An isotope (18O, 15N, and 2H) technique to investigate the metal ion interactions between the phosphoryl group and amino acid side chains by electrospray ionization mass spectrometry

Article abstract of DOI:10.1007/s13361-010-0069-5

Cationic metal ion-coordinated N-diisopropyloxyphosphoryl dipeptides (DIPP-dipeptides) were analyzed by electrospray ionization multistage tandem mass spectrometry (ESI-MSⁿ). Two novel rearrangement reactions with hydroxyl oxygen or carbonyl ox

Full text of DOI:10.1007/s13361-010-0069-5

B American Society for Mass Spectrometry, 2011
J. Am. Soc. Mass Spectrom. (2011) 22:689Y702
DOI: 10.1007/s13361-010-0069-5
RESEARCH ARTICLE
15
2
An Isotope (18O, N, and H) Technique
to Investigate the Metal Ion Interactions
Between the Phosphoryl Group and Amino Acid
Side Chains by Electrospray Ionization Mass
Spectrometry
Xiang Gao,1 Xiaomei Hu,1 Jun Zhu,2 Zhiping Zeng,1 Daxiong Han,3 Guo Tang,1
Xiantong Huang,1 Pengxiang Xu,1 Yufen Zhao1,3
1Department of Chemistry and The Key Laboratory for Chemical Biology of Fujian Province, College of Chemistry and
Chemical Engineering, Xiamen University, Xiamen, 361005, People’s Republic of China
2Department of Chemistry and Fujian Provincial Key Laboratory of Theoretical and Computational Chemistry, College of
Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, People’s Republic of China
3Department of Medicine, Medical College, Xiamen University, Xiamen, 361005, People’s Republic of China
Abstract
Cationic metal ion-coordinated N-diisopropyloxyphosphoryl dipeptides (DIPP-dipeptides) were
analyzed by electrospray ionization multistage tandem mass spectrometry (ESI-MSn). Two
novel rearrangement reactions with hydroxyl oxygen or carbonyl oxygen migrations were
observed in ESI-MS/MS of the metallic adducts of DIPP-dipeptides, but not for the correspond-
ing protonated DIPP-dipeptides. The possible oxygen migration mechanisms were elucidated
through a combination of MS/MS experiments, isotope (18O, 15N, and 2H) labeling, accurate
mass measurements, and density functional theory (DFT) calculations at the B3LYP/6-31 G(d)
level. It was found that lithium and sodium cations catalyze the carbonyl oxygen migration more
efficiently than does potassium and participation through a cyclic phosphoryl intermediate. In
addition, dipeptides having a C-terminal hydroxyl or aromatic amino acid residue show a more
favorable rearrangement through carbonyl oxygen migration, which may be due to metal cation
stabilization by the donation of lone pair of the hydroxyl oxygen or aromatic π-electrons of the
C-terminal amino acid residue, respectively. It was further shown that the metal ions, namely
lithium, sodium, and potassium cations, could play a novel directing role for the migration of
hydroxyl or carbonyl oxygen in the gas phase. This discovery suggests that interactions
between phosphorylated biomolecules and proteins might involve the assistance of metal ions to
coordinate the phosphoryl oxygen and protein side chains to achieve molecular recognition.
Key words: Oxygen migration, Isotope labeling ESI-MS/MS, N-Phosphoryl dipeptide, Metal ion
Introduction
Electronic supplementary material The online version of this article
(doi:10.1007/s13361-010-0069-5) contains supplementary material, which
is available to authorized users.
t has been shown that N-phosphoryl amino acids
I
(NPAAs) and peptides are chemically active species
[1–3], which might be not only related to the prebiotic
synthesis of proteins and nucleic acids [4–6] but also the
Correspondence to: Pengxiang Xu; e-mail: xpengxiang@xmu.edu.cn
Received: 9 January 2010
Revised: 24 December 2010
Accepted: 28 December 2010
Published online: 1 February 2011

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X. Gao, et al.: Isotope Technique to study the Interactions
“Mini-Kinase” model to elucidate the phosphoryl transfer
mechanism for the phosphorylation/dephosphorylation of
proteins [7, 8]. In addition, NPAAs might also be considered
as novel chemical models for the investigation of interac-
tions between phosphoryl groups (P=O) and biomolecules.
For example, in solution phase, N-phosphoryl α-amino acids
could be transformed into both peptides and nucleic acids
via a five-membered cyclic pentacoordinate phosphoric-
amino acid mixed anhydride, while the homo-isomers
N-phosphoryl β- or γ-amino acids are inert [4, 9]. The
β-carboxylic group of N-phosphoryl aspartic acid but not the
γ-carboxylic of N-phosphoryl glutamic acid show neighbor-
ing group effects [10, 11]. Similarly, the catalytic activity of
the side-chain β-OH from serine is higher than the γ-OH of
the homoserine [12, 13]. In addition, several natural and
synthetic N-phosphoryl peptides, such as N-phosphoryl-
leucyl-tryptophan and N-phosphoryl-leucyl-phenylalanine,
are potent reversible inhibitors of metalloproteinases [14].
All these examples illustrate that phosphoryl groups play
significant roles in peptide chemistry. It goes without saying
that phosphoryl groups are common constituents in living
cells and control many biochemical events [15], such as
enzyme regulation, phospho-signaling, and protein/protein
association [16–18]. Hence, studying the properties of
phosphoryl amino acids and peptides as the smallest
chemical models in the liquid phase or the gas phase can
provide some significant clues for understanding the
behavior of phosphorylated biomolecules.
Electrospray ionization tandem mass spectrometry
(ESI-MSn) offers a suitable tool for the study of novel
reactions performed in the gas phase. Since its introduction
by Yamashita and Fenn in 1984 [19], ESI-MS has been
extensively used in biological science, e.g., molecular
recognition and supramolecular chemistry [20], noncovalent
macromolecule–ligand interactions [21], protein phosphor-
ylation [22], and sequencing of peptides or proteins [23, 24].
Furthermore, metal ions are known to play an essential role
in biological systems by interaction with proteins or
phosphorylated proteins. For instance, there have been a
great many studies on the gas phase chemistry of complexes
of the alkali metal ions with amino acids or simple peptides
by ESI-MS in the past decade, such as the dissociation
pathways of cationic peptides [25, 26] and metal ion binding
affinities with a number of amino acids and simple peptides
[27]. However, to the best of our knowledge, there are no
viable model systems for the investigations of the interaction
between the metal ions and phosphorylated biomolecules in
the gas phase.
series of compounds possess a unique advantage of
combining together the metal ion, phosphoryl group (P=O
bond) and amino acid. Therefore, complexes of N-phos-
phoryl amino acids or peptides with metal ions could be
considered as model systems for studies of the metal ion
interactions between the phosphoryl group and various types
of amino acid residue. In this paper, stable-isotope labeling
strategies (18O, 15N, and 2H labeling) were applied to
investigate the novel rearrangement reaction of the alkali
metal ion adducts of N-phosphoryl dipeptides by ESI-MSn.
It was found that metal cations of lithium, sodium, and
potassium could catalyze the hydroxyl oxygen or carbonyl
oxygen migration under collision-induced dissociation (CID)
conditions.
Experimental
Materials and Sample Preparation
H-15N-Gly-OH (98 at% 15N), H218O (98 at% 18O), D2O
(99.9 at% D), and CH3OD (99.9 at% D) were obtained from
Cambridge Isotope Laboratories (Andover, MA, USA).
L-amino acids were purchased from GL Biochem. Ltd.
(Shanghai, China). Compounds 1-17 were synthesized by
literature methods with yields of 60%–70% (Scheme 1)
[33, 34]. DIPP-15N-Gly-Phe was synthesized using H-15N-
Gly-OH as start material. DIPP-Gly-Phe-18OH was prepared
by a one-pot procedure (Scheme 1) [35]: N-diisopropylox-
yphosphoryl Gly-Phe-OH (0.5 mmol) was dissolved in
anhydrous CH2Cl2 (5 mL) under argon atmosphere. To this
solution was slowly added 1, 1'-carbonyldiimidazole (CDI,
0.6 mmol) at room temperature and stirred for about 1 h
until no further CO2 gas evolution was observed. Then,
H218O (2 mmol, 40 μL) was added to the reaction mixture
and stirred for another 2 h. The solvents were removed under
reduced pressure, and the residue was purified by silica gel
chromatography using EtOAc-MeOH (20:1) as eluent. The
DIPP-Gly-Phe-18OH was obtained as a colorless oil in high
yield. All compounds synthesized were identified by 31P
1
NMR, H NMR, 13C NMR, and ESI-MS. NMR data for
DIPP-Gly-Phe (Entry 1): 31P NMR (162 MHz, CDCl3):
1
δ=6.06 ppm. H NMR (400 MHz, CDCl3): δ=1.26–1.32
(m, 12 H), 2.99 (dd, J=6.4, 14.0 Hz, 1 H), 3.19 (dd, J=5.6,
14.0 Hz, 1 H), 3.52–3.58 (m, 2 H), 4.23 (dd, J=8.4, 16.4 Hz,
1 H), 4.50–4.60 (m, 2 H), 4.92 (dd, J=6.0, 14.0 Hz, 1 H),
7.11-7.28 ppm (m, 5 H). 13C NMR (100 MHz, CDCl3):
δ=173.7, 169.6 (d, J=7.4 Hz), 136.5, 129.4, 128.3, 126.8,
72.1 (d, J=5.8 Hz), 72.0 (d, J=5.7 Hz), 52.9, 44.6, 37.8,
23.72, 23.67, 23.60, 23.5 ppm.
In our previous work, multistage ESI-MS combined with
N-phosphoryl derivatization has been used for peptide
sequencing [28, 29]. Several unusual rearrangement reac-
tions for N-phosphoryl amino acids and peptide derivatives
have been observed, such as rearrangement of P–N to P–O
bonds [30], carbonyl oxygen migration [31], and methoxy
group migration [32]. From structural analysis of the N-
phosphoryl amino acids and peptides, it is apparent that this
Mass Spectrometry Conditions
Mass spectra were acquired in the positive ion mode using a
Bruker Esquire 3000Plus ion trap mass spectrometer (Bruker
Daltonics Inc., Billerica, MA, USA) equipped with a gas
nebulizer probe capable of analysis to m/z 6000. Nitrogen

X. Gao, et al.: Isotope Technique to study the Interactions
691
Scheme 1. General strategy for the synthesis of N-diisopropyloxyphosphoryl dipeptides. Reaction conditions: (a) Amino acid
(or 15N labeled amino acid), CCl4, H2O, triethylamine, EtOH, ice-salt bath, 6 h; (b) Amino acid methyl esters, Ph3P, C2Cl6,
triethylamine, CH2Cl2, ice-salt bath, 2 h; (c) 0.5 mol·L–1 NaOH, room temperature, 1 h
gas was used at a flow-rate of 4 L·min–1. The nebulizer
pressure was 8 psi and the electrospray capillary was
typically held at 4 kV. The heated capillary temperature
was 250 °C. Samples in methanol at ~0.01 mg·mL–1 were
ionized by ESI and infused continuously into the ESI
chamber at a flow-rate of 4 μL·min–1 by a Cole-Parmer
74900 syringe pump (Cole-Parmer Instrument Co. Vernon
Hills, IL, USA). The scan range was generally from m/z 50
to 800. Five scans were averaged for each spectrum. The
precursor ions [M+H]+, [M+Li]+, [M+Na]+, and [M+K]+
were selected using an isolation width of 1.0 to 1.5 mass to
charge (m/z) units and analyzed by multistage tandem mass
spectrometry (MSn) through collisions with helium. The
fragmentation amplitude values were 0.3 to 0.8 V and the
fragmentation time was 40 ms.
High-resolution tandem mass spectrum of deuterium
labeled DIPP-Gly-Phe was recorded on a Bruker APEX-
Ultra Fourier transform ion cyclotron resonance mass
spectrometer equipped with an analytic electrospray source
in the positive ion mode. Deuterium-labeled experiment:
DIPP-Gly-Phe (1 mg) was incubated with D2O/CH3OD
(2 mL, 1:1, vol/vol) overnight at room temperature for
exchange of labile protons. An aliquot of the reaction
mixture (10 μL) was diluted with CH3OD (1 mL) and
analyzed by ESI-HRMS/MS. Acquisition parameter: Drying
gas temperature: 180 °C; drying gas flow rate: 5.0 L·min–1;
nebulizer gas flow rate: 1.0 L·min–1; ion accumulation time:
2.0 s. The following voltages were used: capillary entrance
voltage (4.3 kV), end plate (3.8 kV), skimmer 1 (36 V), and
collision energy (13.5 V). The scan range was from m/z 100
to 700. The sample dissolved in CH3OD was introduced into
the source at a flow rate of 4 μL·min–1. The three deuterium
atoms labeled [d3-DIPP-Gly-Phe+Na]+ ion at m/z 412.1718
was isolated with a width of 0.1 mass to charge unit. Twenty
scans were averaged for each spectrum. Other high-
resolution mass spectra were recorded on a Shimadzu liquid
chromatograph/mass spectrometry-ion trap-time-of-flight
(LC-IT-TOF) (Shimadzu, Kyoto, Japan) equipped with an
analytic electrospray source in the positive ion mode. Probe
voltage was 4.5 kV and scan range was from m/z 50 to 800.
The sample in CH3OH was introduced into the source at a
flow rate of 10 μL·min–1. The nebulizing gas flow was
0.5 L·min–1 and the ion accumulation time was 10.0 ms.
Calculations
Molecular geometries of all species in this work were
optimized at the B3LYP/6-31 G(d) level [36–38]. Frequency
calculations at the same level of theory have also been
performed to confirm that all stationary points were minima
(no imaginary frequency) or transition states (one imaginary
frequency). Calculations of intrinsic reaction coordinates
(IRC) [39, 40] were also performed on transition states to
confirm that such structures are indeed connecting two
minima. To examine the effect of basis sets, we also
employed a larger basis set, 6-311 G(d, p), to perform
single-point energy calculations for several selected struc-
tures. The additional calculations show that the basis set
dependence is small. For example, using the smaller basis
set, the relative energies of lithiated IIa, TS-ab, and IIb
(Figure 4) are 0.0, 42.6, and 9.9 kcal·mol–1, respectively.
Using the larger basis set, the relative energies of these
species are 0.0, 42.1, and 8.5 kcal·mol–1, respectively.
Similarly, using the smaller basis set, the relative energies
of sodiated IIa, TS-ab, and IIb (Figure 4) are 0.0, 48.7 and
16.5 kcal·mol–1, respectively. Using the larger basis set, the
relative energies of these sodiated species are 0.0, 47.0, and
14.1 kcal·mol–1, respectively. All calculations were per-
formed with the Gaussian 03 software package [41].
Results and Discussion
The characteristic fragmentations in ESI-MS of N-phos-
phoryl peptides 1–17 are compiled in Table 1. This
illustrates that these group of compounds follow a common
dissociation pathway. As a representative, the ESI-MSn
positive ion mass spectral fragmentation pattern of N-
diisopropyloxyphosphoryl Gly-Phe dipeptide (DIPP-Gly-
Phe, Entry 1) is analyzed in detail. The multiple-stage CID

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X. Gao, et al.: Isotope Technique to study the Interactions
Table 1. Partial ESI-MS/MS data for N-diisopropyloxyphosphoryl dipeptides. The relative abundance percentages are noted in parentheses. (Cat+ = H+, Li+,
Na+, and K+)
Entry and compound
1 DIPP-Gly-Phe
Precursor
ion, m/z
Species
[M+Cat –42]+
[M+Cat –102]+
[M+Cat –164]+
Rearrangement ions
I
[M+Cat]+
II
393
409
425
387
394
410
426
388
[M+Li]+
[M+Na]+
[M+K]+
[M+H]+
[M+Li]+
[M+Na]+
[M+K]+
[M+H]+
[M+Na]+
351(100)
367(100)
383(100)
345(100)
352(100)
368(96)
384(100)
346(100)
370.1243
-
291(3)
307(11)
323(8)
285(16)
292(4)
308(12)
324(9)
286(14)
309.0600
310.0662
309(13)
-
229(0)
245(6)
261(0)
223(24)
257(0)
273(5)
289(21)
251(27)
271(0)
287(7)
303(5)
265(15)
305(5)
321(11)
337(9)
299(17)
305(0)
321(50)
337(2)
299(18)
307(13)
349(5)
349(12)
363(12)
261(7)
275(7)
303(10)
289(10)
229(4)
245(11)
261(0)
223(0)
230(3)
246(11)
262(0)
224(0)
248.1096
-
246(15)
262(24)
278(6)
211(92)
227(99)
243(6)
240(0)
205(0)
2 DIPP-15N-Gly-Phe
247(16)
263(25)
279(7)
212(90)
228(100)
244(7)
241(0)
206(0)
3 d3-DIPP-Gly-Phe
4DIPP-Gly-Phe-18OH
5 DIPP-Ala-Ala
412.1718
264.0954
-
228.0862
229.0925
229(51)
227(48)
149(21)
165(7)
411
[M+Na]+
369(100)
-
247(10)
-
264(13)
262(11)
260(11)
276(24)
292(0)
331
347
363
325
359
375
391
353
373
389
405
367
407
423
439
401
407
423
439
401
409
451
451
465
363
377
405
391
[M+Li]+
[M+Na]+
[M+K]+
[M+H]+
[M+Li]+
[M+Na]+
[M+K]+
[M+H]+
[M+Li]+
[M+Na]+
[M+K]+
[M+H]+
[M+Li]+
[M+Na]+
[M+K]+
[M+H]+
[M+Li]+
[M+Na]+
[M+K]+
[M+H]+
[M+Na]+
[M+Na]+
[M+Na]+
[M+Na]+
[M+Na]+
[M+Na]+
[M+Na]+
[M+Na]+
289(100)
305(100)
321(100)
283(100)
317(100)
333(49)
349(100)
311(97)
331(100)
347(60)
363(100)
325(93)
365(100)
381(96)
397(100)
359(100)
365(100)
381(68)
397(29)
359(100)
367(78)
409(39)
409(100)
423(100)
321(100)
335(89)
363(100)
349(100)
167(5)
183(8)
199(0)
161(0)
195(5)
211(5)
227(45)
189(0)
209(0)
225(8)
241(0)
203(0)
243(3)
259(10)
275(4)
237(0)
243(7)
259(10)
275(3)
164(0)
245(24)
287(14)
287(12)
301(11)
199(8)
213(10)
241(5)
227(7)
181(0)
254(0)
143(0)
6 DIPP-Ala-Val
7 DIPP-Ala-Leu
8 DIPP-Ala-Phe
9 DIPP-Phe-Ala
260(48)
276(40)
292(4)
177(22)
193(5)
209(0)
254(0)
171(0)
260(26)
276(32)
292(12)
254(0)
191(20)
207(6)
223(0)
185(0)
260(16)
276(28)
292(7)
225(97)
241(100)
257(9)
254(0)
219(0)
336(15)
352(23)
368(3)
225(76)
241(55)
257(2)
254(0)
219(0)
10 DIPP-Phe-Gly
11 DIPP-Phe-Val
12 DIPP-Val-Phe
13 DIPP-Leu-Phe
14 DIPP-Ala-Ser
15 DIPP-Ala-Thr
16 DIPP-Leu-Ser
17 DIPP-Val-Ser
352(1)
227(66)
269(57)
269(92)
283(83)
181(76)
195(100)
223(82)
209(95)
352(100)
304(23)
318(19)
276(8)
276(16)
318(10)
304(10)
spectra of native and isotope labeled DIPP-Gly-Phe are
shown in Figure 1 and Figure 2.
by the loss of one or two molecules of propylene, respectively
(Figure 1d). Moreover, a characteristic rearrangement ion for
sodium cationized DIPP-Gly is observed at m/z 163, which
may be produced from a cyclic pentacoordinated phosphoric-
carboxylic intermediate leading to P–N to P–O migration [30].
High-resolution ESI-MS/MS (Table 2) indicates that the exact
mass of ion I was 262.0824, corresponding to elemental
composition C8H18NNaO5P, which could be formed from the
parent ion [M+Na]+ at m/z 409.1486 by the loss of a neutral
C-terminal phenylalanine residue with molecular weight of
147.0662 (Theoretical mass: 147.0684). However, the for-
mation of other novel fragment ion at m/z 227, formed from the
parent ion at m/z 307 by the loss of a neutral molecule 80 Da
(HPO3) as shown in Figure 1c, cannot easily be explained.
High-resolution mass analysis of this ion at m/z 227.0978 Da
indicates an atomic composition of C11H12N2NaO2, which
corresponds within 1 ppm to the proposed structure ion II as
shown in Scheme 7.
The fragmentation patterns of the sodium ion DIPP-Gly-
Phe (1) [M+Na]+ (Figure 1a) at m/z 409 were determined as
shown in Scheme 2. It can be seen that the precursor ion
[M+Na]+ at m/z 409 can lose propylene to form an abundant
daughter ion at m/z 367, which in turn fragments to produce
an ion at m/z 307 by successive loss of propylene (42 Da)
and water (18 Da) through Path A (Figure 1b). The
fragmentation reaction is typical of positive-ion ESI-MS/MS
of DIPP-amino acids and peptides. Furthermore, by Path B,
the fragment ion at m/z 262 (Ion I), corresponding to
[DIPP-Gly+Na]+, could be generated through losing a
C-terminal phenylalanine residue by transfer of carboxyl
oxygen from the original C-terminus of DIPP-Gly-Phe to
the new C-terminal carboxylic acid group of DIPP-Gly. In
order to identify this product ion at m/z 262, its MS3
showed that fragment ions at m/z 220 and 178 were formed

X. Gao, et al.: Isotope Technique to study the Interactions
693
Figure 1. Positive ion ESI tandem mass spectra of DIPP-Gly-Phe (Entry 1). (a) ESI-MS/MS of the sodium adducts of DIPP-Gly-
Phe at m/z 409; (b) ESI-MS3 of the [M+Na – 42]+ ion at m/z 367; (c) ESI-MS4 of the [M+Na – 102]+ ion at m/z 307; (d) ESI-MS3
of the rearrangement ion at m/z 262
In order to clarify the structures of these two novel
product ions at m/z 227 (ion II) and 262 (ion I), isotopic
labeling experiments were conducted. First, 15N-labeled
DIPP-Gly-Phe dipeptide (DIPP-15N-Gly-Phe) was synthe-
sized and analyzed by ESI-MS/MS as shown in Figure 2a.
The signals corresponding to the rearrangement ions I and II,
observed at m/z 262 and 227 with the unlabeled material, are
shifted by 1 u to m/z 263 and 228, respectively, which

694
X. Gao, et al.: Isotope Technique to study the Interactions
Figure 2. Positive ion ESI-MS/MS spectra of the [M+Na]+ of the isotope labeled DIPP-Gly-Phe (18O, 15N, and 2H). (a) ESI-MS/
MS of isotope labeled DIPP-15N-Gly-Phe ion at m/z 410 (Entry 2); (b) High resolution ESI-MS/MS of deuterium-labeled DIPP-
Gly-Phe ion at m/z 412.1718 (d3-labeled, Entry 3); (c) ESI-MS/MS of isotope labeled DIPP-Gly-Phe-18OH ion at m/z 411 (Entry 4)
suggested that both fragment ions contained the nitrogen
atom of the glycine residue derived from parent compound
DIPP-Gly-Phe. Furthermore, according to the general
“N rule” in the elemental analysis, the product ion I should
have one nitrogen atom because of the molecular weight of the
fragment at m/z 262 is odd (239 Da) after subtracting 23 Da of
Na. On the contrary, the Na+ adduct ion II at m/z 227 with even
molecular weight (204 Da) should have two nitrogen atoms.
Secondly, all amino, amide, and acid groups of the
sodiated DIPP-Gly-Phe were labeled with deuterium atoms
via solution-phase hydrogen/deuterium exchange before the
sample was injected into the ESI-FT-ICR-MS. The
d3-labeled [M+Na]+ ion at m/z 412.1718 was subjected to
high resolution ESI-MS/MS. As shown in Figure 2b,
compared with ion I of the native DIPP-Gly-Phe at m/z
262, ion I of d3-labeled DIPP-Gly-Phe appears as a mass
peak at m/z 264.0954 with a 2 u shift, which implies that the
ion I has two active hydrogen atoms. Meanwhile, the
rearrangement ion II could also contain two exchangeable
protons since two mass peaks at m/z 228.0862 (d1-) and m/z
229.0925 (d2-) are observed. As shown in Scheme 3, the
d3-labeled [M+Na – 84]+ ion at m/z 328.0793 could lose a
molecule of HDO and H2O to form two daughter ions at m/z
309.0600 and m/z 310.0662 respectively, which subse-
quently fragmented to produce ions at m/z 228.0862 and
229.0925 by elimination of a molecule of deuterium-labeled
metaphosphate (DPO3).
Thirdly, the C-terminal [18O1]-labeled DIPP-Gly-Phe-18OH
4 was synthesized and analyzed by ESI-MS/MS (Figure 2c
and Figure 3). As shown in Figure 2c, a mass increment
of 2 Da of the precursor ion from m/z 409 to m/z
411 implies the exchange of one 16O with one 18O.

X. Gao, et al.: Isotope Technique to study the Interactions
695
Scheme 2. Fragmentation pathways of the sodium adducts of DIPP-Gly-Phe (Entry 1), DIPP-15N-Gly-Phe (Entry 2), deuterium
labeled d3-DIPP-Gly-Phe (Entry 3), and DIPP-Gly-Phe-18OH (Entry 4)
However, it is possible that the H218O can attack the C=O of
Glycine residue or P=O group and causes 18O labeling at these
sites when carried out the labeled experiments. In order to
specify the location of exchange, the protonated DIPP-Gly-
Phe-18OH at m/z 389 that was shifted 2 Da from the native
protonated DIPP-Gly-Phe at m/z 387 was studied by ESI-MS/
MS (Figure 3). Furthermore, the fragmentation pathways of the
protonated DIPP-Gly-Phe-18OH were compiled in Scheme 4.
Considering the Scheme 4, it was found that the precursor ion
at m/z 389 could lose the neutral H218O+CO or H2O+C18O to
shown in Scheme 5b [42]. Thus, the data from the isotope-
labeled substrates, with respect to retention or elimination of
the isotopes, gives structural information that can unambigu-
ously identify the structures of the product ions.
Based on the experimental results, we propose two
possible rearrangement mechanisms to account for the
formation of ions I and II, respectively (Schemes 6 and 7).
In one mechanism by chelation, the alkali metal ions could
bring the amide carbonyl oxygen and carboxylic oxygen
atom closer through an intermediate seven-membered ring Ia
[43, 44]. Then the oxygen atom of the C-terminal carboxylic
group could attack the carbonyl bond of the adjacent amide
carbonyl nucleophilically to form an anhydride intermediate
Ib with a five-membered ring. Subsequently, by C–N bond
cleavage and by loss of CO and an imine, ion I, metallic ion
adducts of DIPP amino acids, could be formed. Indeed, a
similar reaction mechanism involved the concerted proton
transfer and nucleophilic attack leading to a b-OH type ions
has been investigated extensively [45–48]. The second
mechanism for the generation of ion II is shown in Scheme 7,
the metallic ion might be closely coordinated between the
phosphoryl oxygen, carbonyl oxygen, and phenyl group
produce the single mass peak at m/z 341, indicating that the 18
O
labeling occurred only at the C-terminal carboxylic acid group.
Indeed, because of the equivalence of the two C-terminal
oxygens, it was reasonable to find that ion I appeared at m/z 262
and 264 in a ratio of 1:1, corresponding to unlabeled and
[18O1]-labeled [DIPP-Gly+Na]+ ions, respectively. It indicates
that one of the C-terminal carboxyl oxygens migrates to the
new C-terminus (Scheme 5a). However, it is quite surprising to
find that ion II also appears as two peaks at m/z 227 (unlabeled)
and 229 ([18O1]-labeled) in a ratio of approximately 1:1
(Figure 2c), which might arise from intramolecular 18O
exchange without peptide bond cleavage in the gas phase as

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X. Gao, et al.: Isotope Technique to study the Interactions
Table 2. High-resolution ESI-MS/MS data for the sodium adducts of DIPP-Gly-Phe (Entry 1), DIPP-Ala-Phe (Entry 8), and DIPP-Phe-Ala (Entry 9)
Compound Precursor ion Ion I Ion II
DIPP-Gly-Phe
(1)
m/z
409
262
227
Theoretical mass (m/z)
Measured mass (m/z)
Relative error (ppm)
Chemical formula
m/z
Theoretical mass (m/z)
Measured mass (m/z)
Relative error (ppm)
Chemical formula
m/z
409.1504
409.1486
4.3
262.0820
262.0824
1.5
227.0796
227.0798
0.9
C11H12N2NaO2
241
241.0953
241.0951
0.8
C12H14N2NaO2
241
C17H27N2NaO6P+
423
C8H18NNaO5P+
276
+
+
+
DIPP-Ala-Phe
(8)
423.1661
423.1661
0.0
276.0977
276.0983
2.2
C18H29N2NaO6P+
423
C9H20NNaO5P+
352
DIPP-Phe-Ala
(9)
Theoretical mass (m/z)
Measured mass (m/z)
Relative error (ppm)
Chemical formula
423.1661
423.1661
0.0
352.1290
352.1307
4.8
241.0953
241.0938
6.2
C18H29N2NaO6P+
C15H24NNaO5P+
C12H14N2NaO2
(IIa), then with the strong electron affinity of the phosphoryl
group, the intramolecular oxygen migrates through a five-
membered ring phosphorus intermediate to form IIc, which
in turn breaks down to form ion II by the extrusion of a
fragment HPO3. In fact, the five-membered phosphorus
reaction intermediates of phosphoryl amino acids and
peptides have been trapped and determined by ESI-MS/MS
in our previous work [49, 50].
It is noteworthy that the above rearrangement only
happened with the metallic adducts, but not for the
corresponding protonated DIPP-dipeptides (Table 1). This
result strongly suggested that the activation of metal ion was
essential for the migration and the rearrangement needs the
larger size of Li+ or Na+ to coordinate both P=O and C=O
oxygen atoms to catalyze the oxygen migration via the
unusual and some energetically less favorable cleavage
pathways. For example, the alkali metal ion adducts of
DIPP-Gly-Phe (1) and DIPP-Phe-Ala (9) showed the
presence of the ions I and II. This demonstrates that the
metal ion having multidentate binding ability can preferentially
coordinate with the carbonyl and phosphoryl oxygens and this
might be the energetically preferred reaction intermediate to
make the carbon atom and phosphorus atom more susceptible
to nucleophilic attack. These results are consisted with
literature reports [32, 51, 52]. Furthermore, it is worth noting
that the relatively more intense rearrangement ion II is
observed for Li+ and Na+ adducts of DIPP-dipeptides, which
might be due to the metal ion binding affinity of DIPP-
dipeptides following the order Li+ 9 Na+ 9 K+ [53, 54]. Since
the lithium cation has the highest charge density among all
alkali cations because of its smallest size, the radius of the
cation increases on going down the alkali metal group, the
charge density decreases and the bond distance increases. It
seems that both parameters decrease the stabilizing electro-
static interaction between the DIPP-dipeptides and metal ion,
and subsequently reduce the formation of the metallic
intermediates Ia and IIc (Schemes 6 and 7).
When the ESI-MS/MS data for different DIPP-dipeptides
are compared, it is interesting to note that the side chain
structures of the C-terminal amino acid residues had
considerable effects on the relative abundance of the Li+
and Na+ adducts ion II. For example, in Compounds 14–17
with C-terminal amino acids containing a hydroxyl group,
formation of the Li+ and Na+ adducts ion II is much more
favorable (Table 1). Similarly, the relative intensity of the
Li+ and Na+ adducts ion II is the most abundant (100%)
when the C-terminal amino acids are aromatic amino acids,
such as Compounds 1, 8, 12, and 13. It seems that Li+ and
Na+ presumably coordinate with the hydroxyl or aryl group
of the C-terminal amino acid residues to form an energeti-
cally favorable chelate ring by the donation of aromatic
Scheme 3. Possible fragmentation pathways of the deute-
rium labeled [d3-DIPP-Gly-Phe+Na-42]+ ion at m/z 370.1243

X. Gao, et al.: Isotope Technique to study the Interactions
697
Figure 3. Positive ion ESI-MS/MS spectra of the [M+H]+ ions of the native and isotope labeled DIPP-Gly-Phe. (a) DIPP-Gly-
Phe at m/z 387 (Entry 1); (b) DIPP-Gly-Phe-18OH at m/z 389 (Entry 4)
Scheme 4. Possible fragmentation pathways of the protonated DIPP-Gly-Phe-18OH (Entry 4)

698
X. Gao, et al.: Isotope Technique to study the Interactions
Scheme 5. Fragmentation pathways of the sodium adducts of 18O-labeled DIPP-Gly-Phe-18OH (Entry 4). (a) Formation of the
daughter ions at m/z 262 and 264 (Equilibrium between the C=18O and 18OH); (b) Formation of the daughter ions at m/z 227
and 229 (The oxygen migration of C=18O)
Scheme 6. Proposed rearrangement mechanism for the formation of ion I

X. Gao, et al.: Isotope Technique to study the Interactions
699
Scheme 7. Proposed rearrangement mechanism for the formation of ion II (DIPP-Gly-Phe, Entry 1 as a representative)
π-electrons or lone pair of hydroxyl oxygen atom to the
cationic center respectively [55–58], while in our case,
K+ shows such ability to a moderate extent (Scheme 7).
To gain an insight into this oxygen migration steps, we
carried out DFT calculations at the B3LYP/6-31 G(d) level
by Gaussian 03 program. All species occurring along the
rearrangement for the formation of the ion II are depicted in
Figure 4. Some of the species involved are also displayed
schematically in Scheme 7. The energies (ΔG and ΔE,
kcal·mol-1) of the species involved are given along with the
energy profile during the multistep reaction from IIa to Ion
II···HPO3 (Figure 4). The calculations indicate that the ring
closing step from IIb to IIc, namely concerted proton transfer
and cyclization is the rate-determining reaction step. In
addition, this ring closing step is also a thermodynamically
favorable process in which the relative free energies of this
process are: –2.9 kcal·mo–-1 for Li+, 1.0 kcal·mol–1 for Na+,
and 9.4 kcal·mol–1 for K+. Furthermore, the transition state
Figure 4. Free energies (ΔG), relative energies (ΔE, in parentheses) and the optimized structures (Li+ adducts) for the formation
of ion II corresponded to Scheme 7 (Entry 1, Cat+ = Li+, Na+, and K+). The energies of all different species were relative to that of
IIa for each metal ion complex. The energies are given in kcal·mol–1

700
X. Gao, et al.: Isotope Technique to study the Interactions
Figure 5. Comparison of free energies (ΔG, kcal·mol–1) and relative energies (ΔE, kcal·mol–1, in parentheses) for the formation
of intermediate IIc between Entry 5, 8, and 14. The bottom and right were the optimized structures for the lithiated IIa and
TS-bc, respectively. The energies were relative to that of IIa for each metal ion complex
TS-bc corresponding to this process is located and depicted
in Figure 4. As can be seen, the transferring proton is
somewhere in between the two oxygen atoms, and the
distance between the carbonyl oxygen and phosphorus atom
(1.858Å) for TS-bc is less than that of IIb (3.167Å) but
longer than IIc (1.662Å) (Figure S1), indicating that proton
transfer and nucleophilic attack might happen simulta-
neously as a result of a relatively strong charge transfer
interaction involving the lone pair of oxygen atom and
the partially positively charged phosphorus atom. The
order of the overall barrier is: K+ (56.6 kcal·mol–1)9Na+
(48.0 kcal·mol–1)9Li+ (43.3 kcal·mol–1), in line with the
experimental observations that Li+ can catalyze the
oxygen migration more efficiently than K+. It should be
also pointed here that the relative high free energy of the
Ion II (Ring opening) might be due to the large strain of
the three-membered ring structure, which could further
undergo ring-open process and lead to thermodynami-
cally favorable product containing a N=C–N=C or
N=C–C=N unit depending on which bond (C–C or
C–N bond) is cleaved.
In order to further clarify the effect of the amino acid side
chains, we selectively optimized the rate-determining step in
the reaction mechanism for the lithiated DIPP-dipeptides 5, 8,
and 14 with different C-terminal amino acid residues. As
shown in Figure 5, the order of the calculated barriers for the
formation of the intermediate IIc are: DIPP-Ala-Ala (Entry 5,
51.1 kcal·mol–1)9DIPP-Ala-Ser (Entry 14, 43.1 kcal·mol–1)9
DIPP-Ala-Phe (Entry 8, 40.2 kcal·mol–1), in line with the
observed relative abundance of rearrangement ions II in
ion-trap tandem mass spectrometer (Table 1). Further-
more, the structures of the lithiated intermediates IIa and
transition state TS-bc depicted in Figure 5 demonstrated
that the lithium cation could spatially bind to the phenyl
group (DIPP-Gly-Phe and DIPP-Ala-Phe) or hydroxyl
group (DIPP-Ala-Ser) of the C-terminal amino acid
residues respectively. However, such a stabilization can
not be achieved in DIPP-Ala-Ala with a methyl side
chain. All in all, the calculations suggest that the
formation of ions II depends on the nature of the metal
cations and specific structures of the amino acid residues.
Further evidence for the generation of ion II is shown by
comparing the ESI-MS/MS data of several pairs of formula
isomers, such as DIPP-Ala-Phe (8)/DIPP-Phe-Ala (9), DIPP-
Gly-Phe (1)/DIPP-Phe-Gly (10), and DIPP-Phe-Val (11)/
DIPP-Val-Phe (12) having a phenylalanine residue at the
C- or N-terminal respectively (Table 1). For example, for
compounds 8 and 9, it is notable that both of them yield
the same formula (C12H14N2NaO2, Table 2) for ion II at
m/z 241, but with 100% and 55% abundance respectively.
It means that the C-terminal aromatic amino acid has a
stronger π-system stabilization effect for ion II than that
of the N-terminal one. On the other hand, for the
generation of ion I, there is not much difference between
the corresponding Li+ and Na+ adducts. These data are
consistent with the rearrangement mechanism proposed in
Schemes 6 and 7. Consequently, these results show that
ESI-MS/MS is a useful tool for isomeric differentiation
through collisionally induced dissociation of the metal ion
complexes [59, 60].

X. Gao, et al.: Isotope Technique to study the Interactions
701
9. Gao, X., Liu, Y., Xu, P.X., Cai, Y.M., Zhao, Y.F.: α-amino acid
behaves differently from β- or γ-amino acids as treated by trimetaphos-
phate. Amino Acids 34, 47–53 (2008)
Conclusions
10. Li, Y.M., Zhang, D.Q., Zhang, H.W., Ji, G.J., Zhao, Y.F.: β-carboxyl
catalytic effect of N-phosphoryl aspartic acid. Bioorg. Chem. 20, 285–
295 (1992)
11. Chen, Z.Z., Tan, B., Li, Y.M., Zhao, Y.F.: Activity difference between
α-COOH and β-COOH in N-phosphoryl aspartic acids. J. Org. Chem.
68, 4052–4058 (2003)
12. Xue, C.B., Yin, Y.W., Zhao, Y.F.: Studies on phosphoserine and
phosphothreonine derivatives. Tetrahedron Lett. 29, 1145–1148 (1988)
13. Chen, Z.Z., Li, Y.M., Wang, H.Y., Du, J.T., Zhao, Y.F.: Theoretical study
on the rearrangement of β-OH and γ-OH in ESI mass spectrometry by N-
phosphorylation. J. Phys. Chem. A 108, 7686–7690 (2004)
14. Poncz, L., Gerken, T.A., Dearborn, D.G., Grobelny, D., Galardy, R.E.:
Inhibition of the elastase of Pseudomonas aeruginosa by N-phosphoryl
dipeptides and kinetics of spontaneous hydrolysis of the inhibitors.
Biochemistry 23, 2766–2772 (1984)
15. Westheimer, F.H.: Why nature chose phosphates. Science 235, 1173–
1178 (1987)
16. Hunter, T.: Signaling-2000 and beyond. Cell 100, 113–127 (2000)
17. Johnson, L.N., Lewis, R.: Structural basis for control by phosphor-
ylation. Chem. Rev. 101, 2209–2242 (2001)
18. Adams, J.A.: Kinetic and catalytic mechanism of protein kinases. Chem.
Rev. 101, 227–2290 (2001)
19. Yamashita, M., Fenn, J.B.: Negative ion production with the electro-
spray ion source. J. Phys. Chem. 88, 4671–4675 (1984)
In summary, the collision-induced dissociation reactions of a
variety of N-phosphoryl dipeptides have been studied in
detail using MS/MS experiments, isotope (18O, 15N, and 2H)
labeling, accurate mass measurements and DFT calculations.
The alkali metal cations, especially for Li+ and Na+, can
catalyze a carbonyl oxygen migration through a phosphoryl
group participating five-membered ring intermediate. None
of the oxygen migrations is observed for the corresponding
protonated N-phosphoryl dipeptides. This study indicates
that the Group I metal ions can play an essential role for the
rearrangement of a phosphorylated peptide or protein.
Furthermore, when different N-phosphoryl dipeptides are
compared, it is found that the C-terminal amino acid with
potential coordinating side chains, such as the hydroxyl
group or aromatic ring, the formation of rearrangement ion
for carbonyl oxygen migration is more favorable. These
results show that ESI-MS is a useful tool for structural
determination of phosphorylated dipeptides. In addition, the
discovery of potential metal cation (Li+ or Na+) coordination
between the phosphoryl group and the amino acid residue
side chains may provide new insights into the mechanism
for molecular recognition through the interaction between
the P=O group and protein side chains by the action of
metal ions.
20. Schalley, C.A.: Molecular recognition and supramolecular chemistry in
the gas phase. Mass Spectrom. Rev. 20, 253–309 (2001)
21. Chen, X.L., Qu, L.B., Zhang, T., Liu, H.X., Yu, F., Yu, Y.Z., Liao, X.
C., Zhao, Y.F.: The nature of phosphorylated chrysin–protein inter-
actions involved in noncovalent complex formation by electrospray
ionization mass spectrometry. Anal. Chem. 76, 211–217 (2004)
22. Schlosser, A., Pipkorn, R., Bossemeyer, D., Lehmann, W.D.: Analysis of
protein phosphorylation by a combination of elastase digestion and neutral
loss tandem mass spectrometry. Anal. Chem. 73, 170–176 (2001)
23. Barlow, C.K., O’Hair, R.A.J.: Gas-phase peptide fragmentation: how
understanding the fundamentals provides a springboard to developing
new chemistry and novel proteomic tools. J. Mass Spectrom. 43, 1301–
1319 (2008)
Acknowledgments
The authors acknowledge financial supports from the Major
Program of National Natural Science Foundation of China
(no. 20732004), the National Natural Science Foundation of
China (no. 20972130 and no. 21075103), and startup
funding for J.Z. by Xiamen University. They thank
Professor G. M. Blackburn for helpful discussions and
suggestions.
24. Chu, I.K., Guo, X., Lau, T.C., Siu, K.W.M.: Sequencing of argentinated
peptides by means of electrospray tandem mass spectrometry. Anal.
Chem. 71, 2364–2372 (1999)
25. Lin, T., Payne, A.H., Glish, G.L.: Dissociation pathways of alkali-
cationized peptides: opportunities for C-terminal peptide sequencing. J.
Am. Soc. Mass Spectrom. 12, 497–504 (2001)
26. Lin, T., Glish, G.L.: C-terminal peptide sequencing via multistage mass
spectrometry. Anal. Chem. 70, 5162–5165 (1998)
27. Operti, L., Rabezzana, R.: Gas-phase ion thermochemistry in organo-
metallic systems. Mass Spectrom. Rev. 22, 407–428 (2003)
28. Bao, J.Y., Ai, H.W., Fu, H., Jiang, Y.Y., Zhao, Y.F., Huang, C.: Peptide
sequencing through N-terminal phosphorylation and electrospray ioniza-
tion mass spectrometry. J. Mass Spectrom. 40, 772–776 (2005)
29. Wang, F., Fu, H., Jiang, Y.Y., Zhao, Y.F.: A picomole-scale method for
rapid peptide sequencing through convenient and efficient N-terminal
phosphorylation and electrospray ionization mass spectrometry. J. Am.
Soc. Mass Spectrom. 17, 995–999 (2006)
30. Chen, J., Chen, Y., Jiang, Y., Fu, H., Zhao, Y.F.: Rearrangement of P–
N and P–O bonds in mass spectra of N-diisopropyloxyphosphoryl
amino acids/alcohols. Rapid Commun. Spectrom. 15, 1936–1940 (2001)
31. Chen, J., Chen, Y., Niu, Y.Y., Fu, H., Zhao, Y.F.: A carbonyl oxygen
migration in electrospray ionization mass spectrometry and its applica-
tion in differentiating α- and β-alanyl peptides. J. Mass Spectrom. 37,
934–939 (2002)
References
1. Zhao, Y.F., Cao, P.S.: Phosphoryl amino acids: common origin for
nucleic acids and protein. J. Biol. Phys. 20, 283–287 (1994)
2. Fu, H., Li, Z.Z., Zhao, Y.F., Tu, G.Z.: Oligomerization of N, O-Bis
(Trimethylsilyl)-α-amino acids into peptides mediated by o-phenylene
phosphorochloridate. J. Am. Chem. Soc. 121, 291–295 (1999)
3. Pascal, R., Boiteau, L., Commeyras, A.: From the prebiotic synthesis of
α-amino acids towards a primitive translation apparatus for the synthesis
of peptides. Top. Curr. Chem. 259, 69–122 (2005)
4. Cheng, C.M., Liu, X.H., Li, Y.M., Ma, Y., Tan, B., Wan, R., Zhao, Y.
F.: N-phosphoryl amino acids and biomolecular origins. Orig. Life Evol.
Biosph. 34, 455–464 (2004)
5. Landweber, L.F.: Testing ancient RNA-protein interactions. Proc. Natl.
Acad. Sci. U.S.A. 96, 11067–11068 (1999)
6. Zhou, W.H., Ju, Y., Zhao, Y.F., Wang, Q.G., Luo, G.A.: Simultaneous
formation of peptides and nucleotides from N-phosphothreonine. Orig.
Life Evol. Biosph. 26, 547–560 (1996)
7. Li, W., Ma, Y., Zhao, Y.F.: Penta-coordinated phosphorus structure
analysis on kinases. Sci. China Ser. B Chem. 47, 420–427 (2004)
8. Ni, F., Li, W., Li, Y.M., Zhao, Y.F.: Analysis of the phosphoryl transfer
mechanism of c-AMP dependent protein kinase (PKA) by penta-
coordinate phosphoric transition state theory. Curr. Protein Pept. Sci. 6,
437–442 (2005)
32. Chen, Y., Yin, Y.W., Sun, X.B., Liu, X.H., Wang, H.Y., Zhao, Y.F.: A
novel rearrangement in electrospray ionization mass spectrometry of
amino acids ester cyclohexyl phosphoramidates of AZT. J. Mass
Spectrom. 40, 636–641 (2005)
33. Ji, G.J., Xue, C.B., Zeng, J.N., Li, L.P., Chai, W.G., Zhao, Y.F.:
Synthesis of N-(Diisopropyloxyphosphoryl) amino acids and peptides.
Synthesis 6, 444–448 (1988)
34. Tang, G., Zhou, G.J., Ni, F., Hu, L.M., Zhao, Y.F.: 31P NMR and ESI-
MS studies on some intermediates of the peptide coupling reagents
triphenyl-chlorophosphoranes. Chin. Chem. Lett. 16, 385–388 (2005)

702
X. Gao, et al.: Isotope Technique to study the Interactions
35. Ulibarri, G., Choret, N., Bigg, D.C.H.: Activation of imidazolides using
methyl trifluoromethanesulfonate: a convenient method for the prepa-
ration of hindered esters and amides. Synthesis 11, 1286–1288 (1996)
36. Becke, A.D.: Density-functional thermochemistry. III. The role of exact
exchange. J. Chem. Phys. 98, 5648–5652 (1993)
37. Miehlich, B., Savin, A., Stoll, H., Preuss, H.: Results obtained with the
correlation energy density functionals of Becke and Lee, Yang, and
Parr. Chem. Phys. Lett. 157, 200–206 (1989)
38. Lee, C., Yang, W., Parr, G.: Development of the colle-salvetti
correlation-energy formula into a functional of the electron density.
Phys. Rev. B 37, 785–789 (1988)
39. Fukui, K.: Formulation of the reaction coordinate. J. Phys. Chem. 74,
4161–4163 (1970)
47. Farrugia, J.M., O’Hair, R.A.J.: Involvement of salt bridges in a novel
gas phase rearrangement of protonated arginine-containing dipeptides
which precedes fragmentation. Int. J. Mass Spectrom. 222, 229–242
(2003)
48. Forbes, M.W., Jockusch, R.A., Young, A.B., Harrison, A.G.: Fragmen-
tation of protonated dipeptides containing arginine. Effect of activation
method. J. Am. Soc. Mass Spectrom. 18, 1959–1966 (2007)
49. Qiang, L.M., Liu, R.Y., Xie, Y.L., Cao, S.X., Liao, X.C., Zhao, Y.F.: A
carbonyl oxygen migration of N-phosphoryl dipeptide methyl esters
containing β-alanine or γ-amino butyric acids in electrospray ionization
mass spectrometry. Int. J. Mass Spectrom. 272, 204–207 (2008)
50. Gao, X., Ni, F., Bao, J., Liu, Y., Xu, P.X., Zhao, Y.F.: Formation of
cyclic acylphosphoramidates in mass spectra of N-monoalkyloxyphos-
phoryl amino acids using electrospray ionization tandem mass spec-
trometry. J. Mass Spectrom. 45, 779–787 (2010)
40. Fukui, K.: The path of chemical reactions-the IRC approach. Acc.
Chem. Res. 14, 363–368 (1981)
41. Frisch, M.J., Trucks, G.W., Schlegel, H.B., Scuseria, G.E., Robb, M.A.,
Cheeseman, J.R., Montgomery Jr., J.A., Vreven, T., Kudin, K.N.,
Burant, J.C., Millam, J.M., Iyengar, S.S., Tomasi, J., Barone, V.,
Mennucci, B., Cossi, M., Scalmani, G., Rega, N., Petersson, G.A.,
Nakatsuji, H., Hada, M., Ehara, M., Toyota, K., Fukuda, R., Hasegawa,
J., Ishida, M., Nakajima, T., Honda, Y., Kitao, O., Nakai, H., Klene, M.,
Li, X., Knox, J.E., Hratchian, H.P., Cross, J.B., Bakken, V., Adamo, C.,
Jaramillo, J., Gomperts, R., Stratmann, R.E., Yazyev, O., Austin, A.J.,
Cammi, R., Pomelli, C., Ochterski, J.W., Ayala, P.Y., Morokuma, K.,
Voth, G.A., Salvador, P., Dannenberg, J.J., Zakrzewski, V.G., Dapprich,
S., Daniels, A.D., Strain, M.C., Farkas, O., Malick, D.K., Rabuck, A.D.,
Raghavachari, K., Foresman, J.B., Ortiz, J.V., Cui, Q., Baboul, A.G.,
Clifford, S., Cioslowski, J., Stefanov, B.B., Liu, G., Liashenko, A., Piskorz,
P., Komaromi, I., Martin, R.L., Fox, D.J., Keith, T., Al-Laham, M.A.,
Peng, C.Y., Nanayakkara, A., Challacombe, M., Gill, P.M.W., Johnson,
B., Chen, W., Wong, M.W., Gonzalez, C., Pople, J.A.: Gaussian 03,
revision E.01. Gaussian, Inc, Wallingford (2004)
51. Tang, X.J., Ens, W., Standing, K.G., Westmore, J.B.: Daughter ion
mass spectra from cationized molecules of small oligopeptides in a
reflecting time-of-flight mass spectrometer. Anal. Chem. 60, 1791–1799
(1988)
52. Chen, Y., Zhang, J.C., Chen, J., Cao, X.Y., Wang, J., Zhao, Y.F.:
Sensitivity improvement of amino acids by N-terminal diisopropylox-
yphosphorylation in electrospray ionization mass spectrometry. Rapid
Commun. Spectrom. 18, 469–473 (2004)
53. Abirami, S., Wong, C.C.L., Tsang, C.W., Ma, N.L.: Dissociation of
alkaliated alanine in the gas phase: the role of the metal cation. Chem.
Eur. J. 11, 5289–5301 (2005)
54. Sudha, R., Panda, M., Chandrasekhar, J., Balaram, P.: Structural effects
on the formation of proton and alkai metal ion adducts of apolar, neutral
peptides: electrospray ionization mass spectrometry and Ab Initio
theoretical studies. Chem. Eur. J. 8, 4980–4991 (2002)
55. Hoyau, S., Norrman, K., McMahon, T.B., Ohanessian, G.: A quanti-
tative basis for a scale of na affinities of organic and small molecules in
the gas phase. J. Am. Chem. Soc. 121, 8864–8875 (1999)
56. Ryzhov, V., Dunbar, R.C., Cerda, B.A., Wesdemiotis, C.: Cation-π
effects in the complexation of Na+ and K+ with Phe, Try, and Trp in the
gass phase. J. Am. Soc. Mass Spectrom. 11, 1037–1046 (2000)
57. Wang, P., Wesdemiotis, C., Kapota, C., Ohanessian, G.: The sodium
ion affinities of simple Di-, Tri-, and tetrapeptides. J. Am. Soc. Mass
Spectrom. 18, 541–552 (2007)
42. Ballard, K.D., Gaskell, S.J.: Intramolecular [18O] isotopic exchange in
the gas phase observed during the tandem mass spectrometric analysis
of peptides. J. Am. Chem. Soc. 114, 64–71 (1992)
43. Teesch, L.M., Adams, J.: Fragmentions of gas-phase complexes
between alkali metal ions and peptides: metal ion binding to carbonyl
oxygens and other neutral functional groups. J. Am. Chem. Soc. 113,
812–820 (1991)
44. Grese, R.P., Cerny, R.L., Gross, M.L.: Metal ion–peptide interactions in
the gas phase: a tandem mass spectrometry study of alkali metal
cationized peptides. J. Am. Chem. Soc. 111, 2835–2842 (1989)
45. Feng, W.Y., Gronert, S., Fletcher, K.A., Warres, A., Lebrilla, C.B.: The
mechanism of C-terminal fragmentations in alkali metal ion complexes
of peptides. Int. J. Mass Spectrom. 222, 117–134 (2003)
58. Feng, W.Y., Gronert, S., Lebrilla, C.B.: Lithium and sodium ion
binding energies of N-acetyl and N-glycyl amino acids. J. Am. Chem.
Soc. 121, 1365–1371 (1999)
59. Pingitore, F., Wesdemiotis, C.: Characterization of dipeptide isomers by
tandem mass spectrometry of their mono- versus dilithiated complexes.
Anal. Chem. 77, 1796–1806 (2005)
46. Bythell, B.J., Suhai, S., Somogyi, A., Paizs, B.: Proton-driven amide
bond-cleavage pathways of gas-phase peptide ions lacking mobile
protons. J. Am. Chem. Soc. 131, 14057–14065 (2009)
60. Zhang, J.M., Brodbelt, J.S.: Silver complexation and tandem mass
spectrometry for differentiation of isomeric flavonoid diglycosides.
Anal. Chem. 77, 1761–1770 (2005)