Characterization of amino acid-derived betaines by electrospray ionization tandem mass spectrometry

Article abstract of DOI:10.1002/jms.2029

Betaines belong to the naturally occurring osmoprotectants or compatible solutes present in a variety of plants, animals and microorganisms. In recent years, metabolomic techniques have been emerging as a fundamental tool for biologists because the constellation of these molecules and their relative proportions provide with information about the actual biochemical condition of a biological system. Therefore, identification and characterization of biologically important betaines are crucial, especially for metabolomic studies. Most of the natural betaines are derived from amino acids and related homologues. Although, theoretically, all the amino acids can be converted to corresponding betaines by simple methylation of the amine group, only a few of the amino acid-derived betaines were fully characterized in the literature. Here, we report a combined electrospray ionization tandem and high-resolution mass spectrometry study of all the betaines derived from amino acids, including the isomeric betaines. The decomposition pathway of protonated, sodiated and potassiated molecule ions that enable unambiguous characterization of the betaines including the isomeric betaines and overlapping ionic species of different betaines is distinctive. Copyright

Full text of DOI:10.1002/jms.2029

Research Article
Received: 21 October 2011
Revised: 14 November 2011
Accepted: 15 November 2011
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.2029
Characterization of amino acid-derived
betaines by electrospray ionization
tandem mass spectrometry
V. Naresh Chary,a Ch. Dinesh Kumar,a M. Vairamania,b and S. Prabhakara*
Betaines belong to the naturally occurring osmoprotectants or compatible solutes present in a variety of plants, animals and
microorganisms. In recent years, metabolomic techniques have been emerging as a fundamental tool for biologists because
the constellation of these molecules and their relative proportions provide with information about the actual biochemical
condition of a biological system. Therefore, identification and characterization of biologically important betaines are crucial,
especially for metabolomic studies. Most of the natural betaines are derived from amino acids and related homologues.
Although, theoretically, all the amino acids can be converted to corresponding betaines by simple methylation of the amine
group, only a few of the amino acid-derived betaines were fully characterized in the literature. Here, we report a combined
electrospray ionization tandem and high-resolution mass spectrometry study of all the betaines derived from amino acids,
including the isomeric betaines. The decomposition pathway of protonated, sodiated and potassiated molecule ions that
enable unambiguous characterization of the betaines including the isomeric betaines and overlapping ionic species of differ-
ent betaines is distinctive. Copyright © 2012 John Wiley & Sons, Ltd.
Keywords: betaines; electrospray ionization; protonated and cationized betaines; tandem mass spectrometry; high-resolution mass
spectrometry
spectroscopy that require synthetic standards and significant
purification prior to analysis; this data was usually supported
INTRODUCTION
Betaines are a class of naturally occurring zwitterionic compounds
that possess permanently positively charged quaternary
ammonium or phosphonium group without hydrogen atom and
negatively charged carboxyl group. Betaines are widely
by molecular weight information from mass spectrometry. The
high-pressure liquid chromatography- ultraviolet detection
of aliphatic betaines shows signals at lower wavelengths
(<210 nm);[11] their detection was found to be easy after converting
them as p-bromophenacyl esters that show ultraviolet absorption
max at 262 nm. Because of the permanent positive charge on the
quaternary ammonium moiety, mass spectrometric analysis has
been approached by desorption methods such as fast atom
bombardment (FAB), desorption chemical ionization and plasma
desorption (PD) mass spectrometry.[12–16]
The positive ion desorption chemical ionization mass spectra
of the betaines were dominated with protonated molecule ions
including a few fragment ions resulting from thermal loss of
one of the quaternary methyl groups,[12] but it had drawbacks
of reduced sensitivity and incomplete resolution of complex
mixtures of betaines. The FAB and PD-MS were successfully
applied to analyze the betaines where the major ions observed
were protonated and cluster ions.[13,14] The FAB-MS sensitivity
increased when the samples were derivatized to their n-propyl
or n-butyl esters prior to analysis,[15] and the spectra included
a
a
distributed in a variety of organisms, including animals, plants
and microorganisms.[1–5] Most of the natural betaines are derived
from amino acids in which the nitrogen atom is fully methylated.
The origins and chemistry of betaines have recently been
reviewed.[6] Physiologically, betaines act as either organic osmolytes
or catabolic source of methyl groups via transmethylation for use in
many biochemical pathways.[7] Betaines, as osmolytes, protect cells
from environmental stress such as low water, high salinity and
extreme temperatures.[8,9] In general, inorganic ions tend to
destabilize the folding of proteins, whereas betaines and other
compatible solutes have the opposite effect. Under stress
conditions, betaines play an important role in maintaining the
structural integrity of enzymes, membranes, hormones and other
cellular components. For example, human urine consists of several
betaines including glycine betaine and proline betaine, which
play osmoprotective role for the kidney.[10]
To the best of our knowledge, there were about 25 betaines
detected in plants, animals and aquatic species. Most of the
natural betaines detected belong to quaternary ammonium group
containing betaines, especially derived from amino acids and
related homologues (about 15). In view of the biological role of
betaines, their structural characterization is also equally important
to understand their intrinsic chemistry and function. The structures
of the betaines were determined mainly by proton nuclear
magnetic resonance and carbon-13 nuclear magnetic resonance
*
Correspondence to: S. Prabhakar, National Centre for Mass Spectrometry,
Indian Institute of Chemical Technology, Hyderabad–500 007, India.
E-mail: prabhakar@iict.res.in
a National Centre for Mass Spectrometry, Indian Institute of Chemical Technology,
Hyderabad 500 007, India
b School of Bio-Engineering, SRM University, Chennai 603 203, India
J. Mass. Spectrom. 2012, 47, 79–88
Copyright © 2012 John Wiley & Sons, Ltd.

V. Naresh Chary et al.
fragment ions typically associated with the loss of the alcohol
group. But such derivatization steps can be time-consuming
and may lead to poor recoveries. When betaine mixtures are
analyzed in PD-MS, the resulted ions are expected to overlap
because of formation of different ionic species for each betaine
(protonated, sodiated or potassiated ions). Wood et al. reported
the advantage of electrospray ionization (ESI)-MS over PD-MS[16]
where they applied ESI tandem mass spectrometry (MS/MS) to
discriminate the isomeric ions. Li et al.[17] demonstrated an liquid
chromatography-MS/MS method for separation of seven
betaines present in coral tissues using a pentfluorophenylpropyl
column by adjusting the acidity of the mobile phase, and this
method avoids analyte derivatization prior to analysis.
Mass spectrometry technique with high resolution and tandem
mass spectrometry capabilities is always advantageous over
other spectroscopic techniques to characterize molecules
especially with possibilities of isomeric compounds in a complex
mixture. It is interesting to note that most of the detected
betaines are methylated products of natural amino acids.
Although, theoretically, all the natural amino acids can be
converted to betaines by biochemical methylation in cell or
tissues of both animal and plant sources, to date, only seven of
the betaines derived from amino acids were characterized by
ESI-MS/MS. Because there is a possibility that such amino acid-
based betaines can be encountered in future metabolomic
studies, it is essential to characterize all the amino acid-derived
betaines. This prompted us for synthesis and characterization of
all the betaines derived from natural amino acids and other
isomeric betaines by ESI-MS/MS. Naturally, the cell or tissue
extracts contain high concentrations of sodium or potassium
ions, and the direct ESI-MS analysis of these samples produces
[M+Na]+ and [M+K]+ ions in addition to [M+H]+. Under such
conditions, the m/z value of an ion species of one betaine may
overlap with that of another species from another betaine; for
example, m/z value of [M+Na]+ of serine betaine matches with
[M+K]+ of alanine betaine. Therefore, the analysis of betaines
has two important problems, i.e. isomeric betaines and
overlapping m/z values for certain species. Here, we focused on
characterization of all the betaines by MS/MS and high-resolution
mass spectrometry (HRMS) analyses of all the aforementioned
ionic species expected to be formed under ESI conditions.
vessel was protected from light using aluminum foil, and the reac-
tion was allowed to stir for 16 h at ambient temperature. The
reaction mixture was then evaporated to dryness using rotary
evaporator (bath temperature, 40 ꢀC), and the residue was parti-
tioned using equal volumes of chloroform and distilled water
(5ml each). The upper aqueous layer, which contains betaines,
was collected and stored in a refrigerator till used for mass spec-
trometry analysis. The positive ion ESI-MS analysis of the reaction
products showed only fully methylated products (betaines), and
the partial methylated products (mass value with 14 or 28 u less
than the corresponding betaine) were absent under the used reac-
tion conditions. The methylation at carboxyl group (–COOH) is
prohibited by the use of the base. However, imidazole and the –
SH (but not –OH) groups are methylated. In the case of lysine, only
the e-NH2 is methylated but not the a-NH2, by the fact that the
spectrum of trimetyllysine (24) matches with the reported one.
Mass spectral analysis of all the betaines was performed on a
quadrupole time-of-flight mass spectrometer (Q-Star XL, Applied
Biosystems, USA) under the positive ion ESI conditions using
Analyst software. The betaine samples were diluted appropriately
with methanol before introducing into the source through a
flow injection where methanol was used as the mobile phase at
a flow rate of 30 ml/min. When necessary, the solutions were
acidified with dilute HCl to increase the signals of protonated
molecule ions, [M+H]+. Collision induced dissociation (CID)
spectra were obtained by selecting the mass of ion by the
quadrupole, colliding with nitrogen in the collision cell, followed
by detecting the product ions by the time-of-flight analyzer. The
typical positive ESI conditions were capillary voltage 5.00 kV,
declustering potential 60 V, focusing potential 220 V and
resolution 10 000 (full width at half maximum). All the CID
spectra were recorded at different collision energies (CE = 15,
20, 25, 30 and 35 eV). The abundance precursors and product
ions were varied with type of ion species and structure;
hence, the spectra recorded at suitable CE (where the spectrum
includes maximum product ions) were considered (the spectral
data along with the CE value used was given in the text). For
isomeric compounds, the spectra obtained at the same collision
energy were considered.
The multistage mass spectrometric experiments were
performed with the ion trap mass spectrometer (LCQ Advantage
MAX, Thermo Finnigan, San Jose, CA, USA) equipped with an ESI
source. The data acquisition was under the control of Xcalibur
software (Thermo Finnigan). The typical source conditions were
spary voltage 4 kV, capillary voltage 15–20 V, heated capillary
temperature 200 ꢀC, tube lens offset voltage 20 V and sheath
gas (N2) flow rate 15 arbitrary units, and helium was used as
the damping gas. For the ion trap mass analyzer, the automatic
gain control settings were 2 Â 107 counts for full-scan mass
spectra and product ion mass spectra with a maximum ion injec-
tion time of 200 ms.
EXPERIMENTAL
All the amino acids were obtained from Sigma–Aldrich (St. Louis,
MO, U.S.A). Methyl iodide was purchased from Spectrochem Pvt.
Ltd. (Mumbai, India). All the solvents were of high-pressure liquid
chromatography grade and were purchased from Merck
(Mumbai, India). All the studied betaines were synthesized by
the reaction corresponding amino acid with methyl iodide by
following a known procedure (Scheme 1).[17] Briefly, the amino acid
(1mmol) in 20 ml of 1 : 1 (v: v) methanol and water plus 1 g of
KHCO3 or NaHCO3 was mixed with CH3I (16 mmol). The reaction
All the precursor ion spectra and a few product ion spectra were
performed using a Quattro LC triple quadrupole mass spectrometer
(Micromass, Manchester, UK) using MassLynx software. Samples
Scheme 1. Synthetic procedure for betaines.
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ESI-MS/MS of amino acid betaines
were introduced into the source using a direct infusion pump (Har-
vard Apparatus, Holliston, MA, USA) at a flow rate of 15 ml/min. The
capillary and cone voltages were kept at 3.50 kV and 35 V, respec-
tively. Nitrogen was used as the desolvation gas. The source and
desolvation temperatures were kept at 150ꢀC and 250 ꢀC, respec-
tively. Argon was used as the collision gas, and the collision gas cell
was kept at a pressure of 4.0 Â 10À4 mbar. The collision energy
used was 25 eV.
showed dominant [M+K]+ ions, and those synthesized in the
presence of NaHCO3 showed abundant [M+Na]+ ions. In order to
obtain prevailing [M+H]+ ion, the samples were acidified with dilute
HCl prior to their introduction into the source. The aforementioned
solution conditions were maintained accordingly for successful
generation of the [M+H]+, [M+Na]+ or [M+K]+ ions from all
the betaines, and these ionic species were further subjected for
MS/MS experiments. The MS/MS spectra together with high-
resolution data for each product ion were used for characterization
as well as isomeric differentiation of the studied betaines. The CID
mass spectra recorded for the ionic species from all the betaines
(1–28, Scheme 2) are tabulated in Table 1. Although major
product ions include retention of quaternary ammonium group,
there were other product ions specific to the substituent (–R) of
the amino acids. For convenience, the studied compounds
RESULTS AND DISCUSSIONS
Characterization of betaines is important to understand the
metabolomic picture of a cell or tissue. The positive ion ESI mass
spectra of the betaines synthesized in the presence of KHCO3
Scheme 2. Chemical structures of the studied betaines (1–28).
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V. Naresh Chary et al.
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ESI-MS/MS of amino acid betaines
were divided into five parts that include aliphatic (1–12), aromatic
(13–16), acidic (17–18), amidic or basic (19–24) and cyclic (25–28)
amino acid betaines.
the ions m/z 58 and 59 are negligible (<1%). The spectrum of 4 also
included the product ions at m/z 43 and 45 that resulted from the
further fragmentation of m/z 87 as supported by the CID spectrum
of m/z 87. The 2-amino isobutyric acid betaine (5) is structurally
similar to 2 having an extra methyl group on a-carbon. Like in 2,
the spectrum of 5 showed the product ions at m/z 58, 59 and 60.
The ion at m/z 60, however, is found to be as abundant as m/z 58
and 59 in 5 because the possibility of b-hydrogen migration is
increased by the presence of two methyl groups at a-carbon
(six b-hydrogens). The [MH–N(CH3)3]+ ion (m/z 87) that is dominant
in 4 (100%) is low abundant in 5 (2%).
The compounds 6–8 have an alkyl substituent on a-carbon like
in 2, but they differ in the structure of the alkyl group attached.
As expected, the product ions because of N–C bond cleavage
are dominant in the spectra. The ion at m/z 60 is selectively
dominant in 6 and 8 when compared with 7, which clearly reveals
that the tertiary hydrogen (6 and 8) preferentially migrates to
nitrogen when compared with secondary hydrogen (7). In the
present study, the [M+H]+ ion 6 is isobaric with that of
hydroxyproline betaine (26), but it is possible to discriminate
these species easily by their specific CID spectra (Fig. 1). The
isomeric compounds 7 and 8 can also be discriminated by the
CID spectra in which the ions at m/z 58 and 59 are dominant in
7, whereas the ion at m/z 60 is dominant in 8. The [MH–N(CH3)3]+
ion is not present in 6–8, but further loss of H2O and (H2O+CO)
are found in the spectra. In addition, the compound 6 showed
[MH–NC3H9–CO]+ ion, whereas the compounds 7 and 8 showed
[MH–NC3H9–CH2CO]+ ion. The aforementioned discussed ions
resulted from [MH–NC3H9]+ ion are relatively more abundant in
8 than in 7.
CID OF [M+H]+ IONS
Aliphatic amino acid betaines (1–12)
The CID spectra of [M+H]+ ions from 1 to 8 recorded at CE 25 are
given in Table 1. The smallest betaine derived from amino acids is
glycine betaine (1), which exclude presence of substituents other
than carboxyl and trimethylammonium group. The general
fragmentation pattern of [M+H]+ ions of the acyclic betaines is
shown in Scheme 3. The [M+H]+ ion CID spectrum of 1 that
matches well with the reported spectrum showed the product
ions at m/z 59, 58 and 42 corresponding to C3H9N+., C3H8N+
and C2H4N+.[18] The homolytic cleavage of N–C bond accounts
for the formation of m/z 59. The ion at m/z 42 is formed from
m/z 58 by the loss of CH4 as confirmed by its CID spectrum in
which the ion m/z 42 is the dominant product ion. The spectrum
of alanine betaine (2), with an additional methyl substituent,
resembles with that of 1, except the presence of a low abundant
peak at m/z 60 (1%). The ion m/z 60 may be formed by the
hydrogen transfer from methyl group to nitrogen. The b-alanine
betaine (3), although isomeric to that of 2, resulted in a distinct
spectrum. The spectrum of 3 includes the ions m/z 58, 59 and
60 as that of 2, but their relative abundances are varied when
compared with those from 2. The higher abundance of m/z 60
in 3 compared with 2 may be because of the feasible migration
of secondary hydrogen in 3 instead of primary hydrogen in 2.
Apart from these, 3 showed specific ions at m/z 73, 55 and 45.
The ion at m/z 73 resulted from the direct loss of a neutral
N(CH3)3 from [M+H]+ ion, and the ions m/z 55 and 45 are the
fragment ions from m/z 73 formed by the loss of H2O and CO,
respectively. This is also supported by the CID spectrum of the
ion m/z 73 that showed the ion at m/z 55 as the major product
ion in addition to the ion m/z 45.
The compounds 9–12 have a nucleophilic group (–OH, –SH or
–SCH3) attached to alkyl side chain. As expected, the product ions
because of N–C bond cleavage [m/z 58, 59, 60 and (MH–NC3H9)+]
are found in the spectra. Besides these common ions, the spectra
of 9 and 10 include a specific ion at m/z 118 corresponding to the
loss of CH2O and C2H4O, respectively, which may be formed from
[M+H]+ ion through a McLafferty type rearrangement involving
the migration of the hydroxyl hydrogen to the carbonyl group.
The CID spectrum of the ion at m/z 118 from 9 and 10 showed
the ions m/z 103 and 100 corresponding to a loss of CH3 and
H2O, respectively (spectrum from 10 given in Fig. 2). This
spectrum is very much different from the [M+H]+ ion of the
The betaines 4 and 5 are isomeric to each other, but their CID
spectra were very much distinctive. The g-amino butyric acid
betaine (4) is homologous to 3, and as in 3, the CID spectrum did
show abundant product ion corresponding to [MH–N(CH3)3]+
(m/z 87). But in contrast to 3, the ion m/z 60 is dominant in 4, and
Scheme 3. General fragmentation pattern of [M+H]+ ion of acyclic betaines (1–24).
J. Mass. Spectrom. 2012, 47, 79–88
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V. Naresh Chary et al.
(a)
OH
N
O
[M+H]+
(b)
OH
HO
N
O
[M+H]+
Figure 1. Electrospray ionization tandem mass spectrometry spectra of [M+H]+ ion of (a) valine betaine (6) and (b) hydroxyproline betaine (26).
Figure 2. Electrospray ionization tandem mass spectrometry spectrum of [M+H–C2H4O]+ m/z 118 from threonine betaine (10).
glycine betaine (m/z 118). The CID spectrum of 9 shows an ion at
m/z 86 as well, whose elemental composition matches with
C5H12N. Thus, the ion m/z 86 might have been formed by the loss
of (H2O+CO2) from [M+H]+ ion. Similar type of ion, as expected,
appears at m/z 100 for 10. The compounds 11 and 12 having a
sulfur atom in the side chain showed dominant [MH–NC3H9]+
ion as a base peak at m/z 119 and 133, respectively, whereas this
ion is low abundant in 9 and 10 with an oxygen in the side chain.
The dominant loss of NC3H9 group in 11 and 12 may be because
of SN2-type displacement through
a neighboring group
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ESI-MS/MS of amino acid betaines
participation of sulfur. Interestingly, the 11 and 12 showed the
ion m/z 60 as an abundant ion leaving ions m/z 58 and 59 absent
or low abundant, as similarly found in 4.
(22) specifically showed [MH–CO2]+ in addition to [MH–NC3H9]+
and m/z 60. The spectra of 21 and 22 also included loss of CO
and CO2 from [MH–NC3H9]+. The [MH–NC3H9–CO2]+ ion showed
further loss of a HCN and CH3CN from 21 and 22, respectively,
by the involvement of the imidazole ring. The arginine betaine
(23) showed abundant [MH–NC3H9]+ and subsequent loss of a
CO2. Direct loss of CO2 from [M+H]+ is also present but low
abundant. Similarly, the expected ion at m/z 60 is also low
abundant. Moreover, the compound 23 showed a specific loss
of 42 u from [MH–NC3H9]+, and the HRMS data confirmed that
the loss of 42 u corresponds to the loss of HN=C=H;NH (42 u)
resulted from the guanidine group. The trimethyllysine (24) shows
the ions at m/z 130, 112, 84 (base peak) and 60 corresponding
to [MH–NC3H9]+, [MH–NC3H9–H2O]+, [MH–NC3H9–(H2O+CO)]+
Aromatic amino acid betaines (13–16)
These betaines are derived from the aromatic amino acids in which
an aromatic group (phenyl, indole or imidazole) is present in the
side chain. The compounds 13 and 14 show the ions at m/z 58,
59 and 60, whereas the compounds 15 and 16 show exclusively
one of them, i.e. m/z 60. Even though a b-hydrogen is absent, the
compound 13 shows the ion at m/z 60, wherein the hydrogen from
phenyl group may be transferred to nitrogen. All the compounds
(13–16) show abundant [MH–NC3H9]+ ion may be because the
resulted product ion is well stabilized by the aromatic ring. The
[MH–NC3H9]+ ion (m/z 135) from 13 further showed a consecutive
loss of two CO molecules to result in ions at m/z 107 and 79
(benzonium cation) because of the presence of a phenyl group in
the side chain. This fragmentation process was confirmed by
multistage mass spectrometric experiments and high-resolution
data. Similar fragmentation is also observed for the [MH–NC3H9]+
ion (m/z 149) from 14, where the loss of CH2CO is seen at m/z 107
because of the presence of Ph–CH2 and further loss of a CO resulted
in a benzonium ion C6H+7 (m/z 79). Additionally, loss of CO2, H2O
and (H2O+CO) is observed from [MH–NC3H9]+ of 14. The CID
spectrum of tyrosine betaine (15) showed similar fragmentation
as that of 14, where the characteristic product ion mass values
are shifted by +16 u because of an extra hydroxyl group attached
on the phenyl group in 15. Similarly, tryptophan betaine (16) showed
[MH–NC3H9]+ ion and subsequent loss of H2O and CH2CO from it.
and [HN(CH3)3]+, respectively. The spectrum of 24 is matching well
[19]
the reported spectrum.
Trimethyllysine (24) is isobaric with
glutamine betaine (20), but both of them show distinctive CID
spectra by which they can be discriminated one from another.
Cyclic amino acid betaines (25–28)
The CID spectra of [M+H]+ from all the cyclic amino acid betaines
(25–28) showed the ion m/z 58 corresponding to [H2C=N(CH3)2]+
as observed in other betaines, but in cyclic betaines, this ion
results through a different fragmentation pathway. The ion at
m/z 102 is consistently present in all the cyclic betaines, whose
elemental composition matching with C4H8NO2, irrespective of
the ring size and substituent on the ring. It suggests that all the
cyclic betaines result in a common structure keeping nitrogen
and –COOH functionality intact. Therefore, the ion m/z 102 might
be formed by the loss of C3H6, C3H6O, C2H4S and C4H8 from the
[M+H]+ ion of 25, 26, 27 and 28, respectively. Besides, all the
cyclic amino acid betaines showed [MH–HCOOH]+ ion.
Acidic amino acid betaines (17 and 18)
The CID spectrum of aspartic acid betaine (17) is similar to that of
2 containing the major product ions m/z 58 and 59, but the
spectrum of 17 shows additional low abundant ions (<1%)
corresponding to the loss of H2O, HCOOH and (NC3H9+H2O) from
[M+H]+ ion because of the presence of an additional –COOH
group. The glutamic acid betaine (18), which consists of an extra
methylene group compared with 17, showed the ion at m/z 60 as
the base peak leaving m/z 58 and 59 as low abundant ions. The
spectrum of 18 also showed [MH–H2O]+, [MH–NC3H9]+ and loss
of H2O, CO and HCOOH from [MH–NC3H9]+.
The proline betaine (25) showed an abundant product ion at
m/z 84 as reported by Wood et al. They proposed that this ion
was formed by the loss of (C3H6+H2O) from the [M+H]+ ion but
not supported by HRMS and MS/MS data. In the present study,
however, the high-resolution data showed two peaks for the
ion m/z 84, i.e. 84.0817 (99%) and 84.0446 (1%), where the
elemental composition of the dominant ion matches to the loss
of C2H4O2 (probably CH3COOH) from [M+H]+ and that of low
abundant ion with the loss of (C3H6+H2O) from [M+H]+. The
MS/MS of m/z 102 from 25 showed only m/z 84.0446 as one of
the product ion but not m/z 84.0817, which confirms that
84.0817 is not resulting from m/z 102. The precursor ion
spectrum of the ion m/z 84 showed m/z 144 (protonated
molecule) as the major precursor, which confirms that the ion
at m/z 84 is forming directly from the [M+H]+ ion by the loss of
C2H4O2. Wood et al. showed that the ion at m/z 84 was shifted
to m/z 90 in the d6-proline betaine that can only be explained
retaining two methyl groups on nitrogen intact. It is difficult to
understand the actual mechanism involved for the formation of
m/z 84 from 25; however, a plausible mechanism is shown in
Scheme 4. In addition, the ions m/z 72 and 70 corresponding to
the loss of (CH2=CH–COOH) and (HCOOH+C2H4) from the [M
+H]+ ion, respectively, are also observed for 25. Hydroxyproline
betaine (26) is a hydroxy derivative of proline betaine (25);
consequently, the characteristic product ions found in 25 are
shifted by +16 u in 26. The typical ion m/z 84 in 25 is shifted to
m/z 100 in 26, which further confirms the loss of (C2H4O2) from
[M+H]+. The [MH–(CH2=CH–COOH)]+ ion (m/z 88) is found to be
the dominant ion in 26.
Amidic and basic amino acid-derived betaines (19–24)
The carboxyl group on b-carbon in 17 is changed to amide group
in 19; hence, the spectrum of 19 showed dominant loss of NH3
from [M+H]+ as similar to the H2O loss found in 17. In addition,
the spectrum of 19 showed H2O loss also because of the
presence of a –COOH group as well. A specific loss of CO2 is
found from the [M+H]+ ion 19, and the loss of CO2 is also found
from [MH–NC3H9]+ ion. Interestingly, the ion at m/z 60 is also
abundant in 19, but it is absent in 17. The specific behavior of
19 when compared with 17 demonstrates a special role of
amide group in 19. Addition of an extra methyl group in the
glutamine betaine (20) changed the overall fragmentation. The
compound 20 showed a low abundant [MH–NH3]+ and a
dominant [MH–NC3H9]+ along with abundant ion at m/z 60. The
[MH–NC3H9]+ ion from 20 further resulted in characteristic
fragments corresponding to the loss of CO, HCONH2 and HCOOH.
Like in 19, the histidine betaine (21) and its N-methyl derivative
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V. Naresh Chary et al.
Scheme 4. The plausible mechanism for loss of C2H4O2 in proline betaine (25).
amino acid. Primarily, the ions at m/z 58, 59 and/or 60 reflect
the presence of quaternary ammonium ion, where the m/z 58
and 59 are abundant in all aliphatic amino acids. The cyclic
betaines selectively show m/z 58, whereas the m/z 60 is
dominant where a b-hydrogen to the quaternary ammonium
group is present and also in aromatic amino acid betaines. The
next significant ion is the loss of trimethyl amine group, which
is dominant in b-amino acid and g-amino acid betaines as well
as in heteroatom (S)-containing aromatic amino acid (through
SN2-type displacement). The side chain functional groups are
generally reflected in the further decomposition of [MH–N(CH3)3]+
ion. In a few cases, direct loss of specific fragment ions, diagnostic
to the side chain, is also observed.
Scheme 5. The plausible mechanism for loss of H2O in pipecolic acid
(27) and subsequent fragmentation.
The CID spectrum of pipecolic acid betaine (27, MH+ = m/z 158)
showed all the expected fragment ions, m/z 58, 102,
[MH–HCOOH]+ (m/z 112) and [MH–C2H4O2]+ (m/z 98). Although
it is low abundant, the [MH–H2O]+ (m/z 140) is specifically present
in 27. Further loss of C2H7N and (C2H7N+CO) from m/z 140 is also
found, as confirmed by HRMS and MS/MS of m/z 140. None of
these product ions are present in other cyclic betaines studied.
The H2O loss is special in 27, which may be possible involving
the hydrogen present in d-position with respect to the carbonyl
group through a boat conformation (Scheme 5). The resulted
structure for m/z 140 explains its further fragmentation as
mentioned previously. Thioproline betaine (28) is structurally
similar to proline betaine, where one of the methylene groups
in the five-membered ring is replaced with sulfur. The CID
spectrum of [M+H]+ ion of 28 showed the ion at m/z 58 and
[MH–C2H4S]+ (m/z 102); however, the expected C2H4O2 loss also
matches with the nominal mass m/z 102. But, the HRMS data
confirmed that the ion at m/z 102 exclusively corresponds to
[MH–C2H4S]+ but not [MH–C2H4O2]+. Apart from these, the
spectrum of 28 showed a dominant product ion at m/z 119 (base
peak) corresponding to a loss of 43 u from [M+H]+ ion. There are
three possible species for 43 u, i.e. two radicals (CH3CO and C3H7)
or a neutral molecule (NC2H5, possibly H2C=N–CH3); however, the
accurate mass value (m/z 119.0166) obtained from HRMS data
confirmed that it is the loss of NC2H5. The ions at m/z 77 and
73 are also seen in the spectra that are formed by further
fragmentation of the ion at m/z 119 by the loss of CH2CO and
CH2S, respectively. Similarly, the product ion at m/z 116 can be
expected as HCOOH loss, as seen in other compounds in this
group, but accurate mass value (116.0715) reveals that it is the
loss of CH2S and not of HCOOH.
CID OF [M+Na]+ AND [M+K]+ IONS
The betaines readily produce [M+Na]+ and [M+K]+ ions in the
presence of Na+ and K+ ions, respectively. Sometimes, the nominal
masses of these cationized species can be isomeric or isobaric with
the other cationized or protonated ions of betaines; these
overlapping ions have to be characterized unambiguously when a
mixture of betaines is present in a sample. The CID spectra of
[M+Na]+ and [M+K]+ ions from 1 to 28, recorded at appropriate CE
value, are summarized in Table 1. The CID spectra of all the
[M+Na]+ and [M+K]+ ions characteristically showed Na+ (m/z 23)
and K+ (m/z 39), respectively, by which it is easy confirm whether
the selected ion is sodiated or potassiated. The CID spectra of the
cationized species (Na+ and K+) from all the betaines containing
quaternary trimethyl amine group (1–24, except 9 and 10) showed
the loss of a neutral trimethylamine (NC3H9) by cleavage of the N–C
bond. Further loss of a CO from [(M+Na)–NC3H9]+ is found almost in
all betaines except 3, 4, 19 and 24, whereas similar loss of a CO from
[(M+K)–NC3H9]+ is only observed in a limited number of betaines
(2, 5, 11, 12, 14–16 and 19–22). Direct loss of a CO2 is found from
both [M+Na]+ and [M+K]+ ions, but it is selectively observed in
1, 12, aromatic amino acid betaines (13, 14 and 15), histidine
betaines (21 and 22) and cyclic betaines (25 and 27). The loss of
(NC3H9+CO2) is also seen from [M+Na]+ (13, 14, 17, 18 and 20)
and [M+K]+ ions (17, 19, 21 and 22) of a few betaines. Like in the
case of [M+H]+ ions, the ions m/z 58 and 60 are observed in the
CID of cationized species. The ion at m/z 58 is consistently found
in the decomposition of [M+Na]+ from all the cyclic betaines
(25–28) and 13, whereas in the case of [M+K]+, it is observed only
in 28. Similarly, the ion at m/z 60 resulted from the [M+Na]+ of 5,
6, 8, 15, 17 and 18, but this ion is absent in the CID spectra of
[M+K]+ ions. Apart from these, the ion at m/z 82 corresponding to
[NC3H9+Na]+ (as confirmed by HRMS data) is seen in 6–9, 14, 15
and 23. As expected, the ion at m/z 82 is shifted by +16 u in the case
Thus, the decomposition of [M+H]+ ions of the amino acid-
derived betaines shows a few common ions to reflect the betaine
structure as well as specific ions related to the side chain of
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J. Mass. Spectrom. 2012, 47, 79–88

ESI-MS/MS of amino acid betaines
the loss of (NC3H9+NH3) from [M+Na]+ ions. The compound 23
has a guanidine functional group, consequently showed the spe-
cific product ions corresponding to the loss of (NC3H9+CN2H2)
and (NC3H9+CN3H5) from [M+Na]+ and [M+K]+ ions. The com-
pound 26 showed a loss of 72u from both cationized species that
may correspond to the loss of (CO+C2H4O) involving hydroxyl
group of pyrrolidinium ring. The [M+Na]+ of 28 specifically showed
the loss of C2H4S that appeared at m/z 124. In the present study,
there are a few sets of compounds that result in isobaric ions, i.e.
the nominal m/z value of the sodiated species of one betaine
matches with potassiated species of another betaine. These iso-
baric ions, however, can easily be discriminated as their CID spectra
of distinct from one to another. The CID spectra of [M+Na]+ of 9 and
[M+K]+ of 2 are given in Fig. 3 as an example.
(a)
[M+K]+
CONCLUSIONS
(b)
The ESI-MS/MS analysis of all the amino acid-derived betaines
showed isomer or structure selective fragmentation that enabled
successful characterization of one betaine with another. The het-
erolytic cleavage of C–N bond is the key fragmentation observed
in the CID of all protanted and cationized (Na+ and K+) species of
the betaines. This cleavage produced the ions m/z 58, 59, 60 and
other structure indicative fragment ions depending on the
presence of other functional groups. The acyclic betaines having
a b-hydrogen showed the ion at m/z 60 with an exception of
phenylglycine betaine. The isomeric betaines and other m/z
overlapping species were successfully discriminated. The iso-
meric betaines showed clear-cut differences in their CID spectra.
Similarly, the nature of amino acid (aliphatic, aromatic, acidic and
basic) is also very much reflected in the fragmentation of their
protonated ([M+H]+ CID) species than the cationized species.
The isobaric ions that are possible, when present in a mixture,
can also be distinguished by the accurate mass values as well
as their distinct CID spectra. The isobaric ions are also possible
from same ion species (protonated, sodiated or potassiated) from
different betaines having different elemental composition, which
can also be differentiated by their HRMS data and CID spectra.
This data is very much useful for any future metabolomic studies,
especially in our ongoing metabolomic projects on milk, eggs
and other plant sources. Besides, further study on methyl esters
of betaines and partial methylated products of amino acids is also
in progress.
[M+Na]+
Figure 3. Electrospray ionization tandem mass spectrometry spectra of
(a) [M+K]+ ion of alanine betaine (2) and (b) [M+Na]+ ion of serine betaine (9).
of [M+K]+ ions; however, the [NC3H9+K]+ (m/z 98) ion is found only
in 6–9. The [CO2+Na]+ (m/z 67) ion is observed in the CID spectra of
[M+Na]+ ions from some of the betaines (7, 8, 15 and 16), but the
corresponding [CO2+K]+ ion is absent in the case of [M+K]+ ions.
In addition to the aforementioned discussed ions, the betaines
showed specific product ions reflecting the functional group
present in the substituent at a-carbon of the amino acids.
Like in the CID of [M+H]+ ions, the CID of cationized species
(Na+ and K+) of 9 and 10 shows the loss of CH2O and C2H4O,
respectively, because of McLafferty rearrangement. A subsequent
loss of CO2 from this ion is also observed. Apart from these,
the compounds 9 and 10 showed the loss of (CO2+H2O) from
[M+Na]+ and [M+K]+ ions. The compound 9 shows an ion at
m/z 86 in the CID spectra of [M+Na]+ and [M+K]+ corresponding
to the elemental composition C5H12N, which confirms that cation
is attached to neutral counterpart. The ion m/z 86 is shifted by
+14 u (m/z 100) in 10 in both the cationized species, and the ele-
mental composition of this ion corresponds to C6H14N. These data
suggests that these ions resulted from the direct loss of (OH,
COOX), where X = Na or K from the respective cationized species.
The compound 11 showed the loss of (NC3H9+CH2S) from the CID
of both cationized species (Na+ and K+) because of the presence
of –S–CH3 functional group. Although the same process is expected
in 12, the corresponding ion is absent; instead, it showed the loss
of (NC3H9+CO2). The compound 19 characteristically showed
Acknowledgements
The authors thank the Director, Indian Institute of Chemical Tech-
nology (IICT) for the facilities and encouragement and K. Srinivas,
Senior Scientist, IICT for his valuable discussions on the synthesis
of betaines. The authors V.N.C. and Ch.D.K. thank the Council of
Scientific and Industrial Research, New Delhi for Senior Research
Fellowships.
REFERENCES
[1] M. Adrian romero, S. J. Wilson, G. Blunden, M. H. Yang, A. Carabot
Cuervo, K. Bashir Ahmed. Betaines in coastal plants. Biochem. Syst.
Ecol. 1998, 26, 535.
[2] F. J. Zwart de, S. Slow, R. J. Payne, M. Lever, P. M. George, J. A.
Gerrard, S. T. Chambers. Glycine betaine and glycine betaine
analogues in common foods. Food Chem. 2003, 83, 197.
J. Mass. Spectrom. 2012, 47, 79–88
Copyright © 2012 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jms

V. Naresh Chary et al.
[3] G. Blunden, M. H. Yang, Z. X. Yuan, B. E. Smith, A. V. Patel, J. A.
Cegarra, I. Mathe Jr., G. Janicsak. Betaine distribution in the Labiatae.
Biochem. Syst. Ecol. 1996, 24, 71.
Characterization by Fast Atom Bombardment and Desorption
Chemical Ionization Mass Spectrometry. Plant Physiol. 1991,
96, 892.
[4] G. Blunden, A. V. Patel, N. Armstrong. Betaine distribution in the
Scrophulariaceae and some previously included families. Biochem.
Syst. Ecol. 2003, 31, 359.
[13] G. Blunden, S. M. Gordon, T. A. Crabb, O. G. Roch, M. G. Rowan, B.
Wood. NMR spectra of betaines from marine algae. Magn. Reson.
Chem. 1986, 24, 965.
[5] G. Blunden, A. V. Patel, N. Armstrong, M. A. Romero, P. Melendez.
Betaine distribution in angiosperms. Biochem. Syst. Ecol. 2005, 33,
904.
[6] S. T. Chambers, M. Lever. Betaines and urinary tract infections.
Nephron 1996, 74, 1.
[7] M. Lever, S. Slow. The clinical significance of betaine, an osmolyte
with a key role in methyl group metabolism. Clin. Biochem. 2010,
43, 732.
[14] C. C. Bonham, K. V. Wood, W. J. Yang, A. Nadolska-Orczyk, Y. Samaras, D.
A. Gage, J. Poupart, M. Burnet, A. D. Hanson, D. Rhodes. Identification of
quaternary ammonium and tertiary sulfonium compounds by plasma
desorption mass spectrometry. J. Mass Spectrom. 1995, 30, 1187.
[15] D. Rhodes, P. J. Rich, A. C. Myers, C. C. Reuter, G. C. Jamieson.
Determination of betaines by fast atom bombardment mass Spec-
trometry: identification of glycine betaine deficient genotypes Of
Zea mays. Plant Physiol. 1987, 84, 781.
[8] E. S. Courtenay, M. W. Capp, C. F. Anderson, M. T. Record Jr. Vapor
pressure osmometry studies of osmolyte-protein interactions:
implications for the action of osmoprotectants in vivo and for the
interpretation of “osmotic stress” experiments in vitro. Biochemistry
2000, 39, 4455.
[16] K. V. Wood, C. C. Bonham, D. Miles, A. P. Rothewell, G. Peel, B. C.
Wood, D. Rhodes. Characterization of betaines using electrospray
MS/MS. Phytochemistry 2002, 59, 759.
[17] C. Li, R. W. Hill, A. D. Jones. Determination of betaine metabolites
and dimethylsulfoniopropionate in coral tissues using liquid chro-
matography–time-of-flight mass spectrometry and stable iso-
[9] M. B. Burg, J. D. Ferraris. Intracellular organic osmolytes: function and
regulation. J. Biol. Chem. 2008, 283, 7309.
tope-labeled internal standards. J. Chromatogr.
878, 1809.
[18] J. A. Wyer, L. Feketeova, S. B. Nielsen, R. A. J. O’ Hair. Gas phase
fragmentation of protaonated betaine and its clusters. Phys. Chem.
Chem. Phys. 2009, 11, 8752.
[19] P. Y. Iris Shek, J. Zhao, Y ke, K. W. Michael Siu, A. C. Hopkinson.
Fragmentations of protonated arginine,lysine and their methylated
derivatives; concomitant losses of carbon monoxide or carbon
dioxide and an amine. J. Phys. Chem. A 2006, 110, 8282.
B
2010,
[10] S. T. Chambers, C. M. Kunin. Isolation of glycine betaine and proline
betaine from human urine. Assessment of their role as osmoprotective
agents for bacteria and the kidney. J. Clin. Invest. 1987, 79, 731.
[11] A. Zamarreno, R. G. Cantera, J. M. Garcia-Mina. Extraction and
determination of glycine betaine in liquid fertilizers. J. Agric. Food
Chem. 1997, 45, 774.
[12] K. V. Wood, K. J. Stringham, D. L. Smith, J. J. Volenec, K. L. Hendershot,
K. A. Jackson, P. J. Rich, W. J. Yang, D. Rhodes. Betaines of Alfalfa’
wileyonlinelibrary.com/journal/jms
Copyright © 2012 John Wiley & Sons, Ltd.
J. Mass. Spectrom. 2012, 47, 79–88