Miguel Ribeiro
1,2,3
, Fernando M. Nunes
1,*
, Sofia Guedes
4
, Pedro Domingues
4 , Amélia M.
Silva
5
, Jose Maria Carrillo
6
, Marta Rodriguez-Quijano
6 , Gérard Branlard 7
and Gilberto
Igrejas
2,3
1 CQ-VR, Chemistry Research Centre, Chemistry Department, University of Trás-os-Montes and Alto Douro, 5000-801 Vila Real, Portugal;
2 Department of Genetics and Biotechnology, University of Trás-os-Montes and Alto Douro,
5000-801 Vila Real, Portugal;
3 Functional Genomics and Proteomics Unity, University of Trás-os-Montes and Alto Douro,
5000-801 Vila Real, Portugal;
4 Chemistry Department, University of Aveiro, 3810-193 Aveiro, Portugal;
5 Centre for the Research and Technology of Agro-Environmental and Biological Sciences,
University of Trás-os-Montes and Alto Douro (CITAB-UTAD), 5000-801 Vila-Real, Portugal;
6 Unidad de Genética y Mejora de plantas Departamento de Biotecnología, E.T.S. Ingenieros
Agrónomos Universidad Politécnica de Madrid, 28040 Madrid, España;
7 Institut National de la Recherche Agronomique GDEC/UBP, UMR 1095, 63100 Clermont-
Ferrand, France.
* To whom correspondence should be addressed e-mail: fnunes@utad.pt
Phone: +351259350242

Mw
3.0
a pI
10.0
3.0
b
Mw pI
10.0
Supplementary Figure 1. Two-dimensional electrophoresis pattern (IEF × SDS-
PAGE) of the wheat flour proteins (FRB-50X) . ( a ) unmodified proteins and ( b ) derivatized with n -butylamine. The dashed rectangle indicates protein aggregates.

PQ
19
FEEIR
PSQ
3
Q
4
NPQ
7
AQ
9
GSVQ
13
PQ
15
Q
16
LPQ
19
FEEIR
918.5
100
90
80
70
PSQ
3
Q
4
NPQ
7
AQ
9
GSVQ
13
Q
13
PQ
15
Q
16
LPQ
19
FEEIR
60
50
+1
1350.4
40
+1
1816.6
30
20
10
+1
417.4
+1
441.5
+1
546.4
+1
555.2
+1
693.5
+1
821.3
+1
851.4
979.4
1031.6
y9
1159.5
1222.6
+1
1287.6
0
400 600 800 1000 1200 m/z
+1
1384.6
1400
+1
1512.6
1600
1755.8
1611.7
+1
1698.7
1800
1883.8
1954.8
2000
Supplementary Figure 2. MS/MS analysis of the non-modified α-/β-gliadin derived tryptic digest peptide.
Tandem mass (MS/MS) spectra of non-modified α-/β-gliadin derived tryptic digest peptide [precursor ion at m/z (+2) = 1396.2] from spot 7/8, excised from 2-DE gel of GRB-50X.

100
90
80
70
60
50
40
30
PQ
19
FEEIR
PSQ
3
Q
4
NPQ
7
AQ
9
GSVQ
*
13
PQ
15
Q
16
LPQ
19
FEEIR
918.5
PQ
15
Q
16
LPQ
19
FEEIR
1384.7
PSQ
3
Q
4
NPQ
7
AQ
9
G
1031.6
SVQ
*
13
PQ
15
Q
16
LPQ
19
FEEIR
1504.9
1755.8
1873.7
20
10
417.4
b4 b5
555.5
441.2
+1 y5
693.4
+1
780.3
0
400 600 800
1036.8
1159.6
1287.6
1407.8
1632.9
1760.7
1000 1200 m/z
1400 1600 1800
Supplementary Figure 3. MS/MS analysis of the α-/β-gliadin derived tryptic digest
1940.8
2000 peptide modified at position 13. Tandem mass (MS/MS) spectra of α-/β-gliadin derived tryptic digest peptide [precursor ion at m/z (+2) = 1367.7] from spot 7/8, excised from 2-DE gel of GRB-50X. * indicates the modified glutamine residue.

100
90
80
70
PQ
19
FEEIR
PSQ
3
Q
4
NPQ
7
AQ
9
*
GSVQ
13
PQ
15
Q
16
LPQ
19
FEEIR
918.5
PQ
15
Q
16
LPQ
19
FEEIR
1384.7
GSVQ
13
PQ
15
Q
16
LPQ
19
FEEIR
60
PSQ
3
Q
4
NPQ
7
AQ
9
*
50
1755.8
1873.7
40
PSQ
3
Q
4
NPQ
7
1504.9
1031.6
30
1512.7
1093.6
1036.8
+1
1159.6
1279.3
1407.8
+1
1760.7
20
10
417.4
441.2
555.5
+1
693.4
780.3
0
400 600 800
1611.7
1940.8
1000 1200 m/z
1400 1600 1800
Supplementary Figure 4. MS/MS analysis of the α-/β-gliadin derived tryptic digest
2000 peptide modified at position 9. Tandem mass (MS/MS) spectra of α-/β-gliadin derived tryptic digest peptide [precursor ion at m/z (+2) = 1367.7] from spot 7/8, excised from
2-DE gel of GRB-50X. * indicates the modified glutamine residue.

100
90
80
PQ
19
FEEIR
PSQ
3
Q
4
NPQ
*
7
AQ
9
GSVQ
13
PQ
15
Q
16
LPQ
19
FEEIR
918.5
PQ
15
Q
16
LPQ
19
FEEIR
1384.7
70
AQ
9
GSVQ
13
PQ
15
Q
16
LPQ
19
FEEIR
60
50
40
30
PSQ
3
Q
4
NPQ
*
7
A
PSQ
3
Q
4
N
20
10
+1
417.4
b4
441.2
+1
555.5
0
400 600
+1
693.4
800
+1
908.6
1000
+1
1031.6
1504.9
1755.8
1036.8
+1
1093.6
1159.6
+1
1279.3
+1
1407.8
1512.7
+1
1611.7
+1
1760.7
+1
1873.7
1954.9
1200 m/z
1400 1600 1800 2000
Supplementary Figure 5. MS/MS analysis of the α-/β-gliadin derived tryptic digest peptide modified at position 7.
Tandem mass (MS/MS) spectra of α-/β-gliadin derived tryptic digest peptide [precursor ion at m/z (+2) = 1367.7] from spot 7/8, excised from
2-DE gel of GRB-50X. * indicates the modified glutamine residue.

100
90
PQ
19
FEEIR
+1
918.4
PSQ
3
Q
*
NPQ
7
AQ
9
GSVQ
13
PQ
15
Q
16
LPQ
19
FEEIR
PQ
15
Q
16
LPQ
19
FEEIR
1384.6
80
70
60
AQ
9
GSVQ
13
PQ
15
Q
16
LPQ
19
FEEIR
50
40
30
PSQ
3
Q
*
4
NPQ
7
AQ
9
20
10 +1
417.3
0
400
+1
546.4
+1
693.4
+1
821.0
600 800
+1
1755.7
+1
1031.5
+1
1512.6
+1
1036.4
+1
1159.5
+1
1279.6
+1
1407.6
+1
1611.9
+1
1698.7
+1
1760.7
+1
1873.8
+1
1955.2
1000 1200 m/z
1400 1600 1800 2000
Supplementary Figure 6. MS/MS analysis of the α-/β-gliadin derived tryptic digest peptide modified at position 4. Tandem mass (MS/MS) spectra of α-/β-gliadin derived tryptic digest peptide [precursor ion at m/z (+2) = 1367.7] from spot 7/8, excised from
2-DE gel of GRB-50X. * indicates the modified glutamine residue.

100
90
80
70
740.4
GGSFYPGETTPPQ
13
Q
14
LQ
16
Q
17
R
1479.6
PGETTPPQ
13
Q
14
LQ
16
Q
17
R
GGSFYPGE
60
50
40
+1
896.5
+1
1196.7
30
20
10
0
200
+1
303.2
y3 b5
512.3
431.4
+1 y4
544.6
+1
672.6
+1
795.3
400 600 800
897.7
1000 m/z
+1
1095.2
1200
+1
1319.5
1382.7
1400
+1
1560.5
+1
1816.5
1688.3
1600 1800
Supplementary Figure 7. MS/MS analysis of the of non-modified HMW-GS derived tryptic digest peptide.
Tandem mass (MS/MS) spectra of non-modified
HMW-GS derived tryptic digest peptide [precursor ion at m/z (+2) = 996.0] from spot
1, excised from 2-DE gel of GRB-50X.

a
HNCO
C
4
H
9
m/z 257
C O
+
NHCO
2
Et
-

OEt
CO
2
Et
HNCO
NHCO
2
Et
C
4
H
9
m/z 302 (M +

)
+

-

CO
2
Et
O NH
C
4
H
9
m/z 229
+
NHCO
2
Et
O
H
+
N
NHCO
2
Et
C
4
H
9
-NH
2
CO
2
Et
O
+
N
C
4
H
9
m/z 140 b c
100
80
60
40
20
0
50
84
98
100
140
150
[M+H] +
303
211
257
229
285
[M+C
2
H
5
] +
331
[M+C
3
H
5
] +
343
200 m/z
250 300 350 400
100
80
60
40
20
0
140
183
211
229
257
120 140 160 180 200 220 240 260 280 300 m/z
Supplementary Figure 8.
Confirmation of the identity of

-glutamyl-n-butylamine by
CI-MS and CI-MS 2 . ( a) Fragmentation of

-glutamyln -butylamine under EI ( Fig 4a ).
(b) CI (CH4) mass spectra of

-glutamyln -butylamine. (c) MS
2
of ion m/z 303. The interpretation of the EI mass spectra of

-glutamyln -butylamine and the assignment of various ions were based on the previous knowledge on fragmentation behaviour of the
N-ethoxycarbonyl amino acid ethyl esters (ECEE’s)
1
. In EI spectra of

-glutamyln butylamine ( Fig. 4a ) the molecular ion peak at m/z 302 is absent, a common feature of

the EI mass spectra of ECEE’s. For purpose of molecular weight confirmation, a CI
(CH
4
) mass spectrum was acquired and is shown in (b) , were the ion at m/z 303 was assigned [M+H]
+
, being observed also the ions at [M+29]
+
and [M+41]
+
, representing the adducts with C
2
H
5
+ and C
3
H
5
+ , respectively, as is typical when methane is used as the chemical ionization reagent gas. In order to further confirm the ion m/z 303 as the
[M+H]
+
ion, a MS
2
mass spectra was obtained and is shown in (c) . The EI spectrum in
Fig. 4a shows the base peak at m/z 140 and the peak at the highest value of m/z 257. On
(a) describes the fragmentation route observed for

-glutamyln -butylamine. The peak at m/z 257 is formed by the loss of CH
3
CH
2
O

resulting from the α-cleavage of the ECEE carboxylic ester group. The prevailing route is the loss of the a

CO
2
Et that initiates a sequence reaction leading to the cyclization induced by functional group interaction between -CONH-, and the amino terminus in the EE+ species ( m/z 229 = M –

CO
2
Et) as described for ECEE glutamine fragmentation.
Supplementary references
1. Huang, Z.-H. et al. Characterization of N-ethoxycarbonyl ethyl esters of amino acids by mass spectrometry. J. Chromatogr. A 635 , 271-281 (1993).