Vol. 26, No. 21 (2014)
Characteristics of Early Maillard Reaction Products by Electrospray Ionization Mass Spectrometry 7453
tuning conditions constant. Samples typically comprised 1 µM
amino acid in water/methanol (50:50) solutions containing 1 %
formic acid. The physical parameters of the interface i.e., the
distance between the needle and the hole in the spray shield
(1.2-1.5 cm), the voltage (2.8-3 kV) applied to the stainless-
steel unit and the temperature of the heated capillary (180 °C)-
were optimized at a flow rate of 25 mL/h, using helium as the
collision gas. The injection and activation times for MS2 experi-
ments in the ion trap were 200 ms and 30 ms, respectively.
Fig. 1. Production of Amadori rearrangement products (ARPs)
The goal of the present study was to investigate the gas-phase
CID fragmentation behaviour of sugars and amino acid
moieties ofAmadori rearrangement products in anamino acid-
disaccharide model and to identifydiagnostic fragment ions
for study and understanding of fundamentals of fragmentation
behaviours of Amadori rearrangement products in MS2 and
MS3.
RESULTS AND DISCUSSION
Amadori rearrangement products in amino acid-
disaccharide model: Amino acids and their N-terminal
acetylated derivatives were allowed to react with reducing
sugars at different temperatures (50, 70 and 90 °C) and reaction
times (0.5, 1, 2 and 4 h). Nano-ESI-MS was used to monitor
and characterize theAmadori rearrangement products . In MS
spectra of glycated amino acids with sugars, the main peaks
are protonated amino acids, [M + H]+, with additional peaks
of protonatedAmadori rearrangement products of amino acid
derivatives. The peaks of [M + 324 + H]+ are protonatedAmadori
rearrangement products of amino acids with disaccharides
(lactose and maltose). For example, in the MS spectra, Nα-
acetyl-lysine reacted with lactose and maltose, m/z 189 is the
peak of Nα-acetyl-lysine and m/z 513 is the peak of Amadori
rearrangement products of Nα-acetyl-lysine reacted with sugars
(data not show). ESI-MS results showed that all selected amino
acids could react with sugars noticeably at 90 °C and the peaks
ofAmadori rearrangement products were dominant in the ESI-
MS spectra. However, only Amadori rearrangement products
of lysine and Nα-acetyl-lysine with sugars could be detected at
50 °C for 1 h. It displayed that the ε-NH2 group was more reactive
with sugars than other side-chain amino groups (Table-1).
MS2 of Amadori rearrangement products of sugar
moieties: MS2 spectra of Amadori rearrangement products in
amino acid-disaccharide model showed the main fragmentation
ions of Amadori rearrangement products by the loss of mole-
cule of water and even whole sugar from the sugar moieties as
shown in Fig. 2. The cleavage of sugar moiety occurred with
a mass loss of m/z 162 and m/z 324. The main fragmentation
ions of Amadori rearrangement products with two disaccha-
rides of lactose and maltose were shown in Tables 2 and 3,
respectively and the fragmentations were similar to be differen-
tiated from MS2 data. Among the fragmentation ions, the m/z
EXPERIMENTAL
The amino acids used are lysine (K), arginine (R), aspara-
gine (N), glutamine (Q), histidine (H) and tryptophan (W).
The sugars used are β-lactose.All materials and solvents were
obtained commercially (Aldrich and Sigma, St Louis, MO,
USA).
Sample preparation: The reaction model was set as follo-
wing: 0.1 M of amino acids and acetylated amino acids were
dissolved in 1 M d-glucose solution and got the ratio of 1:10
by molecular weight. Freezed-dry by SC250DDA Speedvac
Plus (Thermo Electron Corporation, Waltham, MA) the
samples to get the whiter power mixture and dry-heating the
samples in sealed vials for 1 h at 50, 70 and 90 °C, respectively
and cooling down the samples in -20 °C immediately after
reaction finished.All samples were dissolved withACN:H2O:
FA (40/60/0.5:v/v/v) just before submitting to MS.
Acetylation of amino acids and purification by HPLC:
A solution of the acetylation reagent was prepared through
the addition of acetic anhydride (250 µL) to methanol (750
µL). The acetylation reagent (1 mL) was added to a mixture
of the amino acid (10 mg) and 50 mM ammonium bicarbonate
(100 µL; pH 7.8). The reaction mixture was stirred for 3 h at
room temperature. The resulting product was dried using a
SC250DDA Speedvac Plus (Thermo Electron Corporation,
Waltham, MA) to obtain a solid product, which was further
purified through HPLC. HPLC purification of acetylated amino
acids (10-20 mg/mL each injection) was carried out in aWaters
Delta 4000 system (Milford, MA 01757) with 2487 UV
detector (λ = 215 nm), using a reverse phase column: Xterra®
C-18 column 19 × 150 mm. The separation was performed
using a mobile phase of double-deionized water with 0.1 %
(v/v) trifluoroacetic acid (isocratic) and the flow rate was main-
tained at 12 mL/min. The LC effluent was dried by SC250DDA
Speedvac Plus (Thermo Electron Corporation,Waltham, MA).
Mass spectrometry: All MS experiments were conducted
in the positive mode of a quadrupole ion trap mass spectro-
meter, LCQ Deca XP Plus (Finnigan LCQ, Thermo Finnigan,
San Jose, CA, USA) equipped with a nanospray ion source.
Ion spray tips were flame-pulled from 150 µm (OD) × 50 mm
(ID) capillaries. The electrospray voltage was typically kept
between 2.8 and 3.0 kV; the inlet capillary was maintained at
180 °C. To obtain MS2 and MSn (n > 2) spectra, the normalized
collision energy was varied while maintaining the other ion
[M-H2O+H]+
453.13
A
Lys-Lac
NCE=22
[M+H]+
[M-2H2O+H]+
471.13
435.07
[M-162+H]+
[M-246+H]+
[M-324+H]+
225.13
309.07
147.20
[M-H2O+H]+
453.12
B
[M+H]+
Lys-Mal
NEC=21
[M-2H2O+H]+ 471.08
[M-246+H]+
435.15
[M-324+H]+
147.09
225.19
Fig. 2. MS2 spectra of Amadori rearrangement products(ARPs) of lysine
reacting with: (A) lactose and (B) maltose
Li, Chuanjiang
