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Non-enzymatic Transglycosylation Reactions Induced by Roasting: New Insights from Models Mimicking Coffee Bean Regions with Distinct Polysaccharide Composition Ana SP Moreira, Joana Simões, Fernando Milheiro Nunes, Dmitry Victorovitch Evtuguin, Pedro Domingues, Manuel A. Coimbra, and Maria Rosario M. Domingues J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00342 • Publication Date (Web): 08 Feb 2016 Downloaded from http://pubs.acs.org on February 13, 2016
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Journal of Agricultural and Food Chemistry
Non-enzymatic Transglycosylation Reactions Induced by Roasting: New Insights from Models Mimicking Coffee Bean Regions with Distinct Polysaccharide Composition
Ana S. P. Moreira,† Joana Simões,† Fernando M. Nunes,‡ Dmitry V. Evtuguin,§ Pedro Domingues,† Manuel A. Coimbra,† M. Rosário M. Domingues†*
†
QOPNA, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal
‡
CQ-VR, Chemistry Research Centre, Department of Chemistry, University of Trás-os-
Montes e Alto Douro, 5001- 801 Vila Real, Portugal §
CICECO, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal
*Corresponding author (Tel: +351 234 370 698; Fax: +351 234 370 084; E-mail:
[email protected])
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Abstract
2
Three mixtures containing different molar proportions of (β1→4)-D-mannotriose
3
and
(α1→5)-L-arabinotriose,
oligosaccharides
structurally
related
to
coffee
4
polysaccharides (galactomannans and arabinogalactans), were roasted at 200 °C for
5
different periods. Electrospray ionization mass spectrometry (ESI-MS) and tandem
6
mass spectrometry (ESI-MSn) analyses of labeled (18O) and unlabeled samples allowed
7
identification of non-hybrid oligosaccharides, but also hybrid oligosaccharides
8
composed by both hexose and pentose units. The identification of hybrid
9
oligosaccharides allowed us to infer the occurrence of non-enzymatic transglycosylation
10
reactions involving both oligosaccharides in the starting mixtures. Also, it was observed
11
that using different proportion of the oligosaccharides in the starting mixtures and
12
extent of the thermal treatment lead to a variation in the composition of the compounds
13
formed. These results have led to the conclusion that, depending on the distribution of
14
the polysaccharides in the bean cell walls and the roasting conditions, different non-
15
hybrid and hybrid structures can be formed during coffee roasting.
16 17
Keywords:
thermal
processing;
18
transglycosylation; isobars; isomers
coffee;
galactomannans;
19
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arabinogalactans;
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INTRODUCTION
21
During the roasting process, galactomannans (GM) and type II arabinogalactans
22
(AG), the most abundant polysaccharides of green coffee beans,1 undergo structural
23
modifications, which are far from being completely elucidated due to the complexity
24
and diversity of the structures formed. However, it is well known that they are
25
depolymerized and debranched.2-5 Together with proteins, chlorogenic acids and
26
sucrose, GM and AG are involved in the formation of melanoidins, which are defined as
27
high molecular weight nitrogenous brown-colored compounds.6, 7 However, their exact
28
structures remain unclear. Also, GM are modified at the reducing end during coffee
29
roasting by the occurrence of dehydration, isomerization, decarboxylation, oxidation,
30
caramelization, and Maillard reactions.8
31
The dry thermal processing of oligosaccharides structurally related to coffee GM,9
32
namely (β1→4)-D-mannotriose (Man3), and (α1→5)-L-arabinotriose (Ara3), which is
33
structurally related to the arabinose side chains of coffee AG,10 promoted the formation
34
of new oligosaccharides. These oligosaccharides have a higher number of
35
monosaccharide units, referred to as degree of polymerization, and new types of
36
glycosidic linkages, showing the occurrence of non-enzymatic transglycosylation
37
reactions (TGR), i.e. the transfer of glycosyl units to the hydroxyl groups of other
38
glycosides, induced by roasting.11, 12 When mixtures of Man3 with Ara3 were roasted,
39
hybrid structures composed by arabinose and mannose residues derived from different
40
origins, Ara3 and Man3, respectively, were formed. The identification of the same type
41
of hybrid structures in roasted coffee polysaccharide-rich fractions supports the
42
hypothesis of the occurrence of non-enzymatic TGR during coffee roasting involving
43
GM and AG side chains.13
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Considering that the distribution of GM and AG in coffee bean cell walls is
45
heterogeneous,14 and they have different vulnerability to roasting-induced degradation,15
46
it is hypothesized that different non-hybrid and hybrid structures can be formed during
47
coffee roasting depending on the distribution of the polysaccharides in the bean cell
48
walls and the roasting conditions. In this work, untreated and thermally treated mixtures
49
with different proportions of Man3 and Ara3, mimicking possible regions within the cell
50
walls with distinct polysaccharide composition, were analyzed. Electrospray ionization
51
mass spectrometry (ESI-MS) and electrospray ionization collision-induced dissociation
52
tandem mass spectrometry (ESI-CID-MSn) analyses were performed by infusing labeled
53
(18O) and unlabeled samples. To verify if the structures formed during the thermal
54
processing of the model mixtures have the same structural features as those formed
55
during coffee roasting, or additional roasting treatments beyond the roasting of the
56
green coffee beans, a GM-rich fraction isolated from spent coffee grounds (SCG),
57
submitted to additional roasting treatments, was treated with an endo-(β1→4)-D-
58
mannanase. The hydrolyzed material was further fractioned by ligand exchange/size-
59
exclusion chromatography (LEX-SEC) and analyzed by ESI-MS and ESI-CID-MSn.
60
The elemental composition of the ions identified in the ESI-MS spectra of both
61
oligosaccharide mixtures and SCG sample was obtained by high resolution and high
62
mass accuracy measurements using an Orbitrap-based mass spectrometer.
63 64
MATERIAL AND METHODS
65 66
Oligosaccharide samples and preparation of the mixtures. Oligosaccharides,
67
Ara3 and Man3, with a purity ≥ 95%, were purchased from Megazyme (County
68
Wicklow, Ireland). As previously described,13 solutions of each oligosaccharide (0.129
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mmol in 200 mL of ultrapure water) were prepared, and then used to prepare three
70
mixtures containing different molar proportions of Ara3 and Man3 as follows: 25%
71
Ara3:75% Man3 (A25M75); 50% Ara3:50% Man3 (A50M50); and 75% Ara3:25% Man3
72
(A75M25). Each mixture (in solution) was freeze-dried, and the resulting solid material
73
was powdered with an agate mortar and pestle, and then stored in a desiccator under a
74
phosphorous pentoxide environment.
75 76
Dry thermal treatments. The thermal treatments were performed on a TGA-50
77
thermogravimetric analyzer (Shimadzu, Kyoto, Japan), using the conditions previously
78
described.11-13 Briefly, three samples of each solid mixture (3-8 mg) were heated from
79
room temperature up to 200 °C, maintained at 200 °C for different periods of time: 0
80
(T1), 30 (T2), and 60 (T3) min. For each condition, the thermal treatment was repeated
81
at least twice in different days. The thermally treated mixtures were recuperated,
82
weighed, and suspended in ultrapure water in a concentration of 5 mg/mL. To facilitate
83
their dissolution, they were then stirred at 37 ºC for 3 h. The water-soluble fractions
84
were separated and kept frozen at -20 °C until analysis by MS. Solutions (1 mg/mL) of
85
each untreated mixture (T0) were also prepared and stored under the same conditions.
86 87
Labeling with oxygen-18. To label with oxygen-18 (18O) the carbonyl oxygen of
88
non-modified reducing sugar residues, or new carbonyl groups formed by dry thermal
89
processing, 50 µL of untreated and thermally treated oligosaccharide mixtures,
90
previously dissolved in water, were dried and redissolved in 50 µL of
91
water (H218O, 97%) (Sigma-Aldrich, St. Louis, MO). Each solution was then kept under
92
stirring at 500 rpm for 4 h at 37 °C in a sealed vial, after which time it was frozen at -20
93
°C until analysis by ESI-MS (4 days). To check the 18O-labeling of a keto group under
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18
O-enriched
Journal of Agricultural and Food Chemistry
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the conditions used because they had previously been used only to label the carbonyl
95
group of aldehydes, 2 µL of 3-octanone (99%) (Aldrich-Chemie, Steinheim, Germany)
96
were diluted in 50 µL of H218O, and then submitted to the same conditions of stirring
97
and freezing before ESI-MS analysis.
98 99
SCG sample, enzymatic hydrolysis and fractionation by LEX-SEC. SCG,
100
obtained after a commercial espresso coffee preparation, were submitted to a roasting
101
pre-treatment at 160 or 220 °C, and then sequentially extracted with water at 90 °C and
102
4 M NaOH solutions at 20, 60 and 120 °C. A GM-rich fraction, containing 89% of
103
mannose, was recovered from SCG roasted at 160 °C upon extraction with 4 M NaOH
104
at 120 °C, and became water insoluble upon neutralization.16 In order to convert this
105
fraction into cold water-soluble material, it was submitted to sequential roasting
106
treatments of 1 h at 200 ºC in a pre-heated oven (Binder). After each roasting procedure,
107
the material was suspended in water at room temperature with stirring during 1h. The
108
suspension was then centrifuged and the cold water-soluble material was recovered and
109
freeze-dried.15 A sample (2.4 mg) of the cold water-soluble material recovered after the
110
second roasting procedure, referred as R2W20sn,15 was hydrolyzed with 0.3 U of a pure
111
endo-(β1→4)-D-mannanase preparation (Aspergillus niger, EC 3.2.1.78) (Megazyme,
112
County Wicklow, Ireland) during 60 h at 37 ºC with continuous stirring in 600 µL of
113
100 mM Na-acetate buffer, pH 5.5, containing 0.02% sodium azide. The hydrolyzed
114
sample was filtrated using a Cronus nylon syringe filter (0.2 µm of pore size and 13 mm
115
of diameter) and then fractioned by LEX-SEC on a high-performance liquid
116
chromatograph equipped with a 300 mm x 20 mm i.d. Shodex sugar KS2002 column
117
from Showa Denko K. K. (Tokyo, Japan). The column was maintained at 30 °C, the
118
injected sample volume was 500 µL, and ultrapure water was used as eluent at a flow
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rate of 2.80 mL/min. A K-2401 refractive index detector (Knauer, Berlin, Germany)
120
was used. All collected fractions were dried, redissolved in 100 µL of ultrapure water,
121
and kept frozen at -20 °C until analysis by MS. To check its LEX-SEC elution profile,
122
the enzyme (0.3 U in 600 µL of 100 mM Na-acetate buffer) was injected and eluted
123
using the same conditions used for the hydrolyzed SCG sample.
124 125
ESI-MS conditions. Immediately prior to ESI-MS analysis, each sample,
126
previously dissolved in water, was diluted in methanol/water (1:1, v/v) containing
127
formic acid (0.1%, v/v).
128
Linear ion trap (LIT) conditions. ESI-MS and ESI-CID-MSn spectra were
129
acquired from all the mixtures and the LEX-SEC fraction obtained from the hydrolyzed
130
SCG sample in positive mode on a LXQ linear ion trap (LIT) mass spectrometer
131
(Thermo Fisher Scientific Inc., Waltham, MA), using the following operating
132
conditions: electrospray voltage, 5 kV; capillary temperature, 275 °C; capillary voltage,
133
1 V; and tube lens voltage, 40 V. Samples were introduced at a flow rate of 8 µL/min
134
into the ESI source. Nitrogen was used as nebulizing and drying gas. ESI-MS spectra
135
were acquired over the range m/z 100-1500. ESI-CID-MSn spectra were acquired with
136
the energy collision set between 19 and 29 (arbitrary units). Data were acquired and
137
analyzed using Xcalibur software.
138
Q Exactive Orbitrap conditions. The Q Exactive hybrid quadrupole-Orbitrap mass
139
spectrometer (Thermo Firsher Scientific, Germany), interfaced with H-ESI II ion
140
source, was employed for accurate mass measurements of the mixtures submitted to the
141
T3 treatment and the A50M50 mixture submitted to the T1 treatment, as well as the
142
LEX-SEC fraction obtained from the hydrolyzed SCG sample. The acquisition method
143
was set with a full scan and 140,000 resolution (relative to m/z 200) in positive mode.
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The method parameters were: AGC, 3e6; IT, 100 ms; scan range, 100-1000; spray
145
voltage, 3.0 kV; sheath gas, 5; aux gas, 1; capillary temperature, 250 °C; S-lens RF
146
level, 50; probe heater temperature, 50 °C; and flow rate, 5 µL/min. The Q Exactive
147
system was tuned and calibrated in positive mode using peaks of known mass from a
148
calibration solution (Thermo Scientific) to achieve a mass accuracy of < 0.5 ppm RMS.
149
The data were processed with Xcalibur 3.0.63 software.
150 151
RESULTS AND DISCUSSION
152 153
Color and water-solubility of each oligosaccharide mixture upon dry heating.
154
The possible occurrence of non-enzymatic TGR involving GM and AG side chains
155
during coffee roasting was investigated using as model solid mixtures of Man3 and
156
Ara3. Three mixtures containing different molar proportions of each oligosaccharide
157
(A25M75, A50M50 and A75M25) were used to mimic possible regions within the cell
158
walls with distinct polysaccharide composition. In order to mimic coffee roasting
159
conditions, the oligosaccharide mixtures were submitted to dry thermal treatments at
160
200 °C. Three different samples of each mixture were heated up to 200 °C and
161
maintained at 200 ºC for different periods: T1 (0 min), T2 (30 min) and T3 (60 min).
162
After dry thermal processing, the mixtures (white or off-white powders) acquired a
163
brown coloration, possibly due to caramelization reactions. In general, the brown
164
coloration was more intense for the longer treatments. However, the coloration of the
165
compounds resulting from the longer treatments (T2 and T3) of the mixtures with
166
higher proportion of Ara3 (A50M50 and A75M25) was darker than that observed when
167
A25M75 mixture was submitted to the same treatments. These observations are in line
168
with the lower thermal stability of Ara3 when compared with Man3,11, 12 which is also
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corroborated by the percentages of mass loss in each treatment from 150 °C, excluding
170
the initial mass loss due to the loss of adsorbed water molecules. Considering the
171
different mixtures, the mass loss percentages from 150 °C were 0.7-3.5% for T1, 11.0-
172
17.8% for T2, and 15.0-19.1% for T3. Also, independently of the starting mixture, the
173
compounds resulting from T1 were completely dissolved in water. However, as
174
observed for Ara3 (but not for Man3), the compounds resulting from T2 treatment of the
175
A75M25 mixture and T3 treatment of all mixtures were only partially dissolved in
176
water. The percentage of water-soluble material was 65.2% for T2 treatment of the
177
A75M25 mixture. For materials resulting from T3, the percentages of water-soluble
178
material were 68.8% for A25M75, 40.7% for A50M50, and 44.4% for A75M25. These
179
values are higher than those obtained with materials resulting from T2 and T3
180
treatments of Ara3 (35.3 and 24.6%, respectively),12 which shows the formation of a
181
higher amount of hydrophobic compounds when the amount of Ara3 is higher.
182
In summary, the observed differences in the coloration and water-solubility of the
183
thermally treated mixtures suggest that the structural modifications occurred in higher
184
extent with increasing of the molar proportion of Ara3 in the starting mixture, and the
185
time at 200 °C. This is in agreement with the higher diversity of ions observed in the
186
matrix-assisted laser desorption/ionization mass spectrometry spectra of the
187
oligosaccharide mixtures subjected to the longer treatments (T2 and T3).13 In order to
188
obtain a deeper insight into the structural modifications induced by dry thermal
189
processing, both untreated samples and water-soluble fractions recovered from thermal
190
treated samples were analyzed by ESI-MS and ESI-MSn.
191 192
Identification of hybrid and non-hybrid compounds upon dry heating. In
193
preliminary testing, we have found that, under ESI-MS conditions, neutral
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oligosaccharides ionize better in positive than in negative mode. For this reason,
195
positive mode was preferred for ESI-MS analysis of both untreated and thermally
196
treated mixtures.
197
As typical of neutral oligosaccharides,11, 12, 17 the oligosaccharides in the starting
198
mixtures (Man3 and Ara3), but also the roasting-induced compounds, were mainly
199
detected as sodium adduct ions ([M+Na]+). Accordingly, the most abundant ions in the
200
ESI-MS spectra of the untreated mixtures were observed at m/z 437 ([Ara3+Na]+) and
201
527 ([Man3+Na]+).
202
After thermal processing, new ions, not observed in the ESI-MS spectra of the
203
untreated mixtures, were identified. Independently of the starting mixture (A25M75,
204
A50M50, or A75M25), the diversity of ions was higher in the ESI-MS spectra obtained
205
after the longer treatments (T2 and T3). Due to the complexity of these ESI-MS spectra,
206
only the ions observed with a relative abundance equal or higher than 15% in at least
207
two ESI-MS spectra acquired from the thermally treated mixtures were considered
208
(Table 1). The assignment of these ions was supported based on their fragmentation
209
pattern under ESI-CID-MSn conditions, as will be later described, and corroborated by
210
the elemental composition obtained from high resolution and high mass accuracy
211
measurements using a hybrid quadrupole-Orbitrap mass spectrometer. Accordingly, the
212
ions identified in the ESI-MS spectra of the thermally treated mixtures were attributed
213
to [M+Na]+ ions of non-hybrid compounds composed by hexose (Hex) or pentose
214
(Pent) residues and derivatives, and hybrid compounds composed by both Hex and Pent
215
residues and derivatives (Table 1). As supported by sugar and glycosidic linkage
216
analyses,13 Pent and Hex were mainly arabinose (Ara) and mannose (Man),
217
respectively. However, new sugar residues, although in minor amounts, were formed
218
during thermal processing, namely ribose (Rib), xylose (Xyl) and lyxose (Lyx) that are
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isomers, i.e. have the same elemental composition, of Ara, and thus they are not
220
distinguishable by MS. Therefore, in Table 1, Pent represents mainly Ara, but also Rib,
221
Xyl and Lyx that are present in minor amounts. Similarly, Hex represents mainly Man,
222
but also isomeric sugars (glucose and galactose) that are present in minor amounts.
223
As observed for Ara3,12 the non-hybrid compounds composed by Pent residues
224
and derivatives identified after thermal treatment of the oligosaccharide mixtures
225
include Pent oligosaccharides (Pentn) with a lower (n=2) and higher (n=4-5) degree of
226
polymerization than that of the Pent oligosaccharide in the starting mixtures (Ara3). The
227
respective monosaccharide was also observed at m/z 173 ([Pent+Na]+). Also, Pentn
228
(n=2-6) derivatives resulting from the formation of a keto group (-2 Da), and
229
dehydration with loss of one, two and three water molecules, as well as resulting from
230
the oxidative scission of a furanose ring with loss of C2H4O2, C3H4O2, C2H6O3, C3H4O3,
231
C3H6O3 and C2H8O5 were identified.
232
As observed for Man3,11 the non-hybrid compounds composed by Hex residues
233
and derivatives identified after thermal treatment of the model mixtures include Hex
234
mono- and oligosaccharides (Hexn, n=1-3). The corresponding dehydrated derivatives
235
formed by loss of one and three up to six water molecules were also observed ([Hexn-
236
xH2O+Na]+; n=1-5; x=1, 3-6). Revisiting the ESI-MS spectra previously acquired from
237
Man3 subjected to T2 or T3 treatment, beyond the derivatives resulting from loss of one
238
and three water molecules previously reported,11 it was possible to observe the ions
239
attributed to dehydrated Hex oligosaccharides resulting from loss of four, five, and six
240
water molecules.
241
In
agreement
with
previous
observation
by
MALDI-MS,13
hybrid
242
oligosaccharides composed by both Hex and Pent units were also identified as [M+Na]+
243
ions in the ESI-MS spectra of the thermally treated mixtures, namely PentHex2 (m/z
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497), PentHex3 (m/z 659) and Pent2Hex2 (m/z 629). The observation of these hybrids
245
corroborates the hypothesis of the occurrence of non-enzymatic TGR involving GM and
246
Ara side chains of AG during coffee roasting. Also, several ions (Table 1) were
247
attributed to dehydrated derivatives formed by loss of one and three up to six water
248
molecules from hybrid oligosaccharides ([PentmHexn-xH2O+Na]+; m, n=1-4; x=1, 3-6).
249
As highlighted in Table 1, among the compounds identified in the ESI-MS spectra
250
of the thermally treated mixtures as [M+Na]+ ions, there are compounds with the same
251
nominal mass (calculated by adding the mass of the predominant isotope of each
252
element contributing to the molecule rounded to the nearest integer value). Some of
253
these compounds are isobaric compounds, i.e. have the same nominal mass but different
254
elemental composition, and thus, different accurate mass; other are isomers, i.e. have
255
the same elemental composition, and thus, the same exact mass. Isobaric and isomeric
256
compounds were differentiated based on specific fragmentation seen in the ESI-CID-
257
MSn spectra.
258 259
Diagnostic neutral losses and product ions observed under ESI-CID-MSn The fragmentation of [M+Na]+ ions of neutral and reducing
260
conditions.
261
oligosaccharides under ESI-CID-MSn conditions results of glycosidic linkage cleavages,
262
cross-ring cleavages, and loss of water. As inferred by
263
oxygen of standard oligosaccharides,12,
264
mainly occur between the anomeric carbon and the glycosidic linkage oxygen, whereas
265
cross-ring cleavages and loss of water occur mainly at the reducing end residue with
266
loss of the carbonyl oxygen. The cross-ring cleavages at the reducing end residue
267
depend on the oligosaccharide structure, giving rise to neutral losses of CH2O (-30 Da),
268
C2H4O2 (-60 Da), C3H6O3 (-90 Da), and C4H8O4 (-120 Da). Based on the knowledge of
18, 19
18
O-labeling of the carbonyl
including Ara3,12 glycosidic cleavages
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the typical fragmentation pathways of non-modified oligosaccharides, but also of
270
derivatives formed when Man3 and Ara3 were individually submitted to the thermal
271
treatments at 200 °C,11,
272
diagnostic of different ion series observed in the ESI-MS spectra of the thermally
273
treated mixtures, which are summarized in Table 2.
12
it was possible to identify neutral losses and product ions
274
The ESI-MSn fragmentation of [M+Na]+ ions of Hex oligosaccharides produces
275
product ions resulting from glycosidic cleavages with loss of a Hex residue (Hexres)
276
(162 Da) and a Hex (180 Da), and diagnostic product ions at m/z 203 ([Hex+Na]+) and
277
185
278
oligosaccharides show product ions resulting from glycosidic cleavages with loss a Pent
279
residue (Pentres) (132 Da) and a Pent (150 Da), and product ions at m/z 173 ([Pent+Na]+)
280
and 155 ([Pentres+Na]+) (Figure 2A). Product ions resulting from the neutral loss of 162
281
Da observed with relative abundance ≤10% in the ESI-MSn spectra of Pent
282
oligosaccharides, as Pent5 and derivatives (Table 3), is not due to the loss of Hexres, but
283
due to the combined loss of Pentres and CH2O (30 Da) from cross-ring cleavage. Neutral
284
losses and product ions observed in the ESI-MSn spectra of non-hybrid oligosaccharides
285
are also observed in the ESI-MSn spectra of Pent-Hex hybrid oligosaccharides.
286
However, the neutral loss of 294 Da, due to the combined loss of Pentres and Hexres, is
287
specific of Pent-Hex hybrid oligosaccharides, as well as the product ion at m/z 335
288
([PentHex+Na]+) (Figure 3A).
([Hexres+Na]+) (Figure
1A).
Similarly,
the
ESI-MSn
spectra
of
Pent
289
When sugar residues are modified, they yield new diagnostic neutral losses and
290
product ions, which can be specific or not of one type of modification. The ESI-MSn
291
fragmentation of an oligosaccharide bearing a (Hex-3H2O) produces a product ion
292
resulting from the neutral loss of 126 Da, corresponding to the loss of (Hex-3H2O), and
293
a product ion at m/z 149 ([Hex-3H2O+Na]+). The loss of 144 Da is observed either in
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294
the ESI-MSn spectra of (Hex-H2O) derivatives, resulting from the loss of (Hex-H2O)res,
295
and of (Hex-2H2O) derivatives, resulting from the loss of (Hex-2H2O). The product ion
296
at m/z 167 is also observed in the ESI-MSn spectra of both (Hex-H2O) and (Hex-2H2O)
297
derivatives, corresponding to [(Hex-H2O)res+Na]+ and [Hex-2H2O+Na]+, respectively.
298
In the case of (Hex-H2O) derivatives, the product ion at m/z 185 can also be due to
299
[Hex-H2O+Na]+, and not exclusively to [Hexres+Na]+. Similarly, the neutral loss of 114
300
Da is observed either in the ESI-MSn spectra of (Pent-H2O) derivatives, resulting from
301
the loss of (Pent-H2O)res, and in the ESI-MSn spectra of (Pent-2H2O) derivatives,
302
resulting from the loss of (Pent-2H2O). In the case of (Pent-H2O) derivatives, the
303
product ion at m/z 155 can also be due to [Pent-H2O+Na]+, and not exclusively to
304
[Pentres+Na]+. The neutral loss of 132 and 162 Da can also be due to the presence a
305
mono-dehydrated sugar, (Pent-H2O) and (Hex-H2O), respectively. Also, the neutral loss
306
of 96 Da can be due either to the loss of (Pent-2H2O)res, or (Pent-3H2O). In the case of
307
oligosaccharides bearing a (Pent-2H), their fragmentation produces a product ion
308
resulting from neutral loss of 148 Da, due to the loss of (Pent-2H), and the product ion
309
at m/z 171 ([Pent-2H+Na]+). The identification of neutral losses and product ions
310
diagnostic of different ion series (Table 2) was essential to disclose the presence of
311
isobaric and isomeric compounds.
312 313
Differentiation of isobaric and isomeric compounds by ESI-CID-MSn. The
314
presence of isobaric/isomeric compounds was inferred by the observation of specific
315
product ions in the ESI-MS2 spectra acquired on the LIT mass spectrometer (Table 3).
316
In most of the cases, the presence of isobaric/isomeric compounds was confirmed by
317
ESI-MSn, n=3-4. As example, it is shown the tandem MS-based strategy to discriminate
318
between the pairs Hex3/(Pent3Hex-4H2O) (m/z 527) and Pent3/(Hex3-5H2O) (m/z 437).
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In the MS analysis of carbohydrate-rich samples, the ion (or product ion) at m/z
320
527 is usually indicative of Hex3. The ESI-MS2 spectrum acquired from the A50M50
321
mixture submitted to the T1 treatment (Figure 1B) is similar to that acquired from the
322
untreated mixture (Figure 1A), suggesting the exclusive presence of the precursor ion
323
[Hex3+Na]+, as corroborated by high resolution and high mass accuracy MS data.
324
However, new product ions, absent in the ESI-MS2 spectrum acquired after T1 (Figure
325
1B), namely the product ions at m/z 401 (-126 Da) and 395 (-132 Da), identified as
326
resulting from the loss of (Hex-3H2O) and Pentres, and the product ions at m/z 287 and
327
263, attributed to [PentPentres+Na]+ and [HexPent-4H2O+Na]+, respectively, were
328
observed in the ESI-MS2 spectra acquired after the longer treatments, T2 (C) and T3
329
(D). These new ions suggest the presence of the precursor ion [Pent3Hex-4H2O+Na]+
330
beyond [Hex3+Na]+, as corroborated by high resolution and high mass accuracy MS
331
data. The relative abundance of the new product ions at m/z 401 and 395 increases with
332
increasing of the treatment time, whereas that at m/z 365 (-162 Da, -Hexres) decreases.
333
These changes suggest the increase of the proportion of [Pent3Hex-4H2O+Na]+ and the
334
decrease of [Hex3+Na]+ ions with increasing of the treatment time.
335
Figure 2 (C-E) shows the ESI-MS2 spectra of the ion at m/z 437 acquired from the
336
A75M25, A50M50, and A25M75 mixtures subjected to the T2 treatment. The product
337
ions at m/z 305 (-Pentres) and 173 (-2 Pentres), also observed in the ESI-MS2 spectrum of
338
the Ara3 (Figure 2A), corroborate the presence of [Pent3+Na]+ as precursor ion. The
339
coexistence of other precursor ion, [Hex3-5H2O+Na]+, is supported by the product ions
340
at m/z 311 (-126 Da), 293 (-144 Da), and 275 (-162 Da), formed respectively by loss of
341
(Hex-3H2O), (Hex-2H2O), and Hexres, as well as those at m/z 203 ([Hex+Na]+), 185
342
([Hexres+Na]+), and 149 ([Hex-3H2O+Na]+). These product ions were also observed in
343
the ESI-MS2 spectrum of (Hex3-5H2O) formed when Man3 was individually subjected
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344
to the treatment T2 (Figure 2B).11 The relative abundance of the product ions at m/z
345
311, 293, and 275 suggests the increase of the proportion of [Hex3-5H2O+Na]+ ions and
346
the decrease of [Pent3+Na]+ ions when the proportion of Man3 increases (Figure 2C-E).
347
As illustrated by the ESI-MS2 spectra of the ions at m/z 527 and 437 in Figures 1
348
and 2, the formation of each isobaric compound, but also of each isomeric compound,
349
was dependent on the thermal treatment and the starting mixture. This suggests that a
350
variety of structures may be formed during the green coffee roasting, depending on the
351
roasting conditions and distribution of the polysaccharides in the coffee beans.
352 353
ESI-MSn analysis after labeling with oxygen-18. Aiming to gain more
354
information about the structure of the compounds formed during thermal processing,
355
thermally treated mixtures were dissolved in H218O before MS analysis. Based on the
356
principles of nucleophilic addition reactions of aldehydes and ketones, they react with
357
water to yield geminal diols (hydrates). The hydration reaction is reversible, and a
358
geminal diol can eliminate water to regenerate an aldehyde or ketone.20 Accordingly,
359
the
360
observed, but also new carbonyl groups, either of ketones or aldehydes, formed by dry
361
thermal processing.
18
O-labeling of the carbonyl oxygen of non-modified reducing sugar residues was
362 363
ESI-MSn of
18
O-labeled Pent-Hex hybrid oligosaccharides. The ESI-MS2
364
spectrum of the ion at m/z 659 acquired from the A50M50 mixture subjected to the T1
365
treatment, and that acquired after
366
accordance to what was previously presented, this species could have the contribution
367
of two isobaric compounds, PentHex3 and (Pent4Hex-4H2O). As corroborated by the
368
ESI-MS2 spectrum before
18
18
O-labeling are shown in Figures 3A and B. In
O-labeling (Figure 3A), namely by the absence of the
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369
product ion at m/z 533, resulting from the neutral loss of (Hex-3H2O), only PentHex3
370
was present.
371
The ESI-MS2 spectrum of
18
O-labeled PentHex3 (Figure 3B) shows the product
372
ion at m/z 529, resulting from the loss of unlabeled Pentres, and the product ion at m/z
373
205, attributed to [M+Na]+ of a 18O-labeled Hex. These product ions indicate that one of
374
the three Hex units, and not the Pent, was 18O-labeled. This means that a Hex is located
375
at the reducing end of PentHex3. The observation of the product ion at m/z 529 also
376
indicates that the three Hex units are linked together, having several possible binding
377
sites for the Pent. The product ion at m/z 337 (-2 Hexres) suggests that the Pent is linked
378
to the Hex located at the reducing end (Figure 3C). However, the product ion at m/z
379
479, formed by loss of the
380
with the Pent not linked to the Hex located at the reducing end (Figure 3D).
18
O-labeled Hex, supports the presence of other structures
381
In any of the possible structures, it is of note that the Pent-Hex glycosidic linkage
382
involves the anomeric carbon of the Pent. The formation of the Pent-Hex glycosidic
383
linkage involving the anomeric carbon of the Pent, and not of the Hex, can be favored
384
by the higher reactivity of pentoses compared to hexoses. In the case of the thermally
385
treated mixtures, the most abundant pentoses and hexoses are Ara and Man,
386
respectively. As also previously observed by glycosidic linkage analysis,13 new types of
387
Man glycosidic linkages, absent in the untreated mixtures, namely (1→2), (1→6),
388
(1→2,3), (1→2,6), (1→3,6), and (1→2,3,6) linkages, were formed during thermal
389
processing of the Ara3-Man3 mixtures. Accordingly, these new Man linkages were
390
identified when a coffee GM-rich fraction was submitted to different dry thermal
391
treatments.15 In both cases, the formation of (1→6) Man linkages was favored over that
392
of other types of Man linkages. As reported for British gums produced by dry heating of
393
starch,21 the formation of (1→6) linkages can be favored over others in terms of
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Page 18 of 34
394
stereochemical and thermodynamic aspects. This can be due to the primary hydroxyl
395
group located at the C6-position, while the other hydroxyl groups of Man are secondary.
396
According to Tomasik et al.,21 anhydrosugars are possible intermediates in the
397
formation of new glycosidic linkages, but the mechanisms involved in the non-
398
enzymatic TGR induced by dry heating are far from being elucidated.
399 ESI-MSn of
400
18
O-labeled oligosaccharides containing a tri-dehydrated hexose.
401
Both non-hybrid and hybrid oligosaccharides containing a (Hex-3H2O) were labeled by
402
dissolving selected thermally treated mixtures in H218O. In Figure 4 are shown the ESI-
403
MS2 spectrum of the ion at m/z 413 ([Pent2Hex-3H2O+Na]+) acquired from the A50M50
404
mixture subjected to the T2 treatment (A), and that acquired after 18O-labeling (B). The
405
product ion observed after 18O-labeling (B) at m/z 287 (-128 Da), resulting from the loss
406
of
407
unlabeled Pentres, attributed to a
408
(Pent2Hex-3H2O) structures with the (Hex-3H2O) located at the reducing end of the
409
corresponding non-modified oligosaccharide.
18
O-labeled (Hex-3H2O), and that observed at m/z 151, formed by loss of two 18
O-labeled (Hex-3H2O), confirm the presence of
410
The occurrence of 18O-labeling at the (Hex-3H2O) suggests that this moiety has a
411
carbonyl group. As previously demonstrated by deuterium-labeling of oligosaccharides
412
bearing a (Hex-3H2O) formed from Man3,11 (Hex-3H2O) may be isomaltol, which has a
413
keto group. Due to the reversibility of the hydration reaction of ketones,20 the
414
labeling of the keto group of isomaltol moieties present in the thermally treated
415
mixtures dissolved in H218O could be expected. However, since the conditions used in
416
this work for 18O-labeling had previously been used only to label the carbonyl group of
417
aldehydes, the ketone 3-octanone was used as standard, confirming the 18O-labeling of a
418
keto group under the conditions used.
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O-
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419
Analysis of a coffee galactomannan-rich fraction. In order to check if the
420
structures formed during thermal processing of the model mixtures have the same
421
structural features as those formed during coffee roasting, or additional roasting
422
treatments beyond the roasting of the green coffee beans, a GM-rich fraction isolated
423
from SCG and submitted to two additional roasting treatments of 1 h at 200 °C was
424
analyzed. First, this GM-rich fraction was treated with an endo-(β1→4)-D-mannanase to
425
selectively cleave the (β1→4)-D-mannan backbone between adjacent (β1→4)-linked
426
Man residues. According to the mechanism of action of this enzyme, the hydrolysis of
427
the (β1→4)-D-mannan backbone is hindered by the presence of substituted Man
428
residues, non-Man residues interspersed in the mannan backbone, modified Man
429
residues, or other Man glycosidic linkages that are not (β1→4), yielding
430
oligosaccharides that contain these structural details. Thus, the hydrolyzed material was
431
further fractionated by LEX-SEC and then analyzed by ESI-MS and ESI-MSn.
432
In accord with previous reports,22, 23 the LEX-SEC fraction that eluted between
433
16-17 min was assigned to the neutral oligosaccharides with the higher molecular
434
weight that resulted from the enzymatic hydrolysis. Due to the enzyme action, these
435
oligosaccharides contain structural details of the coffee GM under study. In the ESI-MS
436
spectrum of this fraction, it was possible to identify the ions at m/z 497, 509, 527, 641
437
and 659, also identified in the mixtures, and other ions at m/z 671, 689, 803, 833, and
438
851. Based on their ESI-CID-MSn fragmentation patterns, and the elemental
439
composition obtained from high resolution and high mass accuracy measurements using
440
a hybrid quadrupole-Orbitrap mass spectrometer, the identification of these ions is as
441
follows: 497, [PentHex2+Na]+; 509, [Hex3-H2O+Na]+; 527, [Hex3+Na]+; 641,
442
[PentHex3-H2O+Na]+; 659, [PentHex3+Na]+; 671, [Hex4-H2O+Na]+; 689, [Hex4+Na]+;
443
803, [PentHex4-H2O+Na]+; 833, [Hex5-H2O+Na]+; 851, [Hex5+Na]+.
19 ACS Paragon Plus Environment
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444
According to the glycosidic linkage composition of the coffee GM-rich fraction
445
under study,15 the observation of Hex3-5 after enzymatic hydrolysis can be related to the
446
presence of substituted Man residues, namely by single Gal residues as occur in green
447
coffee GM; non-mannose residues (Glc and Gal) interspersed in the mannan backbone;
448
and new glycosidic linkages resistant to the enzyme action, such as (1→6) Man
449
linkages, formed during roasting. Since single Ara residues occur as side chains in green
450
coffee GMs,9 PentHex2 and PentHex3 can result from cleavage of the original mannan
451
backbone, without any modification promoted by roasting. Contrarily, the dehydrated
452
derivatives are the result of dehydration reactions occurring during roasting. The ESI-
453
MSn fragmentation of (PentHex3-H2O) and (PentHex4-H2O) suggests that loss of a
454
water molecule occurred at a Hex unit, as inferred by the loss of an intact Pentres (132
455
Da) and (Hex-H2O)res (144 Da). This is in accordance to what was observed with the
456
Ara3-Man3 mixtures, reinforcing the validity of the models used. The absence of hybrid
457
domains formed by non-enzymatic TGR between GM and AG during roasting in the
458
GM-rich fraction analyzed in this study can be related to its original location in the
459
green coffee beans, and the roasting conditions used.
460
In summary, the analysis of the model mixtures containing different molar
461
proportions of Ara3 and Man3, maintained at 200 °C for different periods, showed that
462
different structures can be formed during coffee roasting, depending on the distribution
463
of the polysaccharides in the beans and the roasting conditions. Furthermore, the
464
diversity of isobaric and isomeric compounds formed highlights the importance of a
465
detailed structural characterization when analyzing real roasted carbohydrate-rich
466
matrices such as coffee.
467 468
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469
ABBREVIATIONS USED
470
A25M75 – 25% Ara3:75% Man3
471
A50M50 –50% Ara3:50% Man3
472
A75M25 – 75% Ara3:25% Man3
473
AG – Arabinogalactan
474
Ara3 – (α1→5)-L-arabinotriose
475
GM – Galactomannan
476
Hex – Hexose
477
LEX-SEC – Ligand exchange/size-exclusion chromatography
478
Lyx – Lyxose
479
Man3 – (β1→4)-D-mannotriose
480
Pent – Pentose
481
Rib – Ribose
482
SCG – Spent coffee grounds
483
TGR – Transglycosylation reactions
484
Xyl – Xylose
485 486
ACKNOWLEDGMENT
487
Thanks are due to Fundação para a Ciência e a Tecnologia (FCT, Portugal),
488
European Union, QREN, FEDER, and COMPETE for funding the QOPNA research
489
unit (project PEst-C/QUI/UI0062/2013; FCOMP-01-0124-FEDER-037296), and the
490
Portuguese Mass Spectrometry Network (REDE/1504/REM/2005). Thanks are also due
491
to FCT for the grants of Ana Moreira (SFRH/BD/80553/2011) and Joana Simões
492
(SFRH/BPD/90447/2012).
493
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494
ASSOCIATED CONTENT
495
Supporting Information
496
The Supporting Information is available free of charge on the ACS Publications
497
website.
498
ESI-MS spectra acquired from the A75M25 mixture, before and after thermal 18
499
treatments (Figure S1), ESI-MS spectra of 3-octanone, before and after
500
procedure (Figure S2), ESI-MS2 spectra of [M+H]+ ions of unlabeled and 18O-labeled 3-
501
octanone (Figure S3), LEX-SEC chromatograms (Figure S4), ESI-MS spectrum of F16
502
obtained by LEX-SEC (Figure S5), total mass loss and mass loss from 150 °C during
503
each thermal treatment and water-solubility of the resulting compounds (Table S1),
504
accurate masses found by Q Exactive Orbitrap for the ions identified after heating of the
505
A50M50 mixture submitted to the T3 treatment (Table S2).
O-labeling
506 507
REFERENCES
508
(1)
Bradbury, A. G. W.; Halliday, D. J. Chemical structures of green coffee bean
509
polysaccharides. J. Agric. Food Chem. 1990, 38, 389-392.
510
(2)
511
arabinogalactans from two arabica coffee infusions as affected by the degree of roast. J.
512
Agric. Food Chem. 2002, 50, 1429-1434.
513
(3)
514
characterization of polysaccharides from green and roasted Coffea arabica beans.
515
Carbohydr. Polym. 2003, 52, 285-296.
516
(4)
517
carbohydrate composition of Coffea arabica beans. Carbohydr. Polym. 2003, 54, 183-
518
192.
Nunes, F. M.; Coimbra, M. A. Chemical characterization of galactomannans and
Oosterveld, A.; Harmsen, J. S.; Voragen, A. G. J.; Schols, H. A. Extraction and
Oosterveld, A.; Voragen, A. G. J.; Schols, H. A. Effect of roasting on the
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519
(5)
Redgwell, R. J.; Trovato, V.; Curti, D.; Fischer, M. Effect of roasting on
520
degradation and structural features of polysaccharides in Arabica coffee beans.
521
Carbohydr. Res. 2002, 337, 421-431.
522
(6)
523
melanoidins: structures, mechanisms of formation and potential health impacts. Food
524
Funct. 2012, 3, 903-915.
525
(7)
526
coffee melanoidin formation using modified “in bean” models. J. Agric. Food Chem.
527
2012, 60, 8710-8719.
528
(8)
529
of galactomannan derivatives in roasted coffee beverages. J. Agric. Food Chem. 2006,
530
54, 3428-3439
531
(9)
532
residues as structural features of acetylated galactomannans from green and roasted
533
coffee infusions. Carbohydr. Res. 2005, 340, 1689-1698.
534
(10)
535
Rhamnoarabinosyl and rhamnoarabinoarabinosyl side chains as structural features of
536
coffee arabinogalactans. Phytochemistry 2008, 69, 1573-1585.
537
(11)
538
M. Evaluation of the effect of roasting on the structure of coffee galactomannans using
539
model oligosaccharides. J. Agric. Food Chem. 2011, 59, 10078-10087.
540
(12)
541
Roasting-induced changes in arabinotriose, a model of coffee arabinogalactan side
542
chains. Food Chem. 2013, 138, 2291-2299.
Moreira, A. S. P.; Nunes, F. M.; Domingues, M. R.; Coimbra, M. A. Coffee
Nunes, F. M.; Cruz, A. C. S.; Coimbra, M. A. Insight into the mechanism of
Nunes, F. M.; Reis, A.; Domingues, M. R. M.; Coimbra, M. A. Characterization
Nunes, F. M.; Domingues, M. R.; Coimbra, M. A. Arabinosyl and glucosyl
Nunes, F. M.; Reis, A.; Silva, A. M. S.; Domingues, M. R. M.; Coimbra, M. A.
Moreira, A. S. P.; Coimbra, M. A.; Nunes, F. M.; Simões, J.; Domingues, M. R.
Moreira, A. S. P.; Coimbra, M. A.; Nunes, F. M.; Domingues, M. R. M.
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(13)
Moreira, A. S. P.; Simões, J.; Pereira, A. T.; Passos, C. P.; Nunes, F. M.;
544
Domingues, M. R. M.; Coimbra, M. A. Transglycosylation reactions between
545
galactomannans and arabinogalactans during dry thermal treatment. Carbohydr. Polym.
546
2014, 112, 48-55.
547
(14)
548
Cytochemistry and immunolocalisation of polysaccharides and proteoglycans in the
549
endosperm of green Arabica coffee beans. Protoplasma 2004, 223, 203-211.
550
(15)
551
Thermal stability of spent coffee ground polysaccharides: Galactomannans and
552
arabinogalactans. Carbohydr. Polym. 2014, 101, 256-264.
553
(16)
554
structure of spent coffee ground polysaccharides by roasting pre-treatments. Carbohydr.
555
Polym. 2013, 97, 81-89.
556
(17)
557
R. M. Fragmentation pattern of underivatised xylo-oligosaccharides and their alditol
558
derivatives by electrospray tandem mass spectrometry. Carbohydr. Polym. 2004, 55,
559
401-409.
560
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561
V.; Domingues, M. R. M. Differentiation of isomeric pentose disaccharides by
562
electrospray ionization tandem mass spectrometry and discriminant analysis. Rapid
563
Commun. Mass Spectrom. 2012, 26, 2897-2904.
564
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565
lithium-cationized disaccharides: tandem mass spectrometry and semiempirical
566
calculations. J. Am. Chem. Soc. 1991, 113, 5964-5970.
Sutherland, P. W.; Hallett, I. C.; MacRae, E.; Fischer, M.; Redgwell, R. J.
Simões, J.; Maricato, É.; Nunes, F. M.; Domingues, M. R.; Coimbra, M. A.
Simões, J.; Nunes, F. M.; Domingues, M. R.; Coimbra, M. A. Extractability and
Reis, A.; Coimbra, M. A.; Domingues, P.; Ferrer-Correia, A. J.; Domingues, M.
da Costa, E. V.; Moreira, A. S. P.; Nunes, F. M.; Coimbra, M. A.; Evtuguin, D.
Hofmeister, G. E.; Zhou, Z.; Leary, J. A. Linkage position determination in
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McMurry, J., Organic chemistry - International student edition. 7th ed.;
567
(20)
568
Thomson Brooks/Cole: Belmont, CA, 2008.
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(21)
570
carbohydrates. Part II.* The decomposition of starch. In Adv. Carbohydr. Chem.
571
Biochem., Tipson, R. S.; Derek, H., Eds. Academic Press: 1989; Vol. Volume 47, pp
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279-343.
573
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574
F.; Coimbra, M. A.; Barros, A. I. R. N. A.; Domingues, M. R. M. Oxidation of
575
mannosyl oligosaccharides by hydroxyl radicals as assessed by electrospray mass
576
spectrometry. Carbohydr. Res. 2011, 346, 2603-2611.
577
(23)
578
M.; Domingues, M. R. M. Neutral and acidic products derived from hydroxyl radical-
579
induced oxidation of arabinotriose assessed by electrospray ionisation mass
580
spectrometry. J. Mass Spectrom. 2014, 49, 280-290.
Tomasik, P.; Wiejak, S.; Pałasiński, M., The thermal decomposition of
Tudella, J.; Nunes, F. M.; Paradela, R.; Evtuguin, D. V.; Domingues, P.; Amado,
Moreira, A. S. P.; da Costa, E. V.; Evtuguin, D. V.; Coimbra, M. A.; Nunes, F.
25 ACS Paragon Plus Environment
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FIGURE CAPTIONS
Figure 1. ESI-MS2 spectra of the ion at m/z 527 acquired from the A. untreated A50M50 mixture, and samples of the A50M50 mixture subjected to the B. T1, C. T2, and D. T3 treatments.
Figure 2. ESI-MS2 spectra of the ion at m/z 437 acquired from the A. untreated A75M25 mixture, B. Man3 sample subjected to the T2 treatment, and mixtures subjected to the T2 treatment: C. A75M25, D. A50M50, and E. A25M75.
Figure 3. A. ESI-MS2 spectrum of the ion at m/z 659 ([PentHex3+Na]+) acquired from the A50M50 mixture subjected to T1 treatment, and B. the corresponding ESI-MS2 spectrum acquired after labeling with oxygen-18. The different proposed structures are represented in C and D.
Figure 4. A. ESI-MS2 spectrum of the ion at m/z 413 ([Pent2Hex-3H2O+Na]+) acquired from the A50M50 mixture subjected to the T2 treatment, and B. the corresponding ESIMS2 spectrum acquired after labeling with oxygen-18.
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Journal of Agricultural and Food Chemistry
Table 1. Summary of the [M+Na]+ Ions Identified in the ESI-LIT-MS Spectra Acquired from the Thermally Treated Mixtures. Number (n) of pentose (Pent) units 1 2 3 4 5 6 Non-hybrid compounds composed by pentose residues and derivatives [Pentn+Na]+ 173a 305 †437b †569 †701 + c [Pentn-2H+Na] (-2) 435 [Pentn-H2O+Na]+ (-18) 287 †419 †551 †683 [Pentn-2H2O+Na]+ (-36) 401 [Pentn-3H2O+Na]+ (-54) 383 [Pentn-C2H4O2+Na]+ (-60) 377 †509 †641 + [Pentn-C3H4O2+Na] (-72) †365 †497 †629 (-78) 227 [Pentn-C2H6O3+Na]+ [Pentn-C3H4O3+Na]+ (-88) 217 [Pentn-C3H6O3+Na]+ (-90) †347 †479 †611 ‡743 (-112) 193 325 [Pentn-C2H8O5+Na]+ Proposed assignments
Non-hybrid compounds composed by hexose residues and derivatives 203 †365 †527 [Hexn+Na]+ [Hexn-H2O+Na]+ (-18) 185 †347 †509 (-54) 149 311 473 [Hexn-3H2O+Na]+ [Hexn-4H2O+Na]+ (-72) 293 455 [Hexn-5H2O+Na]+ (-90) †437 [Hexn-6H2O+Na]+ (-108) 257 †419 †581 ‡743 Pentose-hexose hybrid oligosaccharides and derivatives [PentHexn+Na]+ †497 †659 [PentHexn-H2O+Na]+ (-18) 317 †479 †641 [PentHexn-3H2O+Na]+ (-54) 281 443 605 (-72) 425 587 [PentHexn-4H2O+Na]+ [PentHexn-5H2O+Na]+ (-90) †569 (-108) 389 †551 †713 [PentHexn-6H2O+Na]+ [Pent2Hexn+Na]+ †629 [Pent2Hexn-H2O+Na]+ (-18) 449 †611 [Pent2Hexn-3H2O+Na]+ (-54) 413 575 [Pent2Hexn-4H2O+Na]+ (-72) 395 [Pent2Hexn-5H2O+Na]+ (-90) †701 [Pent2Hexn-6H2O+Na]+ (-108) 521 †683 [Pent3Hexn-H2O+Na]+ (-18) †581 ‡743 [Pent3Hexn-3H2O+Na]+ (-54) 545 [Pent3Hexn-4H2O+Na]+ (-72) †527 [Pent4Hexn-H2O+Na]+ (-18) †713 [Pent4Hexn-3H2O+Na]+ (-54) 677 [Pent4Hexn-4H2O+Na]+ (-72) †659 Only the ions observed with a relative abundance equal or higher than 15% in at least two of the ESI-LIT-MS spectra were considered. am/z values. bIons attributed to different compounds having the same nominal mass are marked with a symbol: † for two and ‡ for three compounds. cValues in brackets are the m/z value differences compared to the [M+Na]+ ions of the corresponding non-modified oligosaccharide.
27 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Table 2. Diagnostic Neutral Losses and Product Ions Observed Under ESI-CID-Msn Conditions. Neutral losses and product ions Neutral loss (Da) 96 114 126 132 144 148 150 162 180 294 Product ion (m/z) 149 155 167 171 173 185 203 335
Assignment(s)
(Pent-2H2O)res; (Pent-3H2O) (Pent-H2O)res; (Pent-2H2O) (Hex-3H2O); (Hex-2H2O)res Pentres; (Pent-H2O) (Hex-H2O)res; (Hex-2H2O) (Pent-2H) Pent Hexres; (Hex-H2O); Combined loss of Pentres and CH2O (30 Da) from cross-ring cleavage Hex Combined loss of Pentres and Hexres
[Hex-3H2O+Na]+ [Pentres+Na]+; [Pent-H2O+Na]+ [(Hex-H2O)res+Na]+; [Hex-2H2O+Na]+ [Pent-2H+Na]+ [Pent+Na]+ [Hexres+Na]+; [Hex-H2O+Na]+ [Hex+Na]+ [PentHex+Na]+
28 ACS Paragon Plus Environment
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Journal of Agricultural and Food Chemistry
Table 3. Summary of the Pairs of Isobaric/Isomeric Compounds and One Set of Three Compounds having the Same Nominal Mass Identified as [M+Na]+ Ions in the ESI-MS Spectra of the Thermally Treated Mixtures. Ion (m/z)
Proposed assignments
347
[Pent3-C3H6O3+Na]+ [Hex2-H2O+Na]+
Formula C12H20NaO10
+
Characteristic neutral losses 162 Da 144 Da
132 Da Pentres
Hexres/(Hex-H2O)
365
[Pent3-C3H4O2+Na] [Hex2+Na]+
C12H22NaO11
419
[Pent3-H2O+Na]+ [Hex3-6H2O+Na]+
C15H24NaO12 C18H20NaO10
Pentres/(Pent-H2O)
C15H26NaO13
Pentres
[Pent3+Na] 437
+
[Hex3-5H2O+Na]+
C18H22NaO11
479
[Pent4-C3H6O3+Na]+ [PentHex2-H2O+Na]+
C17H28NaO14
497
[Pent4-C3H4O2+Na]+ [PentHex2+Na]+
C17H30NaO15
509
[Pent4-C2H4O2+Na]+ [Hex3-H2O+Na]+
C18H30NaO15
+
Pentres
(Pentres+CH2O) Hexres
(Hex-H2O)res
a
Hexres
(Hex-3H2O)
Hexres
(Hex-2H2O)
Pentres Pentres
(Pentres+CH2O)a Hexres/(Hex-H2O)
(Hex-H2O)res
Pentres Pentres
(Pentres+CH2O)a Hexres
Pentres
(Pentres+CH2O)a Hexres/(Hex-H2O)
[Hex3+Na] [Pent3Hex-4H2O+Na]+
C18H32NaO16 C21H28NaO14
Pentres
551
[Pent4-H2O+Na]+ [PentHex3-6H2O+Na]+
C20H32NaO16 C23H28NaO14
Pentres/(Pent-H2O) Pentres
+
C20H34NaO17
Pentres
C23H30NaO15
Pentres
Hexres
(Hex-2H2O)
Pentres
Hexres (Hex-H2O)
(Hex-H2O)res
Pentres Pentres
(Pentres+CH2O)a Hexres/(Hex-H2O)
[Pent4+Na]
[PentHex3-5H2O+Na]
+
581
[Hex4-6H2O+Na]+ [Pent3Hex-H2O+Na]+
C24H30NaO15 C21H34NaO17
611
[Pent5-C3H6O3+Na]+ [Pent2Hex2-H2O+Na]+
C22H36NaO18
+
Hexres (Hex-3H2O) Hexres
(Hex-3H2O)
C22H38NaO19
Pentres Pentres
(Pentres+CH2O) Hexres
641
[Pent5-C2H4O2+Na]+ [PentHex3-H2O+Na]+
C23H38NaO19
Pentres Pentres
(Pentres+CH2O)a Hexres/(Hex-H2O) Hexres
[PentHex3+Na] [Pent4Hex-4H2O+Na]+
C23H40NaO20 C26H36NaO18
Pentres Pentres
683
[Pent5-H2O+Na]+ [Pent2Hex3-6H2O+Na]+
C25H40NaO20 C28H36NaO18
Pentres/(Pent-H2O) Pentres
(Hex-H2O)res
a
[Pent5-C3H4O2+Na] [Pent2Hex2+Na]+
659
(Hex-H2O)res (Hex-3H2O)
Hexres
(Hex-3H2O)
+
C25H42NaO21
Pentres
(Pentres+CH2O)
[Pent2Hex3-5H2O+Na]+
C28H38NaO19
Pentres
Hexres
(Hex-2H2O)
713
[PentHex4-6H2O+Na]+ [Pent4Hex-H2O+Na]+
C29H38NaO19 C26H42NaO21
Pentres Pentres
Hexres (Hex-H2O)
(Hex-H2O)res
[Hex5-6H2O+Na]+ [Pent6-C3H6O3+Na]+ [Pent3Hex2-H2O+Na]+
C30H40NaO20
743
[Pent5+Na] 701
a
C27H44NaO22
(Hex-3H2O)/ (Hex-2H2O)res (Hex-3H2O)
629
+
(Hex-3H2O)/ (Hex-2H2O)res
(Hex-H2O)res
527
569
126 Da
a
(Hex-3H2O)
Hexres Pentres Pentres
Hexres/(Hex-H2O)
The corresponding product ion was observed with a relative abundance ≤10%.
29 ACS Paragon Plus Environment
(Hex-3H2O)/ (Hex-2H2O)res
(Hex-3H2O) (Hex-H2O)res
Journal of Agricultural and Food Chemistry
Man3
203
365
Na+
437 185
100
A50M50-T0
60 40 [Hexres+Na]+ 20 0
100
-60 Da
[Hex+Na]+ 185.0 203.0
250
-(Hexres+60 Da) 305.1
300
[M+Na]+ 527.2
-120 Da -90 Da 467.2 407.2 437.1
350 m/z
400
450
500
-Hexres -Hex 347.2 365.2
A50M50 - T1
[Hex3+Na]+
80 60
-H2O 509.2 -60 Da [M+Na]+ -120 Da -90 Da 467.2 527.2 479.2 407.2 437.2
20 0
[Hex+Na]+ -(Hexres+60 Da) 185.0 203.0 305.1
200
C 100
-48 Da
40 [Hexres+Na]+
250
300
350 m/z
-Hex 347.1
[HexPent-4H2O+Na]+
40 20 0
D 100
203.1
[PentPentres+Na]+ 263.0 287.1 305.1
200
250
[Hex+Na]+
300
-Pentres 395.1
400
-Pentres 395.1
80 60
[HexPent-4H2O+Na]+ [PentPentres+Na]+
40 20 0
[Hex+Na]+ 203.1
200
287.1 263.1 305.2 250
300
500
-H2O 509.2
-60 Da -(Hex-3H2O) 467.1 [M+Na]+ 401.1 461.1 -32 Da 527.3 495.1
350 m/z
A50M50 - T3
450
[Hex3+Na]+ and [Pent3Hex-4H2O+Na]+
-Hexres 365.1
A50M50 - T2
80 60
400
-Hexres 365.1 -Hex 347.1
350 m/z
450
401.1 437.2 400
500
[Hex3+Na]+ and [Pent3Hex-4H2O+Na]+
-90 Da
Relative Abundance
B
Relative Abundance
[Man3+Na]+
80
200
Relative Abundance
467
-Hexres -Hex 365.2 347.2
-(Hex-3H2O)
Relative Abundance
A
347
305
[M+Na]+ -H2O 527.2 -60 Da 467.2 509.2
450
Figure 1
30 ACS Paragon Plus Environment
500
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Journal of Agricultural and Food Chemistry
C
347 215
Ara3
173
Na+
305
407
377 245 155
100
A75M25 - T0
-90 Da 347.1
80
-60 Da 377.1
20 0
-Pentres -(Pentres+60 Da) 305.1 -Pent [Pent+Na]+ 287.0 215.0 245.1 155.0 173.0 343.1 -(Pentres+90 Da)
[Pentres+Na]+
150
200
250
300 m/z
350
-30 Da
407.1 -H2O [M+Na]+ 419.1 437.1 400
450
60 40 20 0
-(Pentres+90 Da) -(Pentres+60 Da) -90 Da -Hexres -102 Da 347.1 -60 Da [Pent+Na]+ 377.1 275.0 335.1 155.1 173.0 215.0 245.0
150
100
[Hex-3H2O+Na]+ [Hex+Na]+
40 20 0
-H2O
+Na]+
[Hexres 149.1 185.1 203.1
150
200
-(Hex-2H2O) -90 Da 293.1 -Hex 347.1 257.1 317.2
250
300 m/z
350
419.3 -32 Da + 405.3 [M+Na] 437.4 400
250
300 m/z
350
400
450
[Pent3+Na]+
and [Hex3-5H2O+Na]+
-Pentres 305.1
A50M50 - T2
60 40 20 0
-(Pentres+60 Da) [Hex-3H2O+Na]+ [M+Na]+ -90 Da -60 Da -Hexres -(Pentres+90 Da) 347.1 377.1 -32 Da 437.2 275.1 -(Hex-3H2O) [Pent+Na]+ 405.1 311.1 215.1 245.1 149.0 173.1
100
200
A25M75 - T2
0
300 m/z
350
400
450
[Pent3+Na]+ and [Hex3-5H2O+Na]+
-Hexres 275.1
-(Hex-3H2O) -60 Da -H2O 311.1 -90 Da 377.1 419.1 347.1 -(Hex-2H2O) -32 Da [M+Na]+ 405.1 437.3 293.1 -102 Da -Hex 257.1 335.1
305.1
60 [Hex-3H O+Na]+ 2 [Hex+Na]+ 40 20
250
-Pentres
80
[Hexres+Na]+ 149.1 185.0 203.1
150
450
200
[M+Na]+ 437.2
80
E Relative Abundance
-(Hex-3H2O) 311.2
80 60
[Hex3-5H2O+Na]+
-120 Da
Relative Abundance
100
-Hexres 275.1
Man3 - T2
[Pent3+Na]+ and [Hex3-5H2O+Na]+
[Pentres+Na]+
150
B
-Pentres 305.0
A75M25 - T2
80
D
60 40
100
[Ara3+Na]+
Relative Abundance
A Relative Abundance
287
Relative Abundance
Page 31 of 34
Figure 2
31
ACS Paragon Plus Environment
200
250
300 m/z
350
400
450
20 0
[HexHexres+Na]+ [PentHex+Na]+ [Hex2+Na]+ 335.1
347.1
203.1
275.1 317.1
200
407.1 467.1
300
400
-H2O
641.2 [M+Na]+ 659.2
-60Da 539.2 599.2 -90Da 569.2
365.1
500
m/z 185
D
600
m/z
20 0
[HexHexres+Na]+ [Pent(18Hex)+Na]+
205.1 200
317.1 275.2
[Hex(18Hex)+Na]+
347.1 337.1
300
―O―
m/z 205
m/z 529
m/z 479
479.2 -62Da
367.1 -122Da
437.2 400
539.2 500
-H218O
40
[18Hex+Na]+
Relative Abundance
60
m/z 185
-(18Hex)
80
Na+
Pent
Hex―O―Hex―O―18Hex
-Hexres 529.2 499.2
100
m/z 347
m/z 499 -Pentres
B
529
Hex―O―Hex―O―18Hex
Pent
[M+Na]+
661.5
599.2 641.2 600
m/z
Figure 3
32 ACS Paragon Plus Environment
m/z 529
Na+
m/z 155
40
Na+
Pent
m/z 499 m/z 337
―O―
60
C
―O―
80
-Pentres 527.1 -Hexres 497.1 -Hex 479.1 -120Da
m/z 155
100
[Hex+Na]+
Relative Abundance
A
Page 32 of 34
m/z 155
Journal of Agricultural and Food Chemistry
m/z 367
m/z 205
Hex―O―Hex―O―18Hex m/z 317
m/z 479
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Journal of Agricultural and Food Chemistry
A
m/z 281
m/z 149
Pent―O―Pent―O―(Hex-3H2O) m/z 287
-Pentres 281.0
100 80 60 40 20 0
[Hex-3H2O+Na]+
Relative Abundance
m/z 155
Na+
-(Hex-3H2O) 287.0 [Pentres+Na]+
149.0
155.0
150
+ -90 Da -60 Da -H2O [M+Na] 413.1 323.0 353.0 395.1
-(Pentres+30 Da) 251.0
200
250
300
350
400
m/z
B
m/z 283
m/z 151 Na+
Pent―O―Pent―O―18(Hex-3H2O)
80 60 40 20 0
[18(Hex-3H2O)+Na]+
Relative Abundance
m/z 155
100
151.1 150
m/z 287
-Pentres 283.1
-18(Hex-3H2O) 287.1 [Pentres+Na]+
155.0 200
-90 Da -60 Da 325.1 355.1
-(Pentres+30 Da) 253.1
250
300
350
m/z
Figure 4
33 ACS Paragon Plus Environment
[M+Na]+ 415.2
400
Journal of Agricultural and Food Chemistry
Table of Contents Graphic
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