6
475.7
466.8
873.0
479.7
471.7
465.7
872.2
476.6 477.6
497.6
887
0
m/z
888
466
52Bw4-6-2
468
470
472
474
476
478
480
482
484
486
488
490
492
494
496
498
52Bw4-6-2
RNM_52Bw4-6 198 (18.852) Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (176:231)
TOF MSMS 740.70ES+ RNM_52Bw4-6 198 (18.852) Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (176:231)
14.6 100
187.3
100
TOF MSMS 740.70ES+
13.4
211.3
186.3
213.3
175.3
%
%
183.3
173.3
166.2
212.3
207.3
185.3
209.3
216.3
215.3
169.3
167.3
214.3
176.3
171.3
210.3
181.3
170.3
174.3
177.2
208.3
189.3
182.3
180.3
191.3
188.3
184.3
168.3
162.2
0
RNM_52Bw4-6 198 (18.852) Cm (176:231)
0
TOF MSMS 740.70ES+ RNM_52Bw4-6 198 (18.852) Cm (176:231)
26 100
186.3
100
TOF MSMS 740.70ES+
22
211.3
187.3
211.4
187.3
213.3
186.3
207.3
175.3
%
173.3
166.2
169.3
167.3
165.3
168.3
176.3
171.3
170.3
166.2 167.2
162.3
188.3
173.2
174.3
171.2 171.3
181.3
183.4
186.2
188.3
192.2
0
164
166
168
170
172
174
176
178
180
182
184
186
188
190
208.2
207.4
m/z
162
209.4
208.9 209.2
209.4
214.4
213.4
211.2
212.4
212.8
212.2
214.3
213.4
213.2
212.3
211.2
210.4
216.3
216.4
215.3
216.3
215.4
211.5
213.2
213.2
214.3
214.2
214.2
213
214
215.3
216.3
214.4
215.4
216.2
215.2
215.8
215.1
216.4
216.4
216.4
216.8
216.0
0
m/z
192
208
52Bw4-6-2
211.3
210.3
210.4
210.3
209.4
208.3
207.4
190.3
215.3
214.3
213.3
208.3
191.3
189.2
181.2 181.4 183.2 184.3
178.3 179.3
172.7
213.4
212.3 212.4
209.2
208.3
191.3
184.3
177.2
168.2 169.2
164.3
207.3
183.3 185.2 185.3
171.2
212.3
211.4
187.3
175.2
167.3
162.2
183.3
175.3
166.3
209.3
209.3
209.3
%
185.3
209
210
211
212
215
216
52Bw4-6-2
RNM_52Bw4-6 198 (18.852) Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (176:231)
TOF MSMS 740.70ES+ RNM_52Bw4-6 198 (18.852) Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (176:231)
12.1 100
284.4
100
TOF MSMS 740.70ES+
394.6 8.06
381.5
290.4
385.5
379.5
388.6
389.6
288.4
%
%
282.4
383.5
380.5
286.4
387.5
393.5
382.5
285.4
278.4
391.4
283.4
279.3
289.4
384.5
287.4
0
RNM_52Bw4-6 198 (18.852) Cm (176:231)
0
TOF MSMS 740.70ES+ RNM_52Bw4-6 198 (18.852) Cm (176:231)
20 100
284.4
284.4
100
392.5
390.5
386.6
281.4
280.4
284.4
394.6
381.5
290.4
284.4
TOF MSMS 740.70ES+
394.5 18
381.5
383.5
385.6
281.4
284.3
282.4
279.3
280.4
282.4
282.4
283.4
279.3
280.3
283.3
279.4
280.5 281.3 281.4
283.3
278.5
282.3
283.5 284.3
279.2 279.5
280.5 281.3 281.5
282.5
283.9
280.3
281.3
281.8
279.1
283.2
280.3
281.2
282.3
284.2
279.1
280.2
281.2
283.2
0
278
279
280
281
282
283
288.3
288.4
286.4
284.5
278.4
%
288.3
286.4
285.5
284.5
285.3
287.4
287.3
287.4
286.5 287.3
288.3
286.5
286.3
389.6
288.5
286.9 287.3
286.2
285
288.2
289.5
286
287
288
289
379.4
290.5
379.7
380.6
380.4
290.7
290
52Bw4-6-2
381.6
383.6
383.4
381.7
380.7
380.3
386.5
385.4
384.6
383.6
387.4
388.5
386.5
385.0
387.4
392.5
391.4
390.4
394.4
392.5
390.5
393.4
393.6
393.4
394.4
391.6
388.7
387.6
385.7
391.4
389.6
389.5
388.4
384.4
383.3
388.6
387.6
385.4
382.4
381.3
389.5
387.5
387.5
385.4
382.6
390.3
389.3
391.4
388.4
393.6
393.3
391.7
391.3
394.3
392.4
384.4
381.3
291.0
m/z
291
383.5
382.5
393.5
388.5
383.6
382.5
381.4
380.6
289.5
290.3
289.6
290.0
289.1
381.4
380.4
290.3
288.5
289.3
385.6
380.5
380.5
289.4
285.5
285.1
388.6
% 379.4 379.6
289.4
288.4
389.6
379.5
290.5
285.4
285.3
385.5
290.4
285.4
285.3
284
290.4
393.3
0
m/z
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
52Bw4-6-2
RNM_52Bw4-6 198 (18.852) Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (176:231)
TOF MSMS 740.70ES+ RNM_52Bw4-6 198 (18.852) Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (176:231)
4.78 100
453.6
100
452.6
TOF MSMS 740.70ES+
6.87
521.8
462.6
461.7
513.7
514.7
448.6
460.6
455.6
%
%
451.6
449.6
454.6
450.6
522.7
512.7
463.6
458.6
533.7
524.7
515.7
523.7
456.6
530.8
459.6
505.6
457.6
516.7
506.7
507.7
525.7
518.7
508.7 509.7 510.7
520.7
517.7
511.7
531.7 532.8
526.8
534.7 535.7
527.8
529.6
519.7
0
RNM_52Bw4-6 198 (18.852) Cm (176:231)
0
TOF MSMS 740.70ES+ RNM_52Bw4-6 198 (18.852) Cm (176:231)
15 100
453.6
100
TOF MSMS 740.70ES+
16
521.8
521.8
514.7
452.6
448.6
%
451.7
449.6
449.6
448.6
449.5
449.5
450.5
450.5
452.5
453.7
450.7
449.6 449.7 450.6
448.5
513.7
513.7
462.7
461.7
461.5
462.5
451.6
453.5
451.6
451.5
451.4
453.5
454.7
454.6
454.6
452.5
452.4
455.6
455.6
455.6
456.6
458.6
458.6 458.7
459.5
456.5
455.5
457.5 457.6
456.5 456.7
455.5
456.5
448.4
457.4
460.6
460.6
461.5
460.5
458.5 458.7
458.1
457.4
458.8
462.5
461.4
462.4
505.6
507.8
506.6 507.5
463.7
462.8
463.5
459.4
450
451
452
453
454
455
456
457
458
459
523.7 524.7
524.6 524.7
516.8 517.8
517.7
509.7
510.7
518.6
518.8
519.7
463.9
521.7
530.8 531.8
529.6
525.8
525.8 525.9
520.7
521.6
531.7
528.8
527.8
527.7
530.7
533.7
533.7
532.8
533.8
534.7 535.7
535.7
464.2
m/z
449
511.7 512.6
508.7
463.8
0
448
522.8
515.8
510.7
463.6
463.5 463.6
461.9
460.5
460.5
461.8
521.8
515.7
512.7 513.7
%
462.7
460
461
462
463
464
0
m/z
506
508
510
512
514
516
518
520
522
524
526
528
530
532
534
536
Monrad et al.
page S26
K-525, K(+)QTALVELVK, Transacylation, retention time 22.07 min
B- and Y-ions
B1 (includes
modification)
303.2
303.5
m/z predicted
m/z found
Y2
Y3
Y4
246.2
246.4
359.3
359.6
488.3
488.8
EL
243.1
243.4
ELV
342.2
342.6
Internal ions
AL
185.1
185.3
m/z predicted
m/z found
VE
229.1
229.4
QT
230.1
230.4
VEL
342.2
342.6
LVE
342.2
342.6
ALVE
413.2
413.7
QTAL
414.2
414.6
VELV
441.3
441.7
ALVEL
526.3
526.8
K-525, K(+)QTALVELVK, Transacylation, retention time 22.07 min
52Bw4-8
52Bw4-8
RNM_52Bw4-8 364 (22.032) Cm (329:400)
TOF MSMS 651.40ES+ RNM_52Bw4-8 364 (22.032) Cm (329:400)
7.07e3 100
293.4
100
TOF MSMS 651.40ES+
38
304.5
307.5
307.5
304.5
306.5
%
%
175.3
229.4
653.0
342.6
158.3
185.3
246.4
266.4
300.6
406.6
360.6
150
175
200
225
250
306.4
275
300
325
350
301.4
301.5
375
400
425
450
475
500
525
550
575
600
625
308.5
302.5
303.6
302.6
303.3
302.8
304.6
304.7
304.3
306.6
306.4
305.9
308.5
306.6
306.3
307.6
307.4
308.4
307.6
306.6
308.6
307.3
308.6
308.1
m/z
650
301
52Bw4-8
304.3
303.4
301.7 302.4
301.3
305.5
305.4
305.5
305.4
304.5
304.4
303.5
0
m/z
125
306.5
306.4
301.5
300.6
359.6
294.4
136.2
112.2
0
100
303.5
652.0
265.4
307.4
306.5
304.4
303.5
300.5
302
303
304
305
306
307
308
52Bw4-8
RNM_52Bw4-8 364 (22.032) Cm (329:400)
TOF MSMS 651.40ES+ RNM_52Bw4-8 364 (22.032) Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (329:400)
229.4
610 100
100
TOF MSMS 651.40ES+
1.34e3
%
230.4
0
RNM_52Bw4-8 364 (22.032) Cm (329:400)
%
471.8
488.8
477.7
%
485.8
470.7
472.8
486.8
478.7
504.7 505.8
489.8
0
230.4
0
m/z
470
472
474
476
478
480
482
484
486
488
TOF MSMS 651.40ES+
2.03e3
229.4
100
490
492
494
496
498
500
502
504
m/z
506
225
226
227
228
229
230
231
232
233
234
52Bw4-8
RNM_52Bw4-8 364 (22.032) Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (329:400)
TOF MSMS 651.40ES+
7.37
413.7
100
414.6
%
52Bw4-8
RNM_52Bw4-8 364 (22.032) Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (329:400)
TOF MSMS 651.40ES+
127
243.4
100
415.6
416.6
412.6
0
RNM_52Bw4-8 364 (22.032) Cm (329:400)
%
245.4
0
RNM_52Bw4-8 364 (22.032) Cm (329:400)
413.7
414.7
414.6 414.7
TOF MSMS 651.40ES+
185
243.4
100
414.6
413.8
413.5
412.7
412.6
%
245.9246.0
m/z
243
244
245
414.0
413.1
412.5
413.0
416.6 416.7
416.6
416.7
414.7
413.8
413.4
412.1 412.5
245.4
244.5
414.5
414.5
412.8
244.4
415.7
415.5
%
0
242
TOF MSMS 651.40ES+
14
413.7
413.6
100
244.4
414.7
417.6
416.8
414.8
414.2
414.4
415.5
415.3
415.1 415.3
415.7
416.1
416.0
416.5
417.1 417.2
416.4
418.7
418.7
417.7
417.5
418.5
418.4
417.5
418.6
0
246
m/z
413
414
415
416
417
418
52Bw4-8
RNM_52Bw4-8 364 (22.032) Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (329:400)
52Bw4-8
RNM_52Bw4-8 364 (22.032) Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (329:400)
TOF MSMS 651.40ES+
8.28
441.7
100
TOF MSMS 651.40ES+
3.76
526.8
100
443.7
446.7
442.7
%
%
527.8
528.8
530.8
524.8
529.9
444.7
0
RNM_52Bw4-8 364 (22.032) Cm (329:400)
0
RNM_52Bw4-8 364 (22.032) Cm (329:400)
441.7
TOF MSMS 651.40ES+
9
526.8
TOF MSMS 651.40ES+ 100
15
441.6
441.6
100
443.7
442.6
441.8
442.7
446.7
446.7
443.6
442.7
443.6
442.6
446.6
526.8
446.8
%
442.8
441.5
439.7
441.5
446.6
440.1
440.5
440.0
440.4
440.8
441.2
441.1
441.5
442.5
443.5
442.9
443.9
444.5
444.8
444.7
443.9
445.5
445.4
442.2 442.5
524.7
446.8
445.7
445.7
445.6
524.7
446.0
446.2 446.5
446.9
524.3
m/z
441
442
443
444
445
446
526.7
524.8
525.1 525.2 525.6
525.8
525.7
527.0
525.9
526.1 526.3
527.8
526.6
527.9
530.8
528.9
527.7
530.7
529.9 529.9
527.3 527.6
528.4
529.6
528.5
530.2 530.4
530.7
530.9
531.1
530.6
531.4
447.3 447.3
0
440
528.8
446.8
441.8
441.9
528.8
527.8 527.9
524.8
443.6
442.9
440.6 440.7
527.8
%
443.6
439.7
439.7
526.9
526.7
443.8
447
0
m/z
525
526
527
528
529
530
531
Monrad et al.
page S27
K-534, KQTALVELVK(+), Transacylation, retention time 22.07 min
B- and Y-ions
m/z predicted
m/z found
B1
B2
129.1
129.2
257.2
257.4
Y1 (includes
modification)
321.1
321.5
AL
185.1
185.3
VE
229.1
229.4
QT
230.1
230.4
Internal ions
m/z predicted
m/z found
EL
243.1
243.4
ELV
342.2
342.6
VEL
342.2
342.6
LVE
342.2
342.6
ALVE
413.2
413.7
QTAL
414.2
414.7
VELV
441.3
441.7
ALVEL
526.3
526.8
Mass spectra K-534, KQTALVELVK(+), Transacylation, retention time 22.07 min
52Bw4-8
52Bw4-8
RNM_52Bw4-8 364 (22.032) Cm (337:388)
TOF MSMS 651.40ES+ RNM_52Bw4-8 364 (22.032) Cm (337:388)
6.90e3 100
293.4
100
%
TOF MSMS 651.40ES+
659
136.2
%
652.0
265.4
175.3
229.4
359.6
294.4
136.2
185.3
158.3
112.2
0
100
653.0
342.6
266.4
246.4
130.2
406.6
360.6
125
150
175
200
225
250
275
300
325
350
375
400
425
450
475
500
525
550
575
600
625
650
675
700
0
725
m/z
119
52Bw4-8
137.2
133.2
129.2
m/z
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
52Bw4-8
RNM_52Bw4-8 364 (22.032) Cm (337:388)
TOF MSMS 651.40ES+ RNM_52Bw4-8 364 (22.032) Cm (337:388)
321.5
351 100
100
TOF MSMS 651.40ES+
125
324.5
324.5
321.4 321.5
325.5
325.6
%
%
321.6
324.4
321.6
258.4
0
m/z
254
255
256
257
258
259
260
261
262
321.7
326.5
324.7
323.6
325.7
324.2
326.4
326.6
0
263
m/z
320
52Bw4-8
323.5
323.5 323.6
322.4
320.5
320.4 320.5
257.4
256.4
321
322
323
324
325
326
52Bw4-8
RNM_52Bw4-8 364 (22.032) Cm (337:388)
TOF MSMS 651.40ES+ RNM_52Bw4-8 364 (22.032) Cm (337:388)
1.98e3 100
229.4
100
%
TOF MSMS 651.40ES+
1.02e3
342.6
%
185.3
343.5
186.3
230.4
204.3
202.3
187.3
243.4
215.3
211.3
0
m/z
185
190
195
200
205
210
215
220
225
230
235
240
m/z
337
338
339
340
341
342
343
344
345
346
347
348
52Bw4-8
RNM_52Bw4-8 364 (22.032) Cm (337:388)
100
0
245
52Bw4-8
TOF MSMS 651.40ES+ RNM_52Bw4-8 364 (22.032) Cm (337:388)
441.6 441.7
23 100
410.6
TOF MSMS 651.40ES+
13
443.7
443.7
442.6
441.8
442.6
442.7
411.6
410.7
443.6
414.7
410.7
441.5
414.6
411.6 411.7
411.6
413.5
412.7
442.9
415.7
414.5
413.4
440.7
416.5
415.7416.5
413.8
415.5
416.8
418.7
418.6
417.7
419.6
419.5
420.7
440.8
420.6
m/z
412
413
414
415
416
417
418
419
420
421
RNM_52Bw4-8 364 (22.032) Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (337:388)
TOF MSMS 651.40ES+
6.80
521.8
100
515.8
526.8
%
518.8
516.8
514.7
510.8
522.8
519.8
517.8
520.7
527.8
523.8 524.8
528.8
0
RNM_52Bw4-8 364 (22.032) Cm (337:388)
515.7
515.8
515.9
%
510.8 511.8
512.8
514.7
514.7 515.2
514.5
530.8
TOF MSMS 651.40ES+
15
521.8
100
521.7
521.7 521.9
516.8
518.8
516.8
517.8
518.7
520.7 521.6
526.8
522.7
521.9
526.8 526.9
523.8
519.7 520.7
524.8
526.7
525.9
527.8
528.8
528.9
530.9 531.8
0
m/z
511
512
513
514
515
516
517
518
519
520
521
522
523
524
525
526
527
528
529
530
531
532
441.4
443.5
442.5
442.9
441.5
442.2 442.3 442.5
443.9
443.5
444.1 444.1
0
444.3 444.5
m/z
441
52Bw4-8
441.9
441.2
441.0 441.1
0
411
441.5
416.6
414.7
411.8 412.6
442.8
441.8
415.5
413.8
411.7
443.8
443.6
%
411.7
410.8
443.6
442.7
413.6
413.7
%
443.6
442.6
413.7
442
443
444
Monrad et al.
page S28
The obtained LC–MS/MS data (Table 2 in the manuscript and the K-525 and K-534 MS/MS spectra
above) with m/z 651.4 and a retention time of 22.07 min could in theory be ascribed transacylation
at both lysines 525 and 534. Due to the typical inability of trypsin to tolerate a modification at
lysines at the P1 position we believe that the ion with m/z 651.4 and a retention time of 22.07 min
corresponds to a modification at K-525, but it could in principle be the K-534 modification - or both
simultaneously.
Monrad et al.
page S29
Reactive Accessibility of HSA
The solvent accessibilities at probe sizes 1.0, 1.4 and 2.8 Å calculated using the NACCESS
program5 as the relative % accessibility of each residue as the compared to the accessibility of that
residue in an Ala-xxx-Ala tripeptide6are shown below. The calculations were performed on the A
chain of dimeric HSA; the B strain gave nearly identical values. There is no clear correlation
between reactivity and relative side-chain accessibility. For example, Lys199 has a very low
relative side-chain accessibility; its reactivity might be attributed to a very low pKa of 7.9, which
has been rationalised previously by close interactions with His242.7, 8 Lys525 and Lys534 also have
quite low relative side-chain accessibility.
Monrad et al.
page S30
Monrad et al.
page S31
Control Reaction of HSA with D-glucuronic acid
As a control experiment, D-glucuronic acid (0.5 or 5.0 mM) was incubated with HSA (33 g/L)
according to the general procedure for incubation of AGs with HSA. Workup by size-exclusion
chromatography and subsequent analysis by mass spectrometry (LC/MS) revealed a completely
unmodified spectrum of HSA.
At 0.5 mM
Monrad et al.
At 5 mM
page S32
Monrad et al.
page S33
General Synthetic Methods for AGs
Allyl, benzyl and 4-methoxybenzyl α,β-D-glucuronates, as well as compound 1c ([(2S)-ibuprofen
acyl glucuronide] were prepared as described previously: Tetrahedron, 2007, 63, 7506-7805, ref.
15a in the main MS, and Org. Biomol. Chem., 2009, 7, 2525-2533. Below we summarise the
selective acylation method used for all the 1β-acyl glucuronides and appropriate deprotection
conditions for allyl, benzyl and 4-methoxybenzyl (PMB) esters. Characterisation data are finally
given for all compounds: 1a, 1b, 1c (for ease of comparison with 1b), 1d, 2 and 3.
Selective acylation:
The appropriate carboxylic acid (typically 0.5 mmol) was dissolved with allyl, benzyl or 4methoxybenzyl α,β-D-glucuronate (0.5 mmol) and HATU (0.5 mmol) in anhydrous acetonitrile (5
mL) and stirred with N-methylmorpholine (1.0 mmol) under N2 at 20°C. When the reaction was
judged complete by TLC analysis (10% EtOH-CH2Cl2), neutralization was effected by addition of
Amberlyst A-15 (H+, 2 eq.). The mixture was filtered, then the filtrate was evaporated and the
residue chromatographed on silica, eluting with 5-10% EtOH-CH2Cl2. Appropriate fractions were
combined and evaporated to give the 1β-acyl glucuronide as an allyl, benzyl or PMB ester, typically
as a foam, in 60-80% yield.
Deprotection of allyl esters:
PS–PPh3–Pd(0), purchased from Argonaut Technologies, was stirred in THF–DMF, 1:1 (1 mL per
0.1 g of resin) for 0.5 h, filtered, washed with further THF and dried before use. This material (0.2
eq.), the appropriate allyl ester (1 eq.) and morpholine (1 eq.) were stirred in THF : DMF (4:1, 4 mL
per mmol allyl ester) at 20 °C under N2 for 3 h. Amberlite IR-120 (H+) resin (1.5 eq.) was added
followed by stirring for 0.5 h, then the resin was filtered off and washed with the same solvent (10
mL) followed by evaporation of filtrate and washings to dryness. Chromatography of the crude
product, eluting with 15% and then 50% EtOH–CH2Cl2, afforded the acyl glucuronide as a foam on
evaporation of appropriate fractions.
Deprotection of benzyl esters:
A solution of the appropriate benzyl ester (typically 0.3 mmol) in THF: PriOH, 1:1 (10 mL) was
stirred at 60°C with cyclohexa-1,4-diene (1 mL) and 10% Pd-C (10 mg) for 1.5h. When reaction
was complete by TLC the mixture was cooled and filtered, then the combined filtrate and washings
Monrad et al.
page S34
were evaporated to dryness and the residue was triturated with ether. Removal of ether followed by
rigorous drying afforded the desired acyl glucuronide in high purity.
Deprotection of 4-methoxybenzyl (PMB) esters:
The PMB ester was treated with a solution of 10% CF3CO2H in CH2Cl2 at 0°C and stirred until
deprotection was complete by TLC. Solvent was removed in vacuo and the residue triturated with
CH2Cl2 to deliver the desired acyl glucuronide as a solid, after rigorous drying to remove acid
traces.
Carboxylic acids:
Ibufenac (precursor of 1a) and ponolrestat (precursor of 3) were supplied by Astra Zeneca;
commercial (R, S)-ibuprofen was used to acylate benzyl glucuronate (v. s.) and the diastereomers
were separated at the benzyl ester stage. As noted above, (S)- 1c has been described previously but
its characterisation is given below for clarity along with (R)-1b. [2,2-Dimethyl-(4isobutyl)]phenylacetic acid, the precursor of 1d, was obtained from ibuprofen as described below.
(4-Isobutyl)phenylacetyl 1β-acyl glucuronide (ibufenac acyl glucuronide) 1a
(Via the benzyl ester) 1H NMR (400 MHz, (CD3)2CO): δ 0.88 (6H, d, J = 6.6 Hz, Me2CH), 1.811.86 (1H, m, Me2CH), 2.45 [2H, d, J=7.2 Hz, ArCH2CH(CH3)2], 3.46-3.64 (3H, m, 2´-H, 3´-H and
4´-H), 3.72 (2H, s, ArCH2CO), 3.95 (1H, d, J = 9.5Hz, 5´-H), 5.57 (1H, d, J = 8.1Hz,1´-H), 7.12
(2H, d, J = 8 Hz, ArH) and 7.23 (2H, d, J = 8 Hz, ArH); 13C NMR (100 MHz, (CD3)2CO): δ 21.7,
30.0, 39.8, 44.7, 71.5, 72.4, 75.8, 76.1, 94.5, 128.9, 129.0, 129.3, 131.2, 140.2, 169.5 and 170.3;
+
m/z (ES +ve mode) 391 (MNa ). Found: m/z, 391.1364. C18H24O8Na requires m/z, 391.1369.
[(2R)-2-Methyl-(4-isobutyl)]phenyl]acetyl 1β-acyl [(R)-ibuprofen acyl glucuronide) 1b
(Via the benzyl ester) 1H NMR (400 MHz, (CD3)2CO): δ 0.88 (6H, d, J = 6.5 Hz, Me2CH), 1.47 (3
H, d, J = 6.9 Hz, CH3CH), 1.84 (1H, m, Me2CH), 2.45 [2H, d, J=7.2 Hz, ArCH2CH(CH3)2], 3.41,
3.55 and 3.65 (3H, 3m, 2´-H, 3´-H and 4´-H), 3.81 (1 H, q, J = 6.9 Hz, CH3CH), 3.99 (1 H, d, J =
9.6 Hz, 5´-H), 5.57 (1H, d, J = 8 Hz,1´-H), 7.12 and 7.26 (4 H, dd, ArH); 13C NMR (100 MHz,
(CD3)2CO): δ 18.3, 21.7, 30.1, 44.7, 71.7, 72.5, 75.9, 76.3, 94.6, 127.2, 127.3, 129.1, 137.6, 140.3,
Monrad et al.
page S35
+
169.1 and 172.9; m/z (ES +ve mode) 405 (MNa ). Found: m/z, 405.1519. C19H26O8Na requires
m/z, 405.1525.
[(2S)-2-Methyl-(4-isobutyl)]phenyl]acetyl 1β-acyl [(S)-ibuprofen acyl glucuronide) 1c
(Via the benzyl ester) 1H NMR (400 MHz, (CD3)2CO): δ 0.88 (6H, d, J = 6.8 Hz, Me2CH), 1.45 (3
H, d, J = 7.2 Hz, CH3CH), 1.84 (1H, m, Me2CH), 2.44 [2H, d, J=7.2 Hz, ArCH2CH(CH3)2], 3.43,
3.57 and 3.63 (3H, 3m, 2´-H, 3´-H and 4´-H), 3.81 (1 H, q, J = 7.2 Hz, CH3CH), 3.96 (1 H, d, J =
9.4 Hz, 5´-H), 5.59 (1H, d, J = 8 Hz,1´-H), 7.12 and 7.25 (4 H, dd, ArH); 13C NMR (100 MHz,
(CD3)2CO): δ 18.6, 21.7, 30.0, 44.6, 71.7, 72.5, 75.9, 76.3, 94.6, 127.3, 129.2, 137.7, 140.3, 169.0
+
and 172.8; m/z (ES +ve mode) 405 (MNa ). Found: m/z, 405.1519. C19H26O8Na requires m/z,
405.1525.
[2,2-Dimethyl-(4-isobutyl)]phenyl]acetic acid
Ibuprofen methyl ester (1.42 g, 6.88 mmol) was added in portions under N2 to a stirred suspension
of NaH (1.28 g, 53.6 mmol) in dry THF (50 mL). After stirring at 20°C for 30 min, iodomethane
(2.0 cm3, 32.2 mmol) was added dropwise over 10 min. The mixture was stirred at 20°C for 2 days,
then filtered through Celite, and the filtrate was evaporated. The crude product was dissolved in a
minimum amount of EtOAc and purified by chromatography on SiO2, eluting with a gradient of
3% to 20% of diethylether in hexane. The first eluted product (640 mg, 40%) was the Me ester of
the title compound; it was hydrolysed by dissolving in dioxane: water, 9:1 followed by the addition
of 2M NaOH (13.6 cm3, 27.3 mmol) and heating at 50°C for 16h. After cooling, the bulk of solvent
was removed in vacuo and 2M HCl was added to give a pH of 2, followed by extraction with
diethyl ether (3 x 40 cm3). The combined organic extracts were washed with brine, dried over
MgSO4 and evaporated to dryness. Chromatography of the residue using diethyl ether-hexane
mixtures afforded the title acid (500 mg, 83% yield), Rf 0.15 (90:10, Hex:Et2O); 1H NMR [400
MHz, (CD3)2CO] δ 0.90 (6H, d, J = 6.5 Hz, 2CH3), 1.58 (6H, s, 2CH3), 1.85 (1H, m, CH2CHMe2),
2.44 (2H, d, J = 7.1 Hz, CH2), 7.11 (2H, ddd, J = 2.0, 2.2, 8.3 Hz, ArH), and 7.33 (2H, ddd, J = 2.0,
2.2, 8.3 Hz, ArH);
13
C NMR (100 MHz, (CD3)2CO] δ 22.4, 26.2, 30.1, 44.9, 45.9, 125.5, 129.1,
140.3, 141.0 and 183.2; m/z ESI 238 [M + NH4]+. Found: m/z, 238.18036, C14H24O2N requires
238.18070; found, C 76.22, H 9.19; C14H20O2 requires C, 76.33, H 9.15%). This carboxylic acid
was coupled to benzyl glucuronate, followed by deprotection, as described above.
Monrad et al.
page S36
[2,2-Dimethyl-(4-isobutyl)]phenyl]acetyl 1β-acyl glucuronide 1d
(Via the benzyl ester) 1H NMR [400 MHz, (CD3)2CO]: δ 0.89 (6H, d, J = 6.6 Hz, 2CH3), 1.56 (3H,
s, CH3), 1.59 (3H, s, CH3), 1.85 (1H, m, Me2CHCH2), 2.45 (2H, d, J = 7.1 Hz, CH2), 3.42 (1H, t, J
= 8.0 Hz, CH), 3.55-3.68 (2H, m, CH), 4.01 (1H, d, J = 9.4 Hz, 5´-H), 5.61 (1H, d, J = 8.0 Hz, 1´H), 7.12 (2H, d, J = 8.3 Hz, ArH), and 7.33 (2H, d, J = 8.3 Hz, ArH);
13
C NMR [100 MHz,
(CD3)2CO] : δ 23.1, 27.2, 27.8, 31.3, 45.8, 47.6, 72.9, 73.7, 77.1, 77.6, 96.1, 126.8, 130.2, 141.1,
143.2 and 206.7; m/z (ESI + ve mode) 419 [M+Na]+. Found: m/z, 419.1693; C20H28O8Na requires
m/z, 419.1682.
4-Bromobenzoyl 1β-acyl glucuronide 2
(Via the PMB ester) 1H NMR [400 MHz, (CD3)2CO]: δ 3.60-3.76 ((3H, 3m, 2´-H, 3´-H and 4´-H),
4.11 (1 H, d, J = 9.3 Hz, 5´-H), 5.83 (1H, d, J = 7.6 Hz, 1´-H), 7.75 and 8.02 (4 H, 2d, ArH); 13C
NMR [100 MHz, (CD3)2CO] : δ 72.9, 73.8, 77.1, 77.4, 96.4, 129.4, 129.9, 132.8, 133.1, 160.5 and
-
170.2; m/z (ES, -ve ion mode) 375 (M-H) . Found: m/z, 374.9723. C13H13O879Br requires m/z,
374.9716.
[3-(4-bromo-2-fluorobenzyl)-4-oxo-3H-phthalazin-1-yl]acetyl 1β-acyl glucuronide
(Ponolrestat 1β-acyl glucuronide) 3
(Via the allyl ester) This material was finally purified to homogeneity by preparative HPLC. 1H
NMR [400 MHz, (CD3)2SO]: δ 3.45, 3.58, 3.68 (3 H, 3m, 2´-H + 3´-H + 4´-H), 4.06 (1 H, d, J = 9.5
Hz, 5´-H), 4.16 (2 H, s, ArCH2CO), 4.64 (2 H, m, ArCH2N), 5.64 (1 H, d, J = 7.9 Hz, 1´-H), 7.28 (1
H, m, ArH), 7.36 (1 H, m, ArH), 7.45 (1 H, m, ArH), 7.90-8-08 (3 H, m, ArH) and 8.36 (1 H, m,
-
ArH); m/z (ES –ve mode) 567, 565 [(M-H) for 81Br, 79Br). Found: m/z, 567.0223 and 565.0237;
C23H19N2O9F81Br requires m/z, 567.0237 and C23H19N2O9F79Br requires m/z, 565.0258.
Monrad et al.
page S37
References
1.
2.
3.
4.
5.
6.
7.
8.
D. Bar-Or, R. Bar-Or, L. T. Rael, D. K. Gardner, D. S. Slone and M. L. Craun, Crit Care
Med, 2005, 33, 1638-1641.
R. L. Garlick and J. S. Mazer, J Biol Chem, 1983, 258, 6142-6146.
N. Iberg and R. Fluckiger, J Biol Chem, 1986, 261, 13542-13545.
S. A. Marashi, S. Safarian and A. A. Moosavi-Movahedi, Med Hypotheses, 2005, 64, 881.
S. J. Hubbard and J. M. Thornton, Department of Biochemistry and Molecular Biology,
University College London, 1993.
S. I. van Kasteren, H. B. Kramer, H. H. Jensen, S. J. Campbell, J. Kirkpatrick, N. J. Oldham,
D. C. Anthony and B. G. Davis, Nature, 2007, 446, 1105-1109.
J. T. Gerig and J. D. Reinheimer, J. Am. Chem. Soc., 1975, 97, 168–173.
D. C. Carter and J. X. Ho, Adv. Protein Chem., 1994, 45, 153-203.