A mechanistic study on the fragmentation of peptide-derived Amadori products

Article abstract of DOI:10.1002/jms.1639

Gas phase fragmentation of peptide-derived Amadori products was investigated using synthetic compounds regioselectively deuterated as well as labeled with ¹⁸O at aminofructose moiety. The eliminated molecule CH₂O contains exclusively

Full text of DOI:10.1002/jms.1639

Research Article
Received: 17 April 2009
Accepted: 10 August 2009
Published online in Wiley Interscience: 14 September 2009
(www.interscience.com) DOI 10.1002/jms.1639
A mechanistic study on the fragmentation
of peptide-derived Amadori products
Piotr Stefanowicz,a∗ Katarzyna Kapczynska,a Mariusz Jaremko,b
Łukasz Jaremkob,c and Zbigniew Szewczuka
Gas phase fragmentation of peptide-derived Amadori products was investigated using synthetic compounds regioselectively
deuterated as well as labeled with 18O at aminofructose moiety. The eliminated molecule CH2O contains exclusively protons
attached to carbon C6 of the aminofructose moiety. The hydrogen atoms connected with the carbon C1 of the aminofructose
moiety are partially eliminated as a component of water molecules during the dehydration process and partially dislocated
within the fragmented peptide molecule. The labeled oxygen atom attached to the carbon C2 is eliminated in 100% along with
the first loss of water. The MS3 experiments revealed that the product ion formed by triple dehydration of the Amadori product
does not eliminate the formaldehyde molecule. On the basis of these observations we proposed a hypothetical mechanism of
c
Amadori products’ fragmentation. Copyright ꢀ 2009 John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this article.
Keywords: Amadori rearrangement; glycation; neutral losses; mechanism; nonenzymatic protein modifications; microwave synthesis
Introduction
the peptide-derived Amadori products in collision induced
dissociation (CID) experiments produce a very characteristic
pattern of neutral losses (18, 36, 54, 84 and 162 Da).[9–11] This
series of neutral losses is attributed to the gradual elimination of
1, 2 and 3 molecules of water, followed by the CH2O molecule and
finallythewholehexosemoiety.Thegivenseriesofneutrallossesis
characteristicforAmadoriproductsandenablesthedifferentiation
of these compounds from the products of enzymatic O- or
N-glycosylation.[9] Although the fragmentation behavior of the
glycated peptides is well studied and widely utilized in analysis,
there is limited information on the mechanistic aspects of these
fragmentations.
In the present paper we reinvestigate the fragmentation of
the peptide-derived Amadori products on the basis of the MS2
and MS3 measurements. CID experiments were performed on
the model peptide–glucose conjugates regioselectively labeled
at the sugar moiety with deuterium atoms (attached to the carbon
atoms C1 and C6) as well as 18O atom which was attached to the
C2 carbon. The isotopic labeling allows us to directly monitor the
origin of water and formaldehyde molecules eliminated from the
sugar moiety in the CID experiment. The proposed model was
supported by the similarity of the fragmentation patterns of the
ion produced by the elimination of three water molecules from
the glycated peptide with the synthetically obtained compound
Nonenzymatic glycation is one of the most widely spread
nonenzymatic side-chain-specific posttranslational modifications.
According to Maillard hypothesis, the formation of protein–sugar
conjugates is considered as an important cause of the diabetes
associated complications.[1,2]
The first stage of this reaction involves the interaction
of glucose with the amino group in the protein molecule.
The imines formed in the reversible reaction stage rear-
range to Amadori compounds – N-substituted (1-deoxy-ketos-1-
yl)amines[3,4] – representatives of an important class of Maillard
intermediates. (Fig. 1) These compounds undergo oxidation and
dehydration giving a rise for a diverse group of heterocyclic com-
pounds, called advanced glycation end products (AGEs). There
are numerous pieces of evidence that AGEs mediate diabetes
related pathological processes[5] and aging,[6] mainly by chang-
ing the physicochemical properties of long-living proteins (like
collagen and crystallin) and by the activation of receptors of ad-
vanced glycation end products (RAGEs) resulting in inflammatory
processes.[7]
On the other hand, recent studies indicate that Amadori-
modified proteins of serum may also play a substantive role
in the pathogenesis of vascular complications of diabetes, mainly
arteriosclerosis. The mechanism of this influence is not clear,
however preliminary results suggest that the Amadori-modified
human serum albumin may interact with the smooth muscles of
the blood vessels increasing blood pressure.[8]
∗
Correspondence to: Piotr Stefanowicz, Faculty of Chemistry, University of
Wrocław, F. Joliot-Curie 14, 50-383 Wrocław, Poland.
E-mail: stp@wchuwr.pl
Further understanding of the role of glycation in the diabetic
complications development requires the identification of those
proteins whose glycation may lead to pathological states
as well as the recognition of the amino acid residues that
underwent the modification process. This may be solved by the
combination of enzymatic hydrolysis and liquid chromatography-
mass spectrometry (LC-MS/MS). Earlier studies revealed that
a
b
c
Faculty of Chemistry, University of Wroclaw, F. Joliot-Curie14, 50-383 Wroclaw,
Poland
Institute of Biochemistry and Biophysics, Polish Academy of Sciences,
Pawinskiego 5a, 02-106 Warsaw, Poland
Faculty of Chemistry, Warsaw University, Pasteura 1, 02-093, Warsaw, Poland
c
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Study of peptide-derived Amadori products
Figure 1. Formation of glucose–protein Schiff base and Amadori rearrangement.
corresponding to the postulated product of the dehydration
process.
H-Lys([6,6ꢁ-2H2] Fru)-Ala-Ala-Phe-OH (III)
Glycoconjugate containing two deuterium atoms in the sugar
moiety (III) was synthesized using microwave irradiation on CEM
Discover system. Hundred milligrams of the resin loaded with
peptide was swollen with N,N-dimethylformamide (DMF). Then
200 mg of D-glucose-6,6-d2 in 2 ml of DMF was added to the resin.
Themixturewasirradiatedinamicrowaveovenat40 Wfor20 min.
Then the Fmoc group was removed with 20% piperidine in DMF,
the solution was drained, the resin was washed with DMF (3×),
DCM (3×), MeOH (3×) and dried in vacuum. The resin was treated
with the mixture of TFA : TIS : H2O (95 : 2.5 : 2.5) for 8 h at room
temperature in order to cleave the peptide from the support.
The resin was washed with TFA and the combined filtrates were
evaporated. The final product was purified by preparative high
performance liquid chromatography (HPLC).
Material and Methods
Reagents
Deuterated sodium cyanoborohydride (NaBD3CN), D-glucose-6,6-
2H2 and H218O were purchased from Aldrich. The amino acid
derivativesandcouplingreagent:O-(benzotriazol-1-yl)-N, N, Nꢁ, Nꢁ-
tetramethyluronium tetrafluoroborate (TBTU) were purchased
from Nova-Biochem.
Peptides
Ac-Lys(Fru)-Ala-Ala-Phe-OH (I) and Ac-Lys([1-2H]Fru)-Ala-Ala-Phe-
OH (II) (the abbreviation Fru stands for fructose)
Peptides (I) and (II) were synthesized according to the solid
phase 9-fluorenylmetoxycarbonyl/tert-butyl (Fmoc/tBu) protocol.
Thesynthesishasalreadybeenpresented.[12] Briefly, thelysineside
chain designed for modification was protected by 4-methyltrityl
group. After completing the peptide synthesis the acid-labile
Mtt group was cleaved with 1% triflouoroacetic acid (TFA)
in dichloromethane (DCM) solution. Peptides (I) and (II) were
obtained on solid support by direct alkylation of the lysine
residue’s ε-amino group with 2,3 : 4,5-di-O-isopropylidene-β-D-
arabino-hexos-2-ulo-2,6-pyranose, in the presence of NaBH3CN
and NaBD3CN respectively. All groups protecting amino acids’
side chains as well as isopropylidene groups from the sugar
moiety were removed during the cleavage from the resin using
TFA containing 2.5% of water and 2.5% of triisopropyl silane (TIS)
for 24 h at room temperature.
H-Lys([2-18O] Fru)-Ala-Ala-Phe-NH2 (IV)
The amide H-Lys(Fru)-Ala-Ala-Phe-NH2 was synthesized on the
solid support according to the protocol given in our recent
paper.[12] The purified peptide (1 mg) was dissolved in H218O
(50 µl) and incubated at 25 ◦C. The MS spectrum of the peptide
incubated over 200 h revealed more than 85% of labeling with
18O.
Ac-Lys(HMF)-Ala-Ala-Phe-OH (V)
Peptide (V) was synthesized in solution by the modification of Ac-
Lys-Ala-Ala-Phe-OH. Ac-Lys-Ala-Ala-Phe-OH (5 mg) was incubated
overnight with 5-(hydroxymethyl)furfural (HMF, 5 mg) in 20 µl
of methanol (Fig. 2). The solvent was evaporated and the
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P. Stefanowicz et al.
corresponding fragments of the deuterated and the nondeuter-
ated peptides.
D
0
D(r) = Mav − Mav
D
where D(r) is the number of remaining deuterons, Mav is
the observed average molecular mass of a deuterated peptide
0
fragment; Mav is the observed average molecular mass of a
nondeuterated peptide fragment.
Results
The undeuterated peptide (I) Ac-Lys(Fru)-Ala-Ala-Phe-OH was pre-
pared according to the method described previously.[12] Peptide
(II) Ac-Lys([1-2H]Fru)-Ala-Ala-Phe-OH was obtained by the same
method, using NaBD3CN instead of NaBH3CN. The molecular mass
of the obtained derivative confirmed, that only one deuterium
atom was incorporated into the labeled derivative. Peptide
(III) – H-Lys([6,6ꢁ-2H2] Fru)-Ala-Ala-Phe-OH was obtained by the
method presented previously by Frolov et al.[13] and Stefanowicz
and Szewczuk[14] modified by using microwave activation. The
synthesis was performed on solid support, using a solution of
D-[6,6-2H2]glucose in DMF. The obtained peptide showed the
expected molecular mass indicating incorporation of two D atoms
into the molecule. Peptide (IV) labeled selectively on the oxygen
atom attached to the carbon C2 was prepared by the incubation of
amide H-Lys(Fru)-Ala-Ala-Phe-NH2 with H218O. After 200 h more
than 85% of peptide underwent 16O/18O exchange. The literature
data show that oxygen exchange takes place at the anomeric
carbon atom of some sugars, like glucose, fructose and galactose.
The reaction is attributed to the reactivity of linear sugar structure
which in a water solution forms reversibly the gem-diol hydrate.[15]
The location of isotopically labeled atoms in the hexose moiety
was given in Fig. 3.
Peptide (V) was prepared in solution in the reaction of
hydroxymethylfurfural with the unmodified peptide Ac-Lys(Fru)-
Ala-Ala-Phe-OH according to the procedure given Section on
Material and Methods. The molecular mass of the obtained
conjugates was consistent with the mass calculated on the basis
of the formula.
The identity of obtained peptides including the position of
deuterium atoms is confirmed on the basis of NMR (see the
supporting information) and HR MS. All compounds were purified
on preparative HPLC, and their homogeneity was confirmed by
analytical HPLC. Only compound (V) was not purified and crude
preparation was applied in MS/MS experiments. For this peptide
structure was confirmed by HR-MS.
The nondeuterated peptide (I) was subjected to the MS/MS
experiment. The fragmentation spectrum of the investigated
compound is shown in Fig. 4. The analysis of the spectrum reveals
that the most abundant peaks are formed by neutral losses of
water molecules and the formaldehyde molecule. The elimination
of the hexose moiety and the cleavage of the peptide bonds
are also visible; however, the abundance of such fragments in
relatively low.
The fragments at m/z 622.3124, 604.3015 and 586.2906 were
subjected to the MS3 experiment. The ions at m/z 622.3124 and
604.3015, generated by the elimination of one and two water
molecules respectively, produced in further fragmentation, the
fragment ions at m/z 568.2796 and 556.279, while the ion at m/z
586.2906, arising from the elimination of three molecules of water
produced an ion at 568.2796 (elimination of water) but not an ion
at m/z 556.279 which would be the effect of the elimination of
Figure 2. The synthesis of the peptide (V) Ac-Lys(HMF)-Ala-Ala-Phe-OH.
The abbreviation HMF stands for 5-(hydroxymethyl)furfural.
crude product was used in the MS experiments without further
purification.
Mass spectrometry
ESI-CID
The CID experiments were performed using an Apex-Qe 7T
instrument (Bruker) equipped with dual electrospray ionization
(ESI) source. Spectra were recorded using aqueous solutions
of acetonitrile (50%) and formic acid (1%) at the peptide
concentration 0.5 µM. The potential between the spray needle
and the orifice was set to 4.5 kV. In MS/MS mode, the quadruple
was used to select the precursor ions, which were fragmented
in the hexapole collision cell applying argon as a collision gas.
The product ions were subsequently mass analyzed by the
ion cyclotron resonance (ICR) mass analyzer. For CID MS/MS
measurements, the voltage 20 V over the hexapole collision cell
was applied.
MS3
The initial fragmentation of the Amadori product molecular ions in
the MS3 experiment was achieved by applying a high potential at
the skimmer (70–90 V). The fragments produced by skimmer
fragmentation were selected in the first quadruple (Q1) and
subjected to CID in the second quadruple (Q2). The secondary
product ions were subsequently analyzed in the ICR cell.
Calculation of the extent of deuterium retention
Mav – the average molecular masses of the corresponding deuter-
ated fragments of the peptides (II) and (III) were calculated using
the formula:
Mav = ꢀ(Mi Ai)/ꢀAi
where Mi are the molecular masses for particular isotopic peaks in
the isotopic envelope and Ai are the abundances of corresponding
isotopic peaks.
The deuterium content of deuterated product ions was de-
termined by the difference in the average mass between the
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Study of peptide-derived Amadori products
Figure 3. The location of the isotopic labels in the peptides (II)–(IV).
Figure 4. MS/MS spectrum of [M + H]+ ion of glycated peptide (I) Ac-Lys(Fru)-Ala-Ala-Phe-OH.
formaldehyde. The summary of MS3 experiments was given in the
Fig. 5
Peptide (II) contains only one deuterium atom attached to
the carbon 1 of the fructose moiety. The fragmentation of this
compound was presented in Fig. 6. The first molecule of water
eliminated from this compound did not contain deuterium. The
neutral loss of two, three and four molecules of water as well as
the loss of three molecules of water along with one molecule of
formaldehyde give a rise for the product ions with the deuterium
content (D(r)) 0.82, 0.75, 0.74 and 0.78, respectively. On the other
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P. Stefanowicz et al.
suggest a significant migration of the deuterium atoms attached
to carbon 1 in the course of collision induced fragmentation. The
difference in the deuterium content resulting from the elimination
of the C-terminal Phe is close to an experimental error; however,
additional pieces of evidence (value of D(r) = 0.62, for b1-3H2O
ion and D(r) = 0.11 for the fragment ion produced by the
elimination of hexose moiety) seem to support the hydrogen
transfer hypothesis during the fragmentation process.
The pattern of the fragmentation of the glycoconjugate labeled
on carbon 6 (III) is different (Fig. 7). The elimination of water
does not remove deuterium atoms from the molecule. However,
deuterium is completely removed after the elimination of the
formaldehyde molecule. No deuterium migration in the CID of
compound (III) was observed.
The peptide (IV) was obtained by H218O treatment of amide H-
Lys(Fru)-Ala-Ala-Phe-NH2. The previous studies have proven that
only the anomeric hydroxyl group is susceptible to the isotopic
exchange.[15] The peptide labeled with 18O at C2 was subjected
to the CID experiment. The data presented in Fig. 8 indicate
that the loss of the first water molecule is accompanied by the
elimination of 100% of 18O. Peptide (V) was fragmented (MS2
experiment) and the fragmentation pattern was compared with
the fragmentation of the product ion at m/z 586.2906 generated
by the elimination of three molecules of water from the parent ion
at m/z 640.3232. The comparison revealed the similarity of those
patterns (Fig. 9). The observed minor differences can be explained
by different internal energies of the ions formed in the ion source
and obtained as a result of fragmentation. Interestingly, both ions
(protonated compound (V) and the product ion at m/z 586.2906)
did not produce product ions arising from the elimination of
formaldehyde.
Figure 5. The summary of the products that arise from the MS3
experiments of each of the first-generation product ions formed from
[M + H]+ ion of glycated peptide (I) Ac-Lys(Fru)-Ala-Ala-Phe-OH.
hand, the product ion at 478.2677 resulting from the neutral
loss of a whole hexose moiety shows an increased intensity of
the second isotopic peak (the measured value −39%, while the
calculated one −24% of the intensity of the first isotopic peak).
The deuterium content in this product ion is low (D(r) = 0.11) but
in our opinion out of the range of the experimental error.
The deuterium content for ion at m/z 392.2044 (b3-(3H20, CH2O)
is estimated as 0.65 what indicates, that the elimination of the Phe
moiety reduces the content of D in the peptide. Since Phe is a
C-terminal amino acid in the sequence, distant from the sugar-
baring Lys, the elimination of deuterium with Phe residue may
Discussion
The fragmentation of peptide (II) suggests that the elimination of
water molecules is proton-driven. The first molecule of water is
Figure 6. MS/MS spectrum of [M + H]+ ion of regioselectively deuterated glycated peptide (II) Ac-Lys([1-2H] Fru)-Ala-Ala-Phe-OH.
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Study of peptide-derived Amadori products
Figure 7. MS/MS spectrum of [M + H]+ ion of regioselectively deuterated glycated peptide (III) Ac-Lys([6,6-2H2] Fru)-Ala-Ala-Phe-OH.
Figure 8. MS/MS spectrum of [M + H]+ ion of regioselectively isotopically labeled with 18O glycated peptide (IV) H-Lys([2-18O] Fru)-Ala-Ala-Phe-NH2.
likely to be eliminated as a result of the migration of the proton
attached to the nitrogen atom onto the hydroxyl group attached
to carbon 2.
the migration of the proton is expected to be faster than the water
elimination, proton from the hydroxyl group may be transferred
onto the nitrogen in the lysine side chain and scrambled between
protonation sites within the hexose moiety. Proton localized on
the nitrogen atom can be transferred on the hydroxyl group on
carbon 3. The elimination of the second water molecule (which
is partially deuterated) removed approximately 18% of deuterium
fromthefragmentedion.Consecutiveeliminationofthreeandfour
moleculesofwatercausedtheremovalof25and26%ofdeuterium
respectively. The expected values of D(r) were estimated on the
basis of the following assumptions:
The elimination of water produces an ion with the charge
localized on the oxygen (oxonium ion), which is stabilized by the
transfer of the hydrogen atom from the carbon 1 onto the nitrogen
(the most basic center in the molecule). On the other hand, direct
proton transfer in this case would require a three-center reaction
whatusuallyrequiresahighactivationenergy.[16,17] Thealternative
mechanism involves the solvation of the proton by the remaining
sugar OH group. The hydroxyl group attached to the carbon C2
may serve as an acceptor for the proton eliminated from the C1.
Thisreactionmayproceedasafive-centerprotontransfer. Because
•
The isotopic effects are neglected.
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P. Stefanowicz et al.
Figure 9. Comparison of MS/MS spectrum for [M + H]+ ion of peptide (V) (upper panel) and MS/MS/MS spectrum of product ion obtained by elimination
of three water molecules from the protonated ion of peptide (I) (lower panel).
•
•
•
The hydrogen scrambling takes place within the hexose-
modified lysine side chain.
The migration of the protons to the peptide backbone is
neglected.
Deuterium is equally distributed among all protonation sites in
the glycated side chain of lysine.
There are a number of papers discussing possible mechanisms
of Amadori products fragmentation. Molle et al. analyzed the
fragmentation of lactolated peptides in gas phase. After the elim-
ination of the galactose moiety, the dehydrated aminofructosyl
residue underwent the elimination of a series of water molecules
and of formaldehyde. The authors proposed the oxonium struc-
ture for the ion formed as a result of the elimination of galactose
and two water molecules.[18] The elimination of CH2O gives a
rise for the furylium ion. Jeric and coworkers interpreted the
fragmentation of the protonated Amadori products’ ions, bas-
ing on the pyranose structure of the starting compounds.[11] In
this paper, two fragmentation pathways (oxonium and ammo-
nium) were suggested and the elimination of formaldehyde was
confirmed.
The model of fragmentation proposed herein (Fig. 10) assumes
the furanose form of the aminofructose moiety. Isotopic studies
suggest that the fragmentation of peptide-derived Amadori
products is initiated by the processes similar to the dehydration of
fructose in acid water solution.[19–21] The first step of the reaction
involves the elimination of the hydroxyl group attached to carbon
2. This elimination is initiated by the proton transfer from the
nitrogen atom onto the hydroxyl group. The elimination of the
water molecule gives a rise for the oxonium ion, which potentially
canstabilizebytheindirect,hydroxyl-mediatedtransferofaproton
from carbon C1 onto the nitrogen atom.
The proton attached to the glycated ε-amino group is again
transferred onto the hydroxyl group, initiating the next dehydra-
tion. The loss of 18% of deuterium with the second eliminated
water molecule is in good agreement with the proposed model.
The deuterium attached to the glycated nitrogen atom undergoes
further dislocation, and is transferred onto the other fragments
of the peptide, including the C-terminal phenylalanine (Fig. 11).
The dislocation of the proton in the peptide during CID is
in agreement with the mobile proton model.[22] One of the
experimental consequences of such a process is a hydrogen
scrambling observed in CID of partially deuterated peptides.[23]
For example, the second water molecule is eliminated from
structure E (Fig. 10). The deuterium atom can be localized in five
different positions. Among them position 2 results in elimination
of D2O. Because only 0.5 deuterium atoms is distributed in the
hexose moiety, the expected value of D(r) is 1−(2/5×0.5) = 0.8).
The comparison of D(r) values predicted and measured for the
consecutive losses of water (1/1.04; 0.8/0.82; 0.6/0.75 0.5/0.74)
reveals a significant difference between the expected and the
experimental content of deuterium in the product ions arising
from the elimination of three and four water molecules. This
discrepancy can be explained by the migration of deuterium, as
the hydrogen (deuterium) atom attached to the nitrogen atom in
thelysinesidechainismobileandmayundergofurtherdislocation
onto the peptide backbone (Fig. 10). The migration of the D atoms
is also confirmed by the retention of D in the peptide after
the elimination of the whole hexose moiety. The analysis of the
fragmentation of (II) indicates that D attached to carbon C1 is
shifted onto the heteroatom (most likely N in the side chain of
lysine) and then initiates the loss of water. This proton can also
migrate along the peptide chain. The fragmentation of compound
(III) gives evidence that the protons eliminated with formaldehyde
are attached to carbon C6. In contrast to molecule (II) there is no
proton migration in the peptide (III). Elimination of the protons
connected with carbon C6 along with the formaldehyde molecule
suggeststhattheCH2OmoleculecontainsacarbonC6.Thepeptide
(IV)labeledattheanomericcarbonwith18Oatomeliminates100%
of 18O with the first eliminated water molecule. The elimination
of the hydroxyl group attached to the anomeric carbon C2 at the
beginning of the fragmentation process (reaction B–C according
to Fig. 10) is in good agreement with the proposed model.
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Study of peptide-derived Amadori products
Figure 10. The proposed mechanism of fragmentation of the peptide-derived Amadori products. Bar graphs show the retention of isotopic labels (1-2H;
6-2H; 2-18O) in a consecutive fragmentation steps. The arrows represents the proton transfer within the hexose moiety.
Figure 11. The transfer of protons within the product ion at m/z 587.2970 formed by the elimination of three water molecules from the [M + H]+ ion of
peptide (II).
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P. Stefanowicz et al.
On the other hand, half of the D atoms are permanently bonded
to C1, and can be found in all the stable ions containing furylium
or pyrylium ions.
The fragmentation of peptide (III) indicates that the hydrogens
eliminated along with the formaldehyde molecule were attached
to carbon 6 only. It is also very likely that the carbon eliminated
with CD2O molecule is the C6 atom of fructose. To support the
hypothesis, that the mechanism of gas phase degradation of
aminofructose moiety resembles the acid catalyzed dehydration
of fructose, the peptide (V) (Fig. 2) was synthesized.
inflammatory responses. The Journal of Clinical Investigation 2001,
108, 949.
[8] Y. Hattori, M. Suzuki, S. Hattori, K. Kasai. Vascularsmoothmusclecell
activation by glycated albumin (Amadori adducts). Hypertension
2002, 39, 22.
[9] A. Frolov, P. Hoffmann, R. Hoffmann. Fragmentation behavior of
glycated peptides derived from D-glucose, D-fructose and D-ribose
in tandem mass spectrometry. Journal of Mass Spectrometry 2006,
41, 1459.
[10] P. Stefanowicz, J. Boratynski, U. Kanska, I. Petry, Z. Szewczuk.
Evaluation of high temperature glycation of proteins and peptides
by electrospray ionization mass spectrometry. Acta Biochimica
Polonica 2001, 48, 1137.
[11] I. Jeric, C. Versluis, S. Horvat, A. J. R. Heck. Tracing glycoprotein
structures: electron ionization tandem mass spectrometric analysis
of sugar-peptide adducts. Journal of Mass Spectrometry 2002, 37,
803.
[12] P. Stefanowicz, K. Kapczynska, A. Kluczyk. Z. Szewczuk. A new
procedure for the synthesis of peptide-derived Amadori products
on a solid support. Tetrahedron Letters 2007, 48, 967.
[13] A. Frolov, D. Singer, R. J. Hoffmann. Site-specific synthesis of
Amadori-modified peptides on solid phase. Peptide Science 2006,
12, 389.
[14] P. Stefanowicz, Z. Szewczuk. Studies on glycation of peptides
on solid support. Peptides 2004. Proceedings of the Third
InternationalandTwenty-EighthEuropeanPeptideSymposium.Kenes
International: Israel, 2005, 605.
[15] T. L. Mega, S. Cortez, R. L. Van Etten. The O-18 isotope shift in C-13
nuclearmagnetic-resonancespectroscopy.13.oxygen-exchangeat
the anomeric carbon of deuterium-glucose, deuterium-mannose,
and deuterium-fructose. JournalofOrganicChemistry 1990, 55, 522.
[16] I. P. Csonka, B. Paizs, G. Lendvay, S. Suhai. Suhai Proton mobility in
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Rapid Communications in Mass Spectrometry 2000, 14, 417.
[17] B. Paizs, I. P. Csonka, G. Lendvay, S. Suhai. Proton mobility
in protonated glycylglycine and N-formylglycylglycinamide:
The product of the water elimination has the same M and
elemental composition as the Schiff base obtained in the
reaction of hydroxymethylfurfural with Ac-Lys-Ala-Ala-Phe-OH.
The structure of this compound is the mesomeric form of the ion
postulated by Molle. We found that this ion does not eliminate
the formaldehyde molecule but only H2O, forming the ion at m/z
568.2796. The elimination of CH2O directly from the ions M-3H2O
obtained by the elimination of three molecules of water from
peptide (I) at m/z 586.2906 is not observed. This phenomenon
can be explained assuming that formaldehyde is eliminated
simultaneously with the third molecule of water.
According to nuclear magnetic resonance (NMR) studies
Amadoricompoundsexistinsolutionasanequilibriumoffuranose
and pyranose forms.[24,25] In water solution, the pyranose form is
the predominant one, however in organic solvents [dimethyl
sulfoxide (DMSO)] the most populated form of the amino fructose
moiety is the furanose. There are also suggestions that similar
equilibrium is possible in gas phase.[26] In our model, the furanose
form was chosen, because this formula better explains the
elimination of CD2O from the isotopically labeled peptide (III). The
experiment using model peptide (V) also provides support for the
furanose structure in gas phase. This model is consistent with the
fragmentation mechanism proposed by Molle, and the solution
phase mechanism of the fructose dehydration. On the other
hand, literature indicates that the cyclic compounds containing
the oxonium atom in a six- or five-membered ring undergo fast
and reversible interconvertion.[26] It should also be noted that
in opinion of some authors, the fragmentation of peptide–sugar
conjugates is a complex, multichannel process and most likely
each signal represents a mixture of different isomers.[9]
a
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phy/electrospray tandem mass spectrometry. Analytical Biochem-
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[19] M. J. Antal, W. S. Mok, G. N. Richards. Kinetic studies of the reaction
of ketoses and aldoses in water at high temperatures. 1. Mechanism
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and sucrose. Carbohydrate Research 1990, 199, 91.
[20] Y. Roman-Leskhov, J. N. Chheda, J. A. Dumesic. Phase modifiers
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fructose. Science 2006, 312, 1993.
[21] C. Perez Locas, V. Yaylayan. Isotope labelling studies on the
formation of 5-hydroxymethyl)-2-furaldehyde (HMF) from sucrose
by Py-GC/MS. Journal of Agricultural and Food Chemistry 2008, 56,
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