1
Supporting Information
2
Experimental detailed
3
2.2 Plant material and extraction procedure
4
Broad beans (Granadina cv.) were purchased from local market in their edible stage in
5
April 2012 (Granada, Spain). Then, samples were washed with distilled water, frozen at
6
–25°C, until being lyophilized. Then, 0.5 g of the sample was treated with 20 mL of
7
methanol/water (80:20, v/v), sonicated for 30 min in an ultrasonic bath, and centrifuged
8
at 3800 rpm for 15 min. The supernatant was collected in a round bottom flask and the
9
solvent removed by evaporation using a rotator evaporator in vacuo. Finally, the dry
10
residue was dissolved with 0.5 mL of methanol/water (80:20, v/v) and passed through a
11
0.22 µm syringe filter, previous to its analysis.
12
2.3 Analysis by RP-UHPLC-ESI-QTOF-MS and -MS/MS
13
Analyses were performed using an ACQUITY™ UPLC (Waters, Millford, MA, USA),
14
consisting of a vacuum degasser, an auto-sampler, and a binary pump system. The
15
UHPLC system was coupled to a micrOTOF-Q™ (Bruker Daltonik, Bremen,
16
Germany), equipped with an ESI interface. The analytical conditions were as described
17
previously [23].
18
A reversed-phase C18 analytical column (4.6 × 150 mm, 1.8 μm, Agilent Zorbax
19
Plus) protected by a guard cartridge of the same packing was employed. The mobile
20
phase consisting of acidified water (0.5% acetic acid, v/v) (A) and acetonitrile (B),
21
following the gradient elution program: 0 min, 0% B; 10 min, 20% B; 15 min, 30% B;
22
20 min, 50% B; 25 min, 75% B; 30 min, 100% B; 37 min 0% B; and finally, the initial
23
conditions were maintained for 3 min as a re-equilibration step. The injection volume
24
was 10 μL and the flow rate was set at 0.80 mL/min. The effluent from the UHPLC was
25
splited using a T-type splitter and directed to the mass spectrometer at approximately
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0.2 mL/min. The spectra were acquired in negative-ion mode over a mass-to-charge
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(m/z) range from 50 to 1100. QTOF parameters were set at: capillary voltage, +4.0 kV;
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drying gas temperature, 210°C; drying gas flow, 8.0 L/min; nubilizing gas pressure,
29
2 bar; end-plate offset, –0.5 kV. Moreover, automatic MS/MS was set. Nitrogen was
30
used as drying, nebulising and collision gas. A sodium acetate cluster solution was used
31
for external instrument calibration.
32
The MS and MS/MS data were processed through Data Analysis 4.0 software
33
(Bruker
34
(SmartFormula), simulate isotope patterns, as well as theoretical monoisotopic,
35
nominal, and average masses (IsotopePattern), and build common neutral losses
36
(BuildingBlockEditor). Therefore, the elemental composition of each compound was
37
determined by the SmartFormula tool that generates a list of possible molecular
38
formulae based on a CHNO algorithm, and the other elements which usually occur in
39
smaller numbers could be also added. It provides standard functionalities such as
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minimum/maximum elemental range, electron configuration, and ring-plus double-bond
41
equivalents, the deviation between the measured mass and theoretical mass (error) (Da
42
and ppm), and a sophisticated comparison of the theoretical with the measured isotope
43
patterns (msigma value or m) for increasing the confidence in the suggested molecular
44
formula. The broadly accepted error and m value for confirmation of elemental
45
compositions were set at 5 ppm and at 50, respectively. The use of isotopic-abundance
46
pattern removes more than 95% of false candidates. The molecular formula generated
47
for each compound, its MS/MS spectra and its comparison with spectra found in the
48
literature was the main tool for putative identification of broad bean metabolites. For
49
this, the following chemical structure databases were consulted: PubChem
Daltonics),
which
supplies
tools
to
calculate
molecular
formulae
50
(http://pubchem.ncbi.nlm.nih.gov),
51
SciFinder Scholar (https://scifinder.cas.org), HMDB (http://www.hmdb.ca/), Phenol-
52
Explorer
53
(http://kanaya.naist.jp/knapsack_jsp/top.html), Metlin (http://metlin.scripps.edu), and
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MassBank (http://www.massbank.jp), which is related to KNApSAcK database. The
55
two latter databases, Metlin and MassBank, also include a repository of tandem mass
56
spectrometry data.
57
ChemSpider
(www.phenol-explorer.eu),
(http://www.chemspider.com),
KNApSAcK
Core
System
58
Results detailed
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3.4 Structural prediction of unknown metabolites from V. faba (detailed)
60
Along the consciously profiling of the seed extract, 24 compounds did not match
61
adequately with those found in literature and databases sharing the same molecular
62
formula. Thus, modifications of the previous characterized metabolites through
63
common metabolic reactions [72] were established according to their MS and MS 2 data:
64
hydroxylation (+ O), methylation (+ CH2), conjugation with leucine/isoleucine (+
65
C6H11NO2), and glycosylation (with hexose, C6H10O5, and with pentose, + C5H8O4).
66
These added groups could be assigned directly as fragment ions or indirectly as neutral
67
losses in MS2. The compounds proposed were derivatives of vicine, convicine, eucomic
68
acid, jasmonic acid, and adenosine. To make easy the comprehension, the fragments
69
shared in common or related to those are showed in bold letter in Table S2 (Supporting
70
Information).
71
Several isomers of vicine and convicine conjugated with hexose (compounds A-E)
72
and pentose (compounds F-I) were found. In MS2 the sequential losses of the sugars
73
leaded to the aglycones divicine and isouramil, and, afterwards, the loss of NHO to m/z
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110.0341 and 111.0188 (uracil), respectively. In the case of convicine derivatives, the
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fragment ion at m/z 141.0243 corresponded to the odd ion of isouramil. Moreover, two
76
consecutive losses of CHNO from pyrimidone ring of convicine derivatives were also
77
observed, in agreement with results commented above. On the other hand, the purine
78
nucleoside derivative succinyladenosine (J) was tentatively identified according to the
79
MS and MS/MS data. In this case, the fragmentation pattern was similar to that
80
described above for adenosine and correlated well with our proposal. Thus, the main
81
fragment ions were at m/z 266.0928, 250.0591 and 206.0695, corresponding to the loss
82
of the succinic acid moiety, the ribose moiety and the successive decarboxylation,
83
whereas m/z 134.0464 and m/z 115.0033 were adenine and the succinic acid moiety
84
([C4H6O4 – H2 – H]-), respectively. This compound is a biomarker of purine metabolism
85
disorders in humans [78], so the potential presence in edible vegetables deserves further
86
studies.
87
The other putative metabolites were related to jasmonic acid and dihydrojasmonic
88
acid. In the MS2 spectra of the hydroxylated and glycosylated forms (compounds K-M
89
and P), subsequent neutral losses of H2O according to the number of the OH groups,
90
and/or the hexose moiety was observed, together with the loss of CO2 from the carboxyl
91
group of the jasmonate backbone. In that case, fragment ions at m/z 179.1073
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([C11H16O2 – H]-) from compounds K, L and P and m/z 177.0928 ([C11H14O2 – H]-)
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from compound M resemble fragment ion m/z 181.1224 ([C11H18O2 – H]-) found in
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MS2 spectrum of compound OH-JA-Ile (see Fig. 2C). On the other hand,
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leucine/isoleucine was detected everywhere in compounds S-X and the product ion at
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m/z 142.0874 ([C7H13NO2 – H]-). Alternatively, compounds O and Q gave a major
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fragment ion at m/z 146.0812 with molecular formula C6H13NO3, indicating that could
98
be hydroxyleucine. Regarding to this, 4-hydroxyisoleucine has been described in
99
Fabaceae [79].
100
Two derivatives of eucomic acid were also proposed as methyl eucomic acid
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(compound N, m/z 269.0675) and benzylmalic acid (compound R, m/z 223.0612).
102
According to the fragmentation pattern of eucomic acid, the fragmentation mainly
103
occurred at the malic acid moiety. Thus, compounds N and R showed the major product
104
ions at m/z 209.0462 and 163.0399, respectively, corresponding to the loss of
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CH3−COOH (60.0211 u). Moreover, the loss of H2O and CO2 at m/z 207.0666 and
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161.0601, together with successive losses of C2H4 at m/z 179.0718 and m/z 133.0648, or
107
CO2 at m/z 163.0745 and m/z 117.0702, respectively, reinforce the assignments. In the
108
case of the compound N, the loss of malic acid – H2 (C4H4O5, 132.0059 u) was clearly
109
observed at m/z 137.0603, and subsequently the loss of CH3 at m/z 122.0381.
110
111
Other references
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[78] Ito, T., van Kuilenburg, A. B.P., Bootsma, A. H., Haasnoot, A. J., van Cruchten,
113
A., Wada, Y., van Gennip, A. H., Clin. Chem. 2000, 46, 445–452.
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[79] Jetté, L., Harvey, L., Eugeni, K., Levens, N., Curr. Opin. Investig. Drugs 2009, 10,
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353-358.