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SUPPLEMENTARY INFORMATION
In-source fragmentation and correlation analysis as tools for metabolites
identification exemplified with CE-TOF untargeted metabolomics
Joanna Godzien1; Emily G. Armitage1; Santiago Angulo1; M. Paz Martinez-Alcazar1;
Vanesa Alonso-Herranz1; Abraham Otero2; Angeles Lopez-Gonzalvez1; Coral Barbas1*
1) Centre for Metabolomics and Bioanalysis (CEMBIO), Facultad
Universidad CEU San Pablo, Campus Montepríncipe, Boadilla del
Madrid, Spain;
2) Department of Information and Communications Systems
Universidad CEU San Pablo, Campus Montepríncipe, Boadilla del
Madrid, Spain;
de Farmacia,
Monte, 28668
Engineering;
Monte, 28668
* To whom correspondence should be addressed: Coral Barbas, Pharmacy Faculty,
Campus Monteprincipe, San Pablo-CEU University, 28668 Boadilla del Monte, Madrid,
Spain, tel: 0034913724711, fax: 0034913724712, e-mail: cbarbas@ceu.es
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EXPERIMENTAL DESIGN
Figure 1S: Extracted Ion Chromatograms (EIC) for co-eluting metabolites used in the
experiment;
A) Mix 1: lysine and ornithine; B) Mix 2: threonine and asparagine; C) Mix 3: creatine and
acetylcarnitine; D) Mix 4: valine, serine, leucine and isoleucine; E) Mix 5: glutamine, proline
and phenylalanine; F) tyrosine, betaine, pipecolic acid and aspartic acid;
FRAGMENTOR VOLTAGE ADJUSTMENT
Several fragmentor voltages were tested in the range 150-230V with 20V increments.
Tests were performed using single standard, mix and plasma sample in order to find the
best compromise between high enough signals given by fragments but still high enough
signal given by the “precursor ion”. Data revision revealed that the best voltage for
fragmentation and correlation purposes, was the data obtained with 170V. For many
molecules, 210 and 230V was already too high a voltage and no signals for the
“precursor ion” remained. For standards and mixes, even 190V gave reasonable signals,
both for the ion of interest and its fragments. However for plasma samples, the signal
corresponding to the targeted m/z was very low for all metabolites, therefore 170V was
selected for final data analysis.
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
Figure 2S: Fragmentor voltage selection;
To portray the fragmentor voltage selection, lysine was used. Panels A-C show EICs for lysine
(m/z 147.1125) under different voltages for standard (A), mix (B) and plasma sample (C). As
can be seen, a decrease in the signal was observed with an increase in the voltage due to the insource fragmentation of the molecule. Panels D-G show EICs for lysine and its fragments,
obtained applying 150V (D), 170V (E), 190V (F) and 210V (G).
ADDITIONAL FRAGMENTS
Additional fragments (not listed in the databases) were found for lysine (m/z 101.1064
and m/z 112.0756), ornithine (m/z 88.0774 and m/z 97.0753) and methionine (m/z
135.0281). For lysine, the fragment m/z 101.1064 is formed by the loss of the
carboxylic group (Figure 3S-A), while the product ion m/z 112.0756 is created by
simultaneous loss of ammonia and water (Figure 3S-B). For ornithine, the product ion
m/z 88.0774 is formed by the loss of the carboxylic group (Figure 3S-C), while the
fragment m/z 97.0753 is generated by the simultaneous loss of the carboxylic group and
ammonia (Figure 3S-D). For methionine, the fragment m/z 135.0281 is formed by the
loss of methyl group (Figure 3S-E).
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86
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88
89
90
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92
93
94
95
96
97
98
99
100
101
102
103
104
Figure 3S: Additional fragments formation;
A) formation of fragment m/z 101.1064 for Lysine; B) formation of fragment m/z 112.0756 for
lysine; C) formation of fragment m/z 88.0774 for ornithine; D) formation of fragment m/z
101.1064 for ornithine; E) formation of fragment m/z 135.0281 for methionine.
ADDITIONAL CORRELATION
Six molecules demonstrated additional correlation. This correlation was found based on
the interpretation of the correlation coefficient for the molecules stated in the first
column (Table 1S). Among ions which show strong positive correlation with the
precursor ion, signals which were not fragments of it appeared. These m/z were
searched against the METLIN database through the option Metabolite Search: Multiple
Fragment (http://metlin.scripps.edu/fragment_search_multi.php?&return=yes). This
analysis brought the identification of the molecules listed in the second column (Table
1S).
Table 1S: Additional correlation found between metabolites.
This correlation was found based on the correlation analysis interpretation for metabolites listed
in the first column.
metabolite 1
aspartic acid
betaine
pipecolic acid
serine
valine
phenylalanine
105
metabolite 2
betaine
pipecolic acid
aspartic acid
betaine
valine
serine
citrulline
sample level
mix and plasma
plasma
mix and plasma
plasma
plasma
plasma
plasma
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107
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109
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111
112
113
114
115
116
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118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
Figure 4S: An example of additional correlation occurring between two perfectly coeluting metabolites.
RINGING ARTEFACTS
For some molecules, additional signals, which were found to correlate strongly with the
precursor ion, were not explained. Neither tandem MS database searches nor manual
spectra inspection identified them. The difference in the mass in most cases was around
0.1 Da. Structures of the fragments were inspected with the idea of some groups’
exchange (loss of -OH group instead of -NH3 etc.) or reorganisation, but none of the
additional signals were explained in this way. To investigate the origin of these ions,
further analyses were performed. Pure plasma samples were analysed that were spiked
with each of the metabolites listed in Table 2S. Pure plasma was analysed as a reference
value for both the fragment and its artefact(s), while spiking was performed to test if an
increase was observed either for the fragment alone or for the fragment and artefact
alike. The lack of an increase in the signal of the artefact would prove that it does not
originate from the targeted molecule. Obtained results show an increase for all artefact
signals for all metabolites. Data inspection and literature review allowed the formation
of a hypothesis: that observed artefacts are ringing artefacts. The ringing artefacts arise
from spurious signals close to the main transition in a signal. This hypothesis can be
supported by the fact that the artefact signal is always significantly lower than the
fragment (the ratio between signal of artefact and fragments oscillates between 0.001
and 0.052). Moreover, in the case of more than one artefact observed for one fragment
(leucine, ornithine, phenylalanine and glutamine) the ratio between the delta m/z of the
main signal (fragment) and two subsequent signals (artefacts) is constant at 3.3.
Table 2S: Some examples of observed ringing artefacts
m/z of
m/z of
metabolite
∆ m/z
artefact
fragment
136.1823
136.0755
0.1068
tyrosine
165.1715
165.0545
0.117
72.1596
72.0816
0.078
valine
86.1818
86.097
0.0848
leucine
signal of
artefact
1.22E+04
1.11E+04
1.34E+06
9.75E+04
signal of
fragment
7.31E+05
7.11E+05
2.55E+07
3.66E+06
lysine
ornitnine
phenylalanine
glutamine
133
134
135
136
137
138
139
140
141
142
143
144
145
146
86.2072
130.1908
84.1654
116.1691
116.1996
70.1431
120.1804
120.2111
130.1539
130.1847
84.1291
84.1544
130.0863
84.0814
116.0708
70.066
120.0803
130.0499
84.0449
0.1102
0.1045
0.054
0.0983
0.1288
0.0771
0.1001
0.1308
0.1041
0.1348
0.0792
0.1045
2.06E+04
2.10E+04
2.25E+04
3.25E+04
7.54E+03
7.60E+03
7.96E+04
1.65E+04
1.49E+04
2.81E+03
6.22E+03
3.43E+02
1.07E+06
1.06E+06
1.51E+06
4.61E+05
3.47E+06
7.56E+05
3.96E+05
VALINE AND BETAINE EXAMPLE
Figure 5S: Differentiation between two metabolites with the same monoisotopic mass
by example of valine and betaine;
A) EIC for standard of valine (blue) and betaine (green) and both of them in plasma sample
(black). Panel B and C correspond to valine while D and E to betaine. Panels B and D show
experimental tandem MS spectra for the plasma sample while panels C and E show the library
MS spectra. Red boxes show fragments relevant for ID confirmation based on spectral
comparison.
LEUCINE AND ISOLEUCINE SEMI-QUANTIFICATION
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150
151
152
153
154
155
Figure 6S: Leucine and isoleucine and their fragments;
MS/MS fragmentation spectrum for (A) leucine and (B) isoleucine; (C) EICs for leucine (m/z
132.1018) and its fragment m/z 86.0968 for standard; (D) EICs for isoleucine (m/z 132.1018)
and its fragments m/z 86.0968 and m/z 69.0704 for standard; (E) EICs for co-eluting isoleucine
and leucine (m/z 132.1018) and its fragments m/z 86.0968 and m/z 69.0704 for the mix.
Table 3S: Peak areas for the targeted ion of isoleucine (m/z 132.1025) and its
product ion (m/z 69.0704);
Concentration
[mg/L]
5
10
15
20
25
156
peak area
m/z 132.1025
1,897,224
3,643,672
5,327,077
8,147,814
9,523,011
m/z 69.0704
170,197
292,935
432,322
633,651
721,231
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161
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163
164
165
166
167
168
169
170
171
172
173
Figure 7S: Calibration curve for (A) isoleucine (m/z 132.1025) and its product ion (B)
(m/z 69.0704);
A five point calibration curve was obtained for isoleucine and its fragment. The curve was
obtained with linear regression giving R equal to 0.9948 for isoleucine and 0.9942 for its
fragment.
DATA USED FOR CORRELATION ANALYSIS
Table 4S: Summary of the data used for the correlation analysis;
For each investigated metabolite, detailed information is stated for the standard, mix and plasma
sample. Listed information includes the number of spectra collected across the peak; number of
considered spectra; number of all ions (m/z); number of reproducible ions corresponding to the
ions present in all spectra; number of considered ions restricted to the m/z smaller than targeted
m/z; data standardisation corresponding to the logarithmic transformation and/or auto-scaling;
presence (YES) or absence (NO) of ringing artefacts and presence (YES) or absence (NO) of
additional correlation.
THREONINE
standard
mix
plasma 100s
number of spectra
number of considered
spectra
number of ions
number of reproducible ions
number of considered ions
data standarisation
ringing artefacts
additional correlation
22
16
21
15
21
17
1301
104
52
log + auto-scaling
NO
NO
1222
103
53
log + auto-scaling
NO
NO
1047
68
30
auto-scaling
NO
NO
ASPARAGINE
standard
mix
plasma 100s
number of spectra
number of considered
spectra
number of ions
number of reproducible ions
number of considered ions
data standarisation
ringing artefacts
additional correlation
19
13
19
13
16
12
1299
129
72
log + auto-scaling
NO
NO
1181
123
69
auto-scaling
NO
NO
936
95
46
log + auto-scaling
NO
NO
CREATINE
standard
mix
plasma 100s
number of spectra
number of considered
spectra
number of ions
number of reproducible ions
number of considered ions
data standarisation
ringing artefacts
additional correlation
17
13
16
12
16
12
1096
104
58
auto-scaling
NO
NO
1190
112
62
auto-scaling
NO
NO
906
80
38
auto-scaling
NO
NO
ACETYLCARNITINE
standard
mix
plasma 100s
number of spectra
number of considered
spectra
number of ions
number of reproducible ions
number of considered ions
data standarisation
ringing artefacts
additional correlation
21
15
20
14
16
12
1319
114
86
auto-scaling
NO
NO
1241
112
82
auto-scaling
NO
NO
917
79
44
auto-scaling
NO
NO
ASPARTIC ACID
standard
mix
plasma 100s
number of spectra
number of considered
spectra
number of ions
number of reproducible ions
number of considered ions
data standarisation
ringing artefacts
additional correlation
22
18
23
17
28
22
1377
124
71
auto-scaling
NO
NO
1161
107
64
auto-scaling
NO
YES+ringing artefacts
1280
118
61
auto-scaling
NO
YES+ringing artefacts
TYROSINE
standard
mix
plasma 100s
number of spectra
number of considered
spectra
number of ions
number of reproducible ions
number of considered ions
data standarisation
ringing artefacts
additional correlation
24
18
23
17
24
17
1258
119
88
auto-scaling
YES
NO
1230
119
85
auto-scaling
YES
NO
1090
101
58
auto-scaling
NO
NO
BETAINE
standard
mix
plasma 100s
number of spectra
number of considered
25
19
26
20
24
18
spectra
number of ions
number of reproducible ions
number of considered ions
data standarisation
ringing artefacts
additional correlation
1399
120
51
auto-scaling
NO
NO
1232
102
48
log + auto-scaling
NO
YES
1031
82
28
auto-scaling
NO
NO
PIPECOLIC ACID
standard
mix
plasma 100s
number of spectra
number of considered
spectra
number of ions
number of reproducible ions
number of considered ions
data standarisation
ringing artefacts
additional correlation
19
15
21
17
24
18
1544
130
70
auto-scaling
YES
NO
1481
108
57
auto-scaling
YES
NO
1006
77
34
log
NO
YES+ringing artefacts
SERINE
standard
mix
plasma 100s
number of spectra
number of considered
spectra
number of ions
number of reproducible ions
number of considered ions
data standarisation
ringing artefacts
additional correlation
19
15
16
15
18
14
1270
124
42
auto-scaling
NO
NO
1242
103
41
log
NO
NO
1037
80
28
auto-scaling
NO
YES+ringing artefacts
VALINE
standard
mix
plasma 100s
number of spectra
number of considered
spectra
number of ions
number of reproducible ions
number of considered ions
data standarisation
ringing artefacts
additional correlation
19
15
19
15
19
15
1271
126
56
auto-scaling
YES
NO
1248
106
48
log + auto-scaling
YES
NO
1048
78
30
auto-scaling
YES
YES
LEUCINE
standard
mix
plasma 100s
number of spectra
number of considered
spectra
number of ions
number of reproducible ions
number of considered ions
20
16
21
15
26
18
1518
122
69
1306
111
61
1209
83
39
data standarisation
ringing artefacts
additional correlation
auto-scaling
YES
NO
auto-scaling
YES
NO
auto-scaling
YES
NO
LYSINE
standard
mix
plasma 100s
number of spectra
number of considered
spectra
number of ions
number of reproducible ions
number of considered ions
data standarisation
ringing artefacts
additional correlation
14
10
16
12
14
10
1286
134
81
auto-scaling
YES
NO
1156
112
71
auto-scaling
YES
NO
920
88
52
auto-scaling
YES
NO
ORNITHINE
standard
mix
plasma 100s
number of spectra
number of considered
spectra
number of ions
number of reproducible ions
number of considered ions
data standarisation
ringing artefacts
additional correlation
13
9
14
10
9
9
1159
141
87
log + auto-scaling
YES
NO
1098
115
65
auto-scaling
YES
NO
763
77
45
log
YES
NO
PHENYLALANINE
standard
mix
plasma 100s
number of spectra
number of considered
spectra
number of ions
number of reproducible ions
number of considered ions
data standarisation
ringing artefacts
additional correlation
21
17
32
22
24
18
1469
130
90
auto-scaling
YES
YES
1744
115
84
log + auto-scaling
YES
NO
1120
101
68
auto-scaling
NO
YES
GLUTAMINE
standard
mix
plasma 100s
number of spectra
number of considered
spectra
number of ions
number of reproducible ions
number of considered ions
data standarisation
ringing artefacts
additional correlation
21
17
28
20
22
16
1246
110
73
log
YES
NO
1635
109
66
log + auto-scaling
YES
NO
1167
88
52
log
YES
NO
174
PROLINE
standard
mix
plasma 100s
number of spectra
number of considered
spectra
number of ions
number of reproducible ions
number of considered ions
data standarisation
ringing artefacts
additional correlation
20
16
34
24
22
16
1324
116
51
log + auto-scaling
YES
NO
1889
105
48
log + auto-scaling
YES
NO
1059
94
43
auto-scaling
YES
NO