Characterization of N,N-dimethyl amino acids by electrospray ionization-tandem mass spectrometry

Article abstract of DOI:10.1002/jms.3588

Methylation is an essential metabolic process for a number of critical reactions in the body. Methyl groups are involved in the healthy function of the body life processes, by conducting methylation process involving specific enzymes. In these processes, various amino acids are methylated, and the occurrence of methylated amino acids in nature is diverse. Nowadays, mass-spectrometric-based identification of small molecules as biomarkers for diseases is a growing research. Although all dimethyl amino acids are metabolically important molecules, mass spectral data are available only for a few of them in the literature. In this study, we report synthesis and characterization of all dimethyl amino acids, by electrospray ionization-tandem mass spectrometry (MS/MS) experiments on protonated molecules. The MS/MS spectra of all the studied dimethyl amino acids showed preliminary loss of H₂O+CO to form corresponding immonium ions. The other product ions in the spectra are highly characteristic of the methyl groups on the nitrogen and side chain of the amino acids. The amino acids, which are isomeric and isobaric with the studied dimethyl amino acids, gave distinctive MS/MS spectra. The study also included MS/MS analysis of immonium ions of dimethyl amino acids that provide information on side chain structure, and it is further tested to determine the N-terminal amino acid of the peptides.

Full text of DOI:10.1002/jms.3588

Journal of
MASS
SPECTROMETRY
Research article
Received: 3 December 2014
Revised: 23 February 2015
Accepted: 23 February 2015
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.3588
Characterization of N,N-dimethyl amino acids
by electrospray ionization–tandem
mass spectrometry
V. Naresh Chary,a B. Sudarshana Reddy,a,b Ch. Dinesh Kumar,a R. Srinivasa
and S. Prabhakara,b*
Methylation is an essential metabolic process for a number of critical reactions in the body. Methyl groups are involved in the
healthy function of the body life processes, by conducting methylation process involving specific enzymes. In these processes, var-
ious amino acids are methylated, and the occurrence of methylated amino acids in nature is diverse. Nowadays, mass-
spectrometric-based identification of small molecules as biomarkers for diseases is a growing research. Although all dimethyl
amino acids are metabolically important molecules, mass spectral data are available only for a few of them in the literature. In this
study, we report synthesis and characterization of all dimethyl amino acids, by electrospray ionization–tandem mass spectrome-
try (MS/MS) experiments on protonated molecules. The MS/MS spectra of all the studied dimethyl amino acids showed preliminary
loss of H2O + CO to form corresponding immonium ions. The other product ions in the spectra are highly characteristic of the
methyl groups on the nitrogen and side chain of the amino acids. The amino acids, which are isomeric and isobaric with the stud-
ied dimethyl amino acids, gave distinctive MS/MS spectra. The study also included MS/MS analysis of immonium ions of dimethyl
amino acids that provide information on side chain structure, and it is further tested to determine the N-terminal amino acid of the
peptides. Copyright © 2015 John Wiley & Sons, Ltd.
Additional supporting information may be found in the online version of this article at the publisher’s web site.
Keywords: N,N-dimethyl amino acids; protonated molecules; immonium ions; electrospray ionization; tandem mass spectrometry
potentially causing a betaine insufficiency that could be expected to
have complex consequences in the supply of methyl groups essen-
Introduction
Methylation is the process of controlled transfer of methyl groups
onto small molecules (amino acids, amino alcohols, hydroxyl acids,
etc.), proteins/enzymes and DNA in every cell and tissue of the body
that regulate a number of critical biochemical pathways. Several
articles in the literature have demonstrated that the methylation
process is responsive to environmental conditions and degrades
with age, a process associated with a large variety of age-related
disorders. Methylation process is an important dividing line
between health and disease. In fact, healthy methylation processes
are synonymous with good health, and inability to methylate is
synonymous with symptom expressions and poor health. In biolog-
ical systems, there are several approaches that theoretically could
increase the availability of methyl groups. Methionine, an amino
acid, contains a methyl group that is subsequently used in the
process of methyl donation to many proteins and other molecules.
S-Adenosyl-methionine (SAM) is an important carrier molecule,
which donates methyl groups to a host during biological reactions.
Betaines also acts as methyl donor in biological pathways and used
as a dietary supplement. Lack of methyl group donors (methylation
process) in human body can lead to many diseases like cancer,
autism, diabetes, chronic fatigue and Alzheimer disease. In addi-
tion, methylated derivatives play an essential role in biological
functions which are potential biomarkers of human physiological
processes.[1,2] For example, an abnormal urinary excretion of
methyl donor, betaine has been observed in many patients with
diabetes. As high urinary betaine loss in diabetes mellitus patients,
tial for normal metabolism. Sarcosine was identified as potential
biomarker for prostate cancer,[3] while dimethylglycine was found
to be increased in the plasma of chronic renal failure patients.[4]
The methylated derivatives of arginine, asymmetric dimethylarginine
and symmetric dimethylarginine are markers that are associated with
chronic kidney disease and renal inflammation.[5–8] Kakimoto et al.
reported isolation and identification of the symmetric/asymmetric
dimethylarginines and mono-/di-/tri-methyl derivatives of lysine in
human urine.[9] Identification of N-methylated basic amino acids[10]
and N-methylated acidic amino acids[11] in biological tissues were
also reported in the literature. Bouatra et al. reported methylated
metabolites in human urine using nuclear magnetic resonance spec-
troscopy, gas chromatography–mass spectrometry (GC-MS) and
liquid chromatography–tandem mass spectrometry (LC-MS/MS)
techniques.[12]
As methylation involves specific methylating enzymes, the meth-
ylated derivatives in nature are extremely diverse in association
*
Correspondence to: Prabhakar Sripadi, National Centre for Mass Spectrometry,
CSIR-Indian Institute of Chemical Technology, Hyderabad, 500 007 Telangana,
India. E-mail: prabhakar@iict.res.in
a National Centre for Mass Spectrometry, CSIR-Indian Institute of Chemical
Technology, Hyderabad 500 007 Telangana, India
b Academy of Scientific and Innovative Research, CSIR-Indian Institute of Chemical
Technology, Hyderabad 500 007 Telangana, India
J. Mass Spectrom. 2015, 50, 771–781
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V. Naresh Chary et al.
with small metabolites like amines, acids and alcohols. Amino acids
are one such class of metabolites that can undergo methylation
during metabolism. The methylated amino acid metabolites play
an essential role in understanding the biological reactions, and
thereby the changes in their levels contribute to the development
of new diagnostic and therapeutic methods to diagnose specific
diseases. During the analysis of biological samples, low molecular
weight metabolites like amino acids and their methylated deriva-
tives may also be encountered. For example, in the analysis of the
untreated tissue of Torpedo californica electric organ by mid-
infrared laser ablation electrospray ionization (ESI) mass spectro-
metric method, some of the amino acids and methylated amino
acids like N-methyl and N,N-dimethyl derivatives of same nominal
masses (N-methyl glycine or beta-alanine; dimethylglycine or GABA
and dimethyllysine) were detected.[13]
Characterization of dimethylated amino acids is a part in under-
standing overall methylated metabolites of a cell or tissue. More-
over, nowadays the amino acids in biological matrices have been
detected after converting them into N,N-dimethyl amino acids by
methylation.[14] To the best of our knowledge, the MS/MS spectra
of a few dimethyl derivatives of amino acids, i.e. glycine, histidine,
arginine and lysine were available in the literature and databases
(mass bank, http://www.massbank.jp/index.html; HMDB, http://
www.hmdb.ca/spectra/ms/search and Metlin;http://metlin.scripps.
edu/index.php). So it is important to study the mass spectral char-
acterization of dimethyl derivatives of all the amino acids. In the
previous study,[15] we have characterized all the amino acid derived
betaines. The present study mainly focused on structural character-
ization of N,N-dimethyl amino acids by MS/MS and high-resolution
mass spectrometry (HRMS) analyses under ESI conditions. The
study is also explored to detect the N-terminal amino acid of pep-
tides by applying the MS/MS of immonium ion.
Mass spectral analysis of all the studied compounds (Scheme 2)
were carried out on a quadrupole time-of-flight (Q-TOF) mass spec-
trometer (Q-Star XL, Applied Biosystems, USA), equipped with an
ESI source using Analyst software. The samples were introduced
into the source through a flow injection, where methanol was used
as the mobile phase at a flow rate of 30μl/min. The typical positive
ESI conditions were capillary voltage 5.00 kV, declustering potential
60V, focusing potential 220V, and resolution 10,000 (FWHM).
Collision-induced dissociation (CID) spectra were obtained by
selecting the ion of interest using the quadrupole and then allowed
to fragment in the collision cell by colliding with nitrogen, followed
by detecting the resulted product ions by the TOF analyzer. The CID
spectra were recorded at different collision energies (10, 15, 20 and
25eV). The spectra of isomeric/isobaric ions were obtained at simi-
lar experimental conditions. For recording the CID spectra of
immonium ions of dimethyl amino acids, the immonium ions were
generated in the source by applying a higher declustering potential
(100–160; in-source fragmentation).
Results and discussions
The chemical structures of the methylated amino acids (1–23) were
shown in Scheme 2. The positive ion ESI mass spectra of all the
methylated amino acid solutions, except lysine, showed abundant
[M+ H]+ and [M + Na]+ ions of N,N-dimethyl amino acids (where
α-NH2 group of the amino acids was methylated; hereafter these
compounds are referred as dimethyl amino acids, 1–22). Methyla-
tion of the lysine resulted in the tetramethyllysine (23), where both
α-NH2 and ε-NH2 were methylated. The products due to methyla-
tion of other functional groups like –COOH, –OH, –SH and
guanidinium were not observed. The [M+ H]+ ions of all the meth-
ylated amino acids (1–23) were further subjected to CID experi-
ments to obtain their structural information (spectra are
summarized in Table 1). The CID spectra together with HRMS data
(Tables S1 and S2, see supplementary information) were used to
characterize the dimethyl amino acids. In some cases, CID spectra
recorded for the product ions generated by in-source fragmenta-
tion were also used. Fragmentation pattern of [M+ H]+ ions of di-
methyl amino acids is expected to be different when compared
with that of corresponding free amino acids, because methylation
of amino group tends to increase the proton affinity leading to fac-
ile protonation by mobile proton. For comparison purpose, we have
also recorded the CID spectra of [M+ H]+ ions of corresponding free
amino acids (Table S3). The fragmentation pattern observed for
[M+ H]+ ions of free amino acids was similar to those reported in
the literature.[17–22]
Experimental
All the L-amino acids, 37% formaldehyde and sodium
cyanoborohydrate (NaCNBH3) were obtained from Sigma-Aldrich
(St. Louis, MO, USA). All the solvents were of high performance
liquid chromatography grade, and were purchased from Merck
(Mumbai, India). The dipeptide (Met-Leu) and tetra peptide
(Lys-Ala-Ala-Ala) were obtained from GenPro Biotech (C-39, Sector
10, Noida, Gautham Budh Nagar, Uttar Pradesh-201301, India).
All dimethyl amino acids were synthesized by methylation of free
amino acids using the known procedure (Scheme 1).[16] Briefly, one
equivalent of amino acid was dissolved in 50 mM sodium acetate
buffer (pH 5) and immediately added 10 equivalents of 37% formal-
dehyde and 10 equivalents of sodium cyanoborohydride. The mix-
ture was stirred at ambient temperature for 15–20 min. The
reaction mixture was stored in a refrigerator until it was subjected
to mass spectrometric analysis. The samples were diluted three
times with acetonitrile and acidified with diluted HCl before
injecting into the mass spectrometer. The same procedure was ap-
plied for methylation of the dipeptide (Met-Leu) and tetrapeptide
(Lys-Ala-Ala-Ala).
We observed that the [M+ H]+ ions of all the dimethyl amino
acids were fragmented at higher collision energies than those of
corresponding free amino acids. The dimethyl amino acids showed
primarily the elimination of (H2O + CO) in addition to other specific
product ions due to methyl groups on nitrogen as well as the sub-
stituent (–R) on the α-carbon of the amino acids. For convenience,
these compounds were divided into four groups based on the na-
ture of –R group that include aliphatic (1–12), aromatic (13–16),
acidic (17 and 18) and amidic or basic (19–22) dimethyl amino
acids. The isomeric dimethyl amino acids showed distinct spectra,
which were also discussed subsequently. Some of the dimethyl
amino acids are found to be isomeric with other free amino acids,
and such cases were also included in the discussion. The possible
isomeric and isobaric amino acids and their methyl derivatives were
listed in Table 2.
Scheme 1. Synthetic procedure for N,N-dimethyl amino acids.
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ESI-MS/MS of dimethyl amino acids
Scheme 2. The chemical structures of the studied compounds (1–23).
CID of [M+ H]+ ions
[C2H6N]+ (m/z 44) ions. The CID spectrum of protonated N,N-di-
methyl-β-alanine (3), which is isomeric to 2, showed the base peak
at m/z 58 corresponding to [MH-CH3COOH]+ ion. The other product
ions at m/z 72, 44, 42 and 30 were found to be of low abundance.
The compounds 2 and 3 are also isomeric to valine and glycine
betaine, and they can easily be distinguished based on distinct
CID spectra of their [M+ H]+ ions (Fig. 1). The CID spectrum of
[M+ H]+ of valine showed the [MH-(H2O+ CO)]+ (m/z 72) as the
base peak as found in 2, but it included a specific product ion
[MH-(H2O + CO)-NH3]+ (m/z 55).
Aliphatic dimethyl amino acids (1–12)
Dimethyl glycine (1) is found in blood and urine samples and has a
variety of biological effects.[23,24] The CID mass spectrum of [M + H]+
of 1 showed the [MH-(H2O + CO)]+ (m/z 58) as the base peak (which
is similar to that of glycine, Table S3). The spectrum of 1 included
minor product ions at m/z 86 and 30 corresponding to [MH-H2O]+
and [H2C¼NH2]+ ions, respectively. In real biological samples, de-
tection of dimethylglycine (1) may be challenging because it is iso-
meric with γ-aminobutyric acid (GABA) and 2-aminoisobutyric acid
(2-AIB), which may occur in a given sample. In such cases, MS/MS
analysis of their protonated species can help in peak identification.
Hence, the CID spectra of GABA and 2-AIB were also recorded and
compared with that of 1 (Figure S1). The spectra of [M+ H]+ ions of
2-AIB and 1 showed the base peak at m/z 58. The former included
distinctive product ions at m/z 87, [MH-NH3]+ and m/z 41,
[MH-(H2O + CO)-NH3]+, which are absent for the latter. While the
spectrum of [M+ H] + of 1 showed a specific ion at m/z 30, and that
of GABA displayed [MH-NH3]+ ion as the major product ion in addi-
tion to loss of H2O, (NH3 + H2O), and (H2O + CO) giving rise to ions at
m/z 86, 69 and 58, respectively.
The CID spectrum of [M+ H]+ ion of N,N-dimethyl GABA (4) is
very much different from that of its isomer, N,N-dimethyl-2-amino
isobutyric acid (5) (Fig. 2). The spectrum of 4 showed [MH-HN
(CH3)2]+ (m/z 87; 100%), [MH-H2O]+ (m/z 114; 28%) and other prod-
uct ions at m/z 71, 69, 46, 45 and 43; whereas, the expected [MH-
(H2O + CO)]+ ion was absent. The protonated 5 showed dominant
[MH-(H2O + CO)]+ (m/z 86) and the characteristic product ions of 4
(m/z 114, 87, 86, 46 and 43) were found to be negligible. The com-
pounds 4 and 5 are also isomeric with the free amino acids, leucine
and isoleucine, but they can be easily discriminated based on the
specific fragmentation of their protonated species.
The CID spectra of the protonated N,N-dimethylvaline (6) and N,
N-dimethyl leucine/N,N-dimethylisoleucine (7/8) showed the
[MH-(H2O + CO)]+ ion as the base peak. The anticipated
[MH-(HN(CH3)2)]+ ion, similar to [MH-NH3]+ ion found in free amino
acids, was absent in the spectra of 6–8. Between the isomeric
The CID spectrum of N,N-dimethylalanine (2) showed the
[MH-(H2O + CO)]+ ion (m/z 72) as the major product ion. Other prod-
uct ions were due to further loss of H2, •CH3 radical and CH4 from
[MH-(H2O + CO)]+ ion or corresponding to [C3H8N]+ (m/z 58) and
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Table 1. Collision-induced dissociation mass spectra of [M + H]+ ions of methylated amino acids (1–23)
Comp.
Number m/z (% RA)
[M + H]+
Product ions, m/z (% RA)
[M + H-H2O]+ [M + H-(H2O + CO)]+ [M + H-HN(CH3)2]+
Other ions
1
2
3
4
5
6
7
8
9
10
104 (46)
118 (100)
118 (6)
86 (1)
100 (1)
100 (2)
114 (28)
114 (1)
58 (100)
72 (40)
44 (2), 43 (1), 42 (1), 30 (2)
70 (3), 57 (2), 44 (1)
58 (100), 46 (1), 44 (2), 42 (1), 30 (1), 28 (1)
72 (1)
132 (18)
132 (38)
146 (13)
160 (13)
160 (16)
134 (20)
148 (18)
86 (6)
87 (100)
87 (2)
71 (3), 69 (5), 46 (29), 45 (16), 44 (9), 43 (28), 41 (2)
46 (2)
86 (100)
100 (100)
114 (100)
114 (100)
88 (100)
102 (51)
46 (2), 44 (4)
72 (3), 58 (3)
69 (2), 58 (1), 46 (3)
116 (46)
104 (2), 98 (27), 74 (25), 73 (2), 72 (17), 58 (3), 57 (6), 46 (2), 44 (13), 30 (1)
112 (4), 104 (30), 89 (2), 88 (18), 86 (19), 85 (3), 84 (52), 74 (19), 72 (8), 70
(6), 58 (8), 56 (2), 46 (4), 45 (5), 44 (11)
130 (100)
11
12
13
14
15
150 (12)
178 (8)
104 (14)
132 (16)
134 (23)
148 (100)
164 (52)
105 (21)
133 (100)
135 (80)
149 (2)
116 (6), 87 (32), 78 (2), 59 (16), 58 (15), 46 (100)
147 (2), 130 (14), 118 (3), 105 (2), 102 (8), 87 (7), 85 (1), 84 (15)
121 (3), 120 (1), 107 (100), 91 (2), 79 (54), 77 (19)
131 (7), 107 (3), 102 (5), 58 (3), 46 (4)
180 (3)
194 (36)
210 (62)
165 (100)
147 (28), 123 (37), 121 (1), 119 (15), 103 (1), 102 (1), 95 (1), 59 (2), 58 (5),
46 (98), 30 (1)
16
233 (5)
187 (1)
188 (63)
202 (41), 169 (3), 160 (11), 158 (4), 146 (16), 144 (5), 129 (2), 117 (2), 115
(2), 102 (100), 101 (2), 97 (4), 88 (3), 79 (13), 57 (3), 46 (5)
102 (100), 74 (1), 56 (1)
17
18
19
20
21
22
162 (6)
176 (23)
161 (26)
175 (8)
184 (2)
203 (23)
116 (15)
130 (100)
115 (6)
158 (10)
185 (3)
145 (8), 144 (10), 116 (2), 98 (44), 85 (5), 84 (4), 72 (1), 70 (6), 46 (10)
144 (1), 102 (100)
129 (53)
138 (100)
157 (3)
130 (100)
158 (21)
158 (43), 112 (3), 102 (7),85 (5), 84 (21), 46 (9)
140 (1), 123 (8), 102 (4),95 (74),82 (2)
161 (3), 144 (100), 130 (9), 125 (4), 116 (6), 114 (34), 112 (15), 100 (52),
97 (5), 72 (6), 70 (8), 58 (3), 46 (2), 43 (3)
140 (1), 130 (6), 114 (18), 112 (3), 58 (1), 46 (1)
23
203 (12)
157 (7)
158 (100)
Collision energy (CE) = 20 eV, except 19 where CE = 15 eV.
RA, relative abundance.
compounds 7 and 8, the spectrum of latter showed specific prod-
uct ions at m/z 69 and 46 corresponding to [MH-(H2O + CO)-
HN(CH3)2]+ and [HN(CH3)2 + H]+, respectively. The McLafferty-type
rearrangement product ions from [MH-(H2O + CO)]+ ion was
observed in the spectra of 7 and 8 (m/z 72 and 86, respectively),
but they were of low abundance. In our previous study on
N-substituted (acetyl, benzoyl and pivaloyl) leucine and
isoleucines,[25] specific product ions were observed from their
[MH-COOH]+• ions. However, the [MH-COOH]+• ions were absent
in the dimethylleucine/isoleucine (7 and 8).
The CID spectra of protonated N-dimethylserine (9) and N,N-
dimethylthreonine (10) showed loss of H2O (Scheme 3) and
(H2O + CO) due to the presence of hydroxyl group on the side
chain. The spectrum of 9 included the product ions produced by
the loss of H2O (18 u), CH2CO (42 u), NC2H5 (43 u), and CO2 (44 u)
from the ion [MH-H2O]+ at m/z 98, 74, 73 and 72, respectively, which
can be explained from the proposed linear structure (Scheme 3).
The m/z values of these product ions are shifted by +14u in the
CID spectrum of protonated 10; it also showed an additional prod-
uct ion at m/z 70 due to the loss of CH3COOH from [MH-H2O]+ ion.
The spectrum of protonated 9 included the product ions at m/z 98,
74, 73 and 72, because of further loss of H2O (18 u), CH2CO (42 u),
NC2H5 (43 u), and CO2 (44 u), respectively, from the ion [MH-H2O]+
(Scheme 3); these product ions are shifted by +14 u in the spectrum
of protonated 10. The spectrum of 10 showed an additional
product ion at m/z 70 due to the loss of CH3COOH from [MH-H2O]+
ion. The spectra of 9 and 10 showed a common product ion at
m/z 104 corresponding to the loss of CH2O and C2H4O, respec-
tively, which may be formed from [M+ H]+ ions through a
McLafferty-type rearrangement involving migration of the hydro-
gen from hydroxyl group to the carbonyl group. The [M+ H]+
ion of compound 10 is isobaric with the [M + H]+ ion of glutamic
acid (same nominal mass); however, these two compounds can
easily be distinguished by their distinct CID spectra (Fig. 3).
The compounds N,N-dimethylcysteine (11) and N,N-
dimethylmethionine (12) have sulfur atoms in their side chains.
CID spectra of their protonated species generated the
[MH-(H2O + CO)]+ ions. In addition, the spectra displayed specific
[MH-HN(CH3)2]+ ion in the CID spectra of 11 and 12 that may be
because of sulfur atom involving cleavage of C–N bond. The com-
pound 11 showed loss of H2S from [M+ H]+ ion (6%), whereas this
loss was negligible in free cysteine (<1%). Other product ions,
appeared at m/z 87 and 59 in the spectrum of 11, were formed
by loss of H2O from [MH-HN(CH3)2]+ and loss of dimethylamine
from [MH-(H2O + CO)]+, respectively. The spectrum of 12 showed
the [MH-CH3SH]+ (m/z 130) and [MH-C3H8S]+ ion (m/z 102) that re-
flect the structure of its side chain. The fragmentation pattern of the
[M+ H]+ of 12 was found to be similar to that observed for the
[M+ H]+ of free methionine (Figure S2). The N,N-dimethylcysteine
(11) is isomeric with free methionine, but their CID spectra are
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SPECTROMETRY
ESI-MS/MS of dimethyl amino acids
Table 2. Possible isomeric (same formula) and isobaric compounds
(different formula with same nominal mass) among amino acids and
their methyl derivatives
m/z of [M + H]+ ions
(chemical formula)
Possible isomeric and
isobaric compounds
m/z 104 (C4H10NO2)
γ-Aminobutyric acid (GABA) (C4H10NO2)
2-Aminoisobutyric acid (2-AIB) (C4H10NO2)
N-Methylalanine (C4H10NO2)
N-Methyl-β-alanine (C4H10NO2)
N,N-Dimethylglycine (DMG) (C4H10NO2)
Valine (C5H12NO2)
m/z 118 (C5H12NO2)
N-Methyl GABA (C5H12NO2)
N-Methyl-2-AIB (C5H12NO2)
N,N-Dimethylalanine (C5H12NO2)
N,N-Dimethyl-β-alanine (C5H12NO2)
Glycine betaine (C5H12NO2)
Leucine (C6H14NO2)
m/z 132 (C6H14NO2)
Isoleucine (C6H14NO2)
N-Methylvaline (C6H14NO2)
N,N-Dimethyl GABA (C6H14NO2)
N,N-Dimethyl-2-AIB (C6H14NO2)
Alanine betaine (C6H14NO2)
β-Alanine betaine (C6H14NO2)
Aspartic acid (C4H8NO4)
m/z 134
(C5H12NO3/ C4H8NO4)
N-Methylthreonine (C5H12NO3)
N,N-Dimethylserine (C5H12NO3)
Glutamic acid (C5H10NO4)
m/z 148
(C6H14NO3/C5H10NO4)
N-Methylaspartic acid (C5H10NO4)
N,N-Dimethylthreonine (C6H14NO3)
Arginine (C6H15N4O2)
m/z 175 (C7H15N2O3/
C8H19N2O2/C6H15N4O2)
N,N-Dimethylglutamine (C7H15N2O3)
N,N-Dimethyllysine (C8H19N2O2)
Asparagine betaine (C7H15N2O3)
Figure 1. Collision-induced dissociation mass spectra of (a) N,N-
dimethylalanine, (b) N,N-dimethyl-β-alanine and (c) valine at collision
energy of 20 eV.
very much distinctive (Figure S2), where loss of NH3 and HN(CH3)2
is characteristic to free amino acid and dimethyl amino acid,
respectively.
Aromatic dimethyl amino acids (13–16)
The CID spectra of all the protonated aromatic dimethyl amino
acids, i.e. N,N-dimethylphenylglycine (13), N,N-dimethyl phenylala-
nine (14), N,N-dimethyltyrosine (15) and N,N-dimethyltryptophan
(16) showed [MH-HN(CH3)2]+ ion (63–100%, except 14 where it
was 2%), which may be well stabilized by the aromatic ring. All
these compounds, except 13, displayed the product ion at m/z 46
retaining charge on dimethyl amine part. The [MH-HN(CH3)2]+ ion
(m/z 135) from 13 showed the ions at m/z 107 and 79 because of
consecutive losses of two CO molecules; this was further confirmed
by the CID of m/z 135. Similarly, consecutive loss of CH2CO and CO
from [MH-HN(CH3)2]+ ion was found in the case of 14 because of
the presence of a benzyl group at the α-position. The compound
15 showed a similar fragmentation as that of 14, where mass values
of all the characteristic product ions were shifted by +16 u because
of the hydroxyl group on the phenyl group in 15. The compound
16 also showed loss of CH2CO from [MH-HN(CH3)2]+ ion because
–CH2-(imidazole ring) group is attached to an α-carbon. The
compounds 15 and 16 showed the loss of CO2 from the
[MH-HN(CH3)2]+ ion. Apart from these, [MH-(H2O + CO)]+ ion was
present in all these compounds (23–100% in 13–15, and 1% in
Figure 2. Collision-induced dissociation mass spectra of [M + H]+ ion (a) N,
N-dimethyl GABA (4) and (b) N,N-dimethyl 2-AIB (5) at collision energy of
20 eV.
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product ions. The [MH-HN(CH3)2]+ ion of 20 further fragmented to
result in characteristic product ions corresponding to the loss of
H2O, CO, (H2O + CO), and HCONH2. The nominal mass of [M+ H]+
ion of 20 matches with that of free amino acid arginine (isobaric);
but they can be easily discriminated by their distinct CID spectra,
which clearly reflect the functional groups (Figure S3).
The CID spectrum of protonated N,N-dimethylhistidine (21)
showed [MH-(H2O+ CO)]+ ion and a specific product ion corre-
sponding to [MH-CO2]+ ion. The spectrum also included the
[MH-CO2-NC2H5]+ (m/z 95) and the m/z 102 formed by the loss
of side chain group (H3C-imidazole) from [M+ H]+ ion. The CID
spectrum of N,N-dimethylarginine (22) showed the major
product ions corresponding to [MH-(HN(CH3)2)]+ (m/z 158) and
[MH-(HN¼C(NH2)2)]+ (m/z 144; 100%) (base peak). The spectrum
also included [MH-H2O]+, [144-CO2]+ and other product ions due
to further losses of NH3, CO, CO2 and HCOOH from [MH-HN(CH3)
2]+ ion. The spectrum of 22 is found to match well with the reported
spectrum by Shek et al.[26] Appearance of the specific product ion at
m/z 144 in 22 rules out methylation on guanidinium group,
because this ion known to be present in the CID mass spectra of
asymmetric and symmetric dimethylarginines.[27]
Scheme 3. Plausible mechanism for the formation of [MH-H2O]+ of
compounds 9 (R¼H), 10 (R¼CH3).
The CID spectrum of [M+ H]+ ion of Nα,Nα,Nε,Nε-tetramethyllysine
(23) showed loss of HN(CH3)2 and HCOOH from [M+ H]+ ion.
Further loss of H2O, CO, CO2 and HCOOH were also found from
[MH-HN(CH3)2]+ ion. This spectrum well matched with the spec-
trum reported by Shek et al.[26] The tetramethyllysine (23) and
dimethylarginine have the same nominal mass (isobaric); however,
they can be well discriminated with their [M+ H]+ ion CID spectra.
Figure 3. Collision-induced dissociation mass spectra of [M + H]+ ion of (a)
N,N-dimethylthreonine (10) and (b) glutamic acid at collision energy of
20 eV.
CID of immonium ions
The MS/MS spectra of protonated peptides show specific product
ions including immonium ions (RCH¼NH+2) in the low mass range
that are characteristic of the amino acids present in the
peptide.[28–30] Recently, Hohmann et al.[31] reported that the
immonium ion can be used as a quantitative indicator. The precur-
sors to the immonium ions were examined by reaction intermedi-
ate scans sequence in peptide.[32] The selective decomposition of
immonium ions from free amino acid were also used to confirm
the side chain of the amino acid. Moreover, methylation is one of
the post translation modifications of proteins; in this process,
N-terminal amino acid may undergo methylation and yield mono
and/or dimethylated derivative.[33] In such a case, the N-terminal
modified protein/peptide produces corresponding immonium
ion, and further fragmentation of this ion provide the structural in-
formation of the modified amino acid. Thus, it is essential to study
the CID of immonium ions of all the dimethyl amino acids to enable
their identification as a methylated free amino acid or amino acid
located at N-terminal.
In this study, the immonium ions of all the dimethyl amino acids
(except 3, 4 and 16 because of their low abundance), were gener-
ated by in-source fragmentation of their [M+ H]+ ions. These
immonium ions were further subjected to CID experiments for their
structural characterization. The resulting immonium ion was de-
noted as Xi, where X is the dimethyl amino acid number and i refer
to the immonium ion generated from X (for example, 1i refers to
immonium ion of 1). The general structure of immonium ion
formed from the dimethyl amino acids is shown in Scheme 4. The
CID mass spectra of the immonium ions of all the dimethyl amino
acids are tabulated in Table 3 and HRMS data is shown in Table
S2. In the study of immonium ions, also a few isomeric/isobaric
immonium ions can be formed from dimethyl amino acids and free
16), while [MH-(H2O + CO)-HN(CH3)2]+ was present in 14 and 15.
The compound 16 selectively showed an abundant product ion
at m/z 102 corresponding to the loss of side group with abstrac-
tion of hydrogen (loss of H3C-imadazole ring); similar product
ion (m/z 102) was observed in 14 and 15 but low in abundance.
Acidic dimethyl amino acids (17 and 18)
The CID spectrum of [M+ H]+ of N,N-dimethylaspartic acid
(17) showed [MH-CH3COOH]+ (m/z 102;100%), [MH-(H2O + CO)]+
(m/z 116) and further losses of H2O, CO, (H2O + CO) from the
[MH-CH3COOH]+ ion (m/z 84, 74 and 56, respectively), which con-
firms the presence of a second carboxylic group in the structure.
The N,N-dimethylglutamic acid (18) showed expected losses
of H2O (m/z 158), (H2O + CO) (m/z 130, 100%) and CH3COOH
(m/z 116) from [M+ H]+ ion. The other product ions seen in the
spectrum of 18 were due to the loss of H2O, (H2O + CO), and HN
(CH3)2 from the [MH-(H2O + CO)]+ ion at m/z 112, 84, and 85,
respectively, and [MH-(CH3COOH + H2O)]+ ion (m/z 98).
Amidic/basic dimethyl amino acids (19–22) and tetramethyllysine
The [M+ H]+ ion CID spectrum of N,N-dimethylasparagine (19)
showed an abundant ion at m/z 102 by loss of CH3CONH2
(59 u), which inferred the presence of amide group in the side
chain in 19. The spectrum also included the [MH-NH3]+ and
[MH-(H2O + CO)]+ ions, whereas the N,N-dimethylglutamine (20)
showed dominant [MH-HN(CH3)2]+ ion (m/z 130), and [MH-NH3]+
(m/z 158) and [MH-(H2O +CO)]+ (m/z 129) were next two abundant
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ESI-MS/MS of dimethyl amino acids
(Fig. 4). The ion 2i is isomeric to the immonium ion of valine. The
immonium ion of valine showed the product ion at m/z 57 (loss
of •CH3) as the base peak (Fig. 4) and specific product ions ions at
m/z 55 and 53 due to the loss of NH3 and (NH3 + H2), respectively.
5i+ (m/z 86). The CID spectrum of immonium ion of N,N-dimethyl-
2-amino isobutyric acid (5i, m/z 86) showed product ions at m/z 84,
71, 70, 56, 43 and 41 corresponding to the losses of H2, •CH3, CH4,
C2H6, (N,C2,H5) and (HN(CH3)2), respectively. The ion 5i + is isomeric
to the immonium ions of leucine and isoleucine. The CID of 5i + and
the immonium ions of leucine and isoleucine were shown in Figure
S5. The spectrum of leucine immonium ion showed the product
ions at m/z 69 (7%), 44 (base peak) and 43 corresponding to the
losses of NH3, C3H6 (through a McLafferty-type rearrangement),
and •C3H7 radical, respectively. The spectrum of isoleucine
immonium ion showed the product ions at m/z 69 (66%), 67, 58,
57, 41 and 30 corresponding to the losses of NH3, (NH3 + H2),
C2H4 (through McLafferty-type rearrangement), •C2H5 radical,
(C2H4 + NH3) and C4H8 (loss of side chain with hydrogen migration),
respectively.
Scheme 4. Formation of immonium ions from N,N-dimethyl amino acids.
amino acids. Hence, we have also studied the CID of immonium
ions generated from free amino acids for comparison purpose,
and the spectra thus obtained were tabulated in Table S4.
Characteristic ions resulting from immonium ions of dimethyl
amino acids were discussed subsequently as per their m/z values.
The isomeric/isobaric immonium ions were included at appropriate
paragraph.
1i+ (m/z 58). The CID spectrum of immonium of N,N-
dimethylglycine (1i; m/z 58) showed the product ions because of
•
losses of H2 (m/z 56), CH3 radical (m/z 43) and CH4 (m/z 42;
100%). It also displayed other low mass product ions at m/z 30
(H2CꢀNH2)+, 29 (+C2H5), 28 (HNꢀCH)+, and 15 (+CH3). The ion 1i +
is isomeric with the immonium ion of 2-AIB; however, their CID
spectra were distinctive (Figure S4). The immonium ion of 2-AIB
showed specific losses of NH3 and (NH3 + H2) resulting in the prod-
uct ions at m/z 41 and 39, respectively.
6i+ (m/z 100). The CID spectrum of immonium ion of N,N-
dimethylvaline showed the product ions at m/z 98, 85 (base peak),
84, 70, 58, 57 and 55 corresponding to the losses of H2, •CH3 radical,
•
CH4, C2H6, C3H6 (by McLafferty-type rearrangement), C3H7 radical
and HN(CH3)2, respectively. The spectrum also showed an abun-
2i+ (m/z 72). The CID spectrum of immonium ion of N,N-
dimethylalanine (2i, m/z 72) showed product ions at m/z 70, 57,
56, 44, 42 and 29 corresponding to the losses of H2, •CH3 radical,
CH4, C2H4, C2H6 and (N,C2,H5; may be H2C¼N-CH3), respectively
dant product ion at m/z 44 corresponding to [C2H6N]+.
7i+ and 8i+ (m/z 114). The CID spectra of the immonium ions of N,
N-dimethylleucine (7i) and N,N-dimethylisoleucine (8i) showed
Table 3. Collision-induced dissociation mass spectra of immonium ions of dimethyl amino acids
Immonium ion;
DP*
Collision
Product ions, m/z (% RA)
m/z (% RA)
value (V) energy (eV)
1i; 58 (20)
140
120
140
140
180
140
160
140
100
150
140
25
20
15
25
25
25
20
20
20
15
30
56 (2), 44 (2), 43 (42), 42 (100), 32 (2), 30 (38), 29 (8), 28 (7), 27 (4), 15 (1)
70 (16), 57 (14), 56 (6), 44 (30), 42 (29), 41 (1),30 (2)
2i; 72 (100)
5i; 86 (100)
6i; 100 (31)
7i; 114 (7)
84 (4), 71 (12), 70 (1), 56 (20), 46 (1), 44 (9), 43 (2), 42 (1)
85 (100), 84 (27), 70 (72), 58 (13), 55 (54), 53 (2), 44 (90), 43 (5), 42 (7), 41 (7)
72 (68), 71 (20), 70 (4), 58 (23), 57 (9), 56 (3), 55 (1), 44 (5), 43 (100), 41 (23), 30 (1)
99 (3), 86 (5), 85 (100), 84 (41), 72 (7), 71 (4), 70 (52), 69 (17), 58 (48), 46 (11), 44 (21), 43 (2), 42 (9), 41 (36), 30 (2)
86 (3), 73 (5), 70 (14), 59 (3), 58 (100), 56 (3), 55 (3), 46 (21), 45 (23), 44 (59), 43 (12), 42 (12), 31 (13), 30 (29)
87 (2), 84 (9), 74 (22), 72 (1), 60 (2), 58 (44), 57 (3), 56 (100), 46 (19), 45 (1), 44 (18), 42 (33), 30 (16), 29 (2), 28 (18)
89 (1), 86 (2), 78 (2), 77 (1), 71 (100), 70 (3), 58 (15), 56 (20), 46 (7), 44 (5)
114 (1), 84 (100), 61 (17), 42 (2)
8i; 114 (18)
9i; 88 (83)
10i; 102 (44)
11i; 104 (23)
12i; 132 (27)
13i; 134 (95)
132 (6), 119 (13), 118 (76), 117 (3), 116 (3), 106 (6), 105 (3), 104 (7), 103 (3), 94 (8), 93 (6), 91 (100), 88 (3), 86 (5),
77 (9), 74 (8), 74 (8), 70 (3), 67 (3), 65 (8), 63 (3), 56 (14), 53 (4), 46 (8), 44 (4)
147 (2), 146 (4), 133 (100), 132 (43), 118 (8), 117 (2), 116 (4), 115 (2), 105 (38), 104 (18), 103 (9), 91 (62), 79 (11),
77 (4), 71 (4), 67 (2), 58 (2), 56 (4), 44 (4), 42 (7), 41 (1)
14i; 148 (25)
15i; 164 (69)
17i; 116 (19)
140
150
130
30
25
20
162 (2), 149 (100), 148 (7), 147 (2), 135 (2), 134 (3), 132 (3), 121 (31), 119 (7), 108 (7), 107 (22), 105 (2), 103 (5),
94 (2), 93 (10), 91 (15), 77 (3), 72 (2), 71 (18), 58 (3), 56 (4), 46 (12), 45 (7), 44 (6)
98 (100), 88 (25), 84 (8), 74 (76), 73 (3), 72 (42), 71 (48), 70 (42), 58 (4), 57 (36), 56 (53), 55 (12), 46 (22), 45 (14),
44 (51), 43 (21), 42 (78), 37 (11), 33 (4), 28 (59)
18i; 130 (6)
19i; 115 (19)
20i; 129 (7)
21i; 138 (6)
22i; 157 (7)
23i; 157 (21)
150
160
140
150
170
140
20
20
20
25
20
20
98 (6), 84 (100), 71 (7), 70 (20), 58 (1), 56 (4), 46 (4), 44 (2)
98 (100), 72 (97), 70 (10), 57 (13), 44 (11)
112 (2), 86 (1), 84 (100), 83 (2), 69 (1), 55 (2), 46 (2), 42 (6)
123 (12), 122 (2), 106 (1), 96 (4), 95 (100), 81 (1), 68 (12), 44 (2), 41 (4), 30 (1)
112 (22), 111 (7), 100 (4), 98 (22), 84 (7), 70 (100), 52 (7), 44 (4), 42 (7), 41 (4)
114 (100), 112 (63), 97 (3), 84 (31), 58 (5), 46 (1), 44 (1)
RA, relative abundance.
* Declustering potential (DP) value used to generate the immonium ion in the ESI source.
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(CH3)+2 at m/z 69 (17%), and the similar product ion in 7i + was neg-
ligible (<1%).
9i+ (m/z 88) and 10i+ (m/z 102). The CID spectrum of immonium
of N,N-dimethylserine (9i; m/z 88) showed the product ions at m/z
86, 73, 70, 58 (base peak), 55, 45, 44, 43 and 42 corresponding to
the losses of H2, •CH3 radical, H2O, CH2O (due to McLafferty-type re-
arrangement), (H2O + •CH3), (N,C2,H5), (H2O + C2H2), HN(CH3)2 and
(CH2O + CH4), respectively. The CID spectrum of immonium ion of
N,N-dimethyl threonine (10i; m/z102) included the product ions at
m/z 100, 87, 84, 74, 72, 58, 57, 56 (base peak), 44, 43 and 42 corre-
sponding to the losses of H2, •CH3 radical, H2O, CO, C2H6, C2H4O
(due to McLafferty-type rearrangement), HN(CH3)2, (H2O + C2H4),
(C2H6 + CO), (C2H4O + •CH3), and (C2H4O + CH4), respectively. The
loss of CO was specifically found from 10i+ and the immonium of
threonine, while it was not found from 9i + and the immonium of
serine.
11i+ (m/z 104) and 12i+ (m/z 132). The CID spectrum of
immonium ion of N,N-dimethyl cysteine (11i; m/z 104) showed
the product ions at m/z 89, 71 (base peak), 70, 58 and 56 corre-
•
sponding to the losses of •CH3, SH, H2S, CH2S (due to McLafferty-
type rearrangement), and (CH2S + H2), respectively. The other prod-
uct ions appeared in the low mass region at m/z 46, 44 and 43. The
CID spectrum of 12i + (m/z 132) showed characteristic product ions
at m/z 84 (loss of CH3SH), 61 [H2C¼S-CH3]+ and 42 [H2C¼N¼CH2]+.
Figure 4. Collision-induced dissociation mass spectra of immonium ions of
(a) N,N-dimethylalanine and (b) valine at collision energy of 20 eV.
13i+ (m/z 134), 14i+ (m/z 148) and 15i+ (m/z 164). The CID spectra
of immonium ions of N,N-dimethyl phenyl glycine (13i), N,N-di-
methyl phenyl alanine (14i) and N,N-dimethyl tyrosine (15i)
showed the common product ions corresponding to the loss
distinct product ions (Fig. 5). The spectrum of 7i + included the
product ions at m/z 72, 71, 58 and 43 (base peak) corresponding
to the losses of C3H6, (due to McLafferty-type rearrangement),
•C3H7 radical, C4H8 and (C4H8 + •CH3), respectively. The spectrum
of 8i + showed the specific product ions at m/z 99, 85 and 84 corre-
•
of H2, CH3 radical, CH4, (N,C2,H5) and HN(CH3)2 at respective
m/z values. The spectrum of 13i+ showed product ions at m/z 91
(+C7H7), 77 (+C6H5) and 65 (+C5H5) confirming presence of a phenyl
ring. Similarly, the spectrum of 14i+ showed m/z 105 (C8H9+) and 79
(C6H+7) due to benzyl group and that of 15i + showed m/z 121
(C8H10O+) and 107 (C7H8O+) due to -CH2PhOH group.
•
•
sponding to the losses of CH3 radical, C2H5 radical and C2H6, re-
spectively. Additionally, the spectrum of 8i + included an
abundant product ion at m/z 70 because of the loss of a •CH3 radical
from [8i-C2H5]+• ion or •C2H5 radical from [8i-CH3]+• ion. The spec-
trum of 8i + also displayed abundant product ion because of HN
17i+ (m/z 116), 18i+ (m/z 130). The CID spectrum of immonium ion
of N,N-dimethylaspartic acid (17i+) showed the characteristic prod-
uct ions at m/z 98 (base peak), 88, 74, 73, 72, 71, 70, 57, 56 and 42
corresponding to the losses of H2O, CO, CH2CO, (C2, N, H5), CO2,
HN(CH3)2, (H2O + CO), (CO2 + •CH3), (CO2 + CH4), and (CO2 + C2H6),
respectively. The CID spectrum of immonium of N,N-
dimethylglutamic acid (18i) showed the product ions at m/z 86,
84, 71, 70, 56 and 42 corresponding to the losses of CO2,
•
(H2O + CO), CH2COOH radical, CH3COOH, (H2O + CO+ C2H4), and
(CH3COOH + C2H4), respectively.
19i+ (m/z 115), 20i+ (m/z 129). The CID spectrum of immonium ion
of N,N-dimethyl asparagine (19i +) showed characteristic product
ions at m/z 98, 72, 70, 57, 55, 44 and 42 corresponding to the losses
of NH3, (O¼C¼NH), (HCONH2; specific), (O¼C¼NH + •CH3),
(HCONH2 + •CH3), (O¼C¼NH+ C2H4) and (HCONH2 + C2H4), respec-
tively. The immonium ion of asparagine showed the loss of NCO,
but this loss was absent in the spectrum of 19i +. The spectrum of
immonium ion of N,N-dimethylglutamine (20i +) included the char-
acteristic product ions at m/z 112, 101 and 84 corresponding to the
losses of NH3, CO and HCONH2, respectively. The loss of HCONH2
was predominant in the spectrum of 20i+, while the loss of NH3
was dominant from the immonium ion of glutamine.
21i+ (m/z 138). The CID spectrum of immonium ion of N,N-
dimethylhistidine (21i +) showed product ions at m/z 123, 122
Figure 5. Collision-induced dissociation mass spectra of immonium ions of
m/z 114 (a) N,N-dimethylleucine and (b) N,N-dimethylisoleucine at collision
energy of 25 eV.
•
and 95 (dominant) corresponding to the losses of CH3, CH4 and
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ESI-MS/MS of dimethyl amino acids
Figure 6. Collision-induced dissociation mass spectra of (a) Met-Leu dipeptide inset shows after dimethylation of dipeptide, (b) immonium ion of m/z 132
formed at declustering potential of 150 V from N,N-dimethyl Met-Leu dipeptide and (c) immonium ion from N,N-dimethylmethionine.
(C2, N, H5), respectively. Consecutive loss of two HCN moieties from
the ion m/z 95 were also present that reflects the presence of imid-
azole group in the side chain. The immonium ion of histidine specif-
ically showed loss of NH3, HCN and (2HCN).
Further, CID experiments on the immonium ions provide the infor-
mation on the methylated N-terminal amino acid. To evaluate the
methodology of methylation followed by immonium ion CID exper-
iment for the determination of N-terminal amino acid in the pep-
tides, we have selected a dipeptide and a tetrapeptide and
performed the experiments. The two peptides (Met-Leu and
Lys-Ala-Ala-Ala) were subjected to methylation by applying a simi-
lar procedure used for methylation of free amino acids. The ESI-MS
spectrum of the original dipeptide (Met-Leu) showed the ion at
m/z 263 corresponding to [M + H]+ ion, and this ion was shifted
by +28 u after methylation reaction and appeared at m/z 291, which
indicates that the peptide was dimethylated at N-terminal amino
acid. Similarly, the [M + H]+ ion of the tetrapeptide (Lys-Ala-Ala-
Ala) shifted from m/z 360 to m/z 416 (+56u) upon methylation,
which correspond to a tetramethylated peptide. As lysine is the
N-terminal amino acid, both the amines might have undergone
methylation resulting in a tetramethylated product.
22i+ and 23i+ (m/z 157). The CID spectrum of immonium ion of N,
N-dimethylarginine (22i) showed product ions at m/z 112, 98 and
70 (base peak) corresponding to the losses of HN(CH3)2,
HN¼C(NH2)2 and [HN(CH3)2 + HN¼C¼NH], respectively, due to
guanidine group. The spectrum of 23i + showed product ions at
m/z 114 (base peak), 112 and 84 corresponding to losses of (C2, N,
H5), HN(CH3)2 and (HN(CH3)2 + C2H4), respectively. The immonium
ion of Nα,Nα,Nε,Nε-tetramethyllysine (23i) is isobaric with the 22i+,
but their CID spectra were distinctive.
Determination of N-terminal amino acids of the peptides
The immonium ions generated during peptide fragmentation can
also provide information about the posttranslational modifications,
especially the N-terminal modifications. For example, N-terminal
modified peptides (methylated/acetylated) showed the immonium
ions with a mass shift of +28/+42u, when compared with unmod-
ified peptides.[34,35] As methylation of protein/peptide at the amino
group of N-terminal amino acids is known in the literature, it is ex-
pected to get corresponding N-methylated immonium ions during
the CID experiments on methylated peptides. In such cases, the CID
of immonium ions of methylated amino acids discussed in the ear-
lier section would be useful in characterization of the actual
N-terminal amino acid methylated in a protein/peptide. In this
experiment, initially the target protein/peptide is subjected to
methylation by which the amino group of N-terminal amino acid
undergoes dimethylation; in the next step, the methylated
protein/peptide is subjected to in-source fragmentation in which
the immonium ion of N-terminal modified amino acid is generated.
When the methylated peptides were subjected to in-source frag-
mentation, the methylated dipeptide showed specific immonium
ion at m/z 132, whose CID spectrum matched with the immonium
ion of N,N-dimethyl methionine (Fig. 6). Similarly, the modified tetra
peptide showed the specific immonium ion at m/z 157; the CID
spectrum of this ion matched well with that of the immonium ion
of tetramethyllysine. These experiments on the peptide clearly con-
firmed that the free amino groups of the peptide (including
N-terminal amino acid) undergo methylation, and it is possible to
determine the N-terminal amino acid of an unknown peptide based
on the CID spectra of corresponding immonium ions (methylated).
Conclusions
In this study, dimethyl α-amino acids, including a few β-amino acids
and γ-amino acids were successfully synthesized and characterized
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V. Naresh Chary et al.
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by ESI-MS and ESI-MS/MS. The MS/MS of protonated N,N-dimethyl
amino acids and their immonium ions showed structure indicative
product ions. This study also shows that isomeric/isobaric natural
amino acids can be clearly differentiated from dimethyl amino
acids in biological matrices. The nature of amino acid (aliphatic,
aromatic, acidic and basic) and methyl groups on nitrogen is very
much reflected in the fragmentation of protonated N,N-dimethyl
amino acids and their immonium ions. The immonium ions
formed from the isomeric N,N-dimethyl amino acids and free amino
acids also showed clear-cut differences in their CID spectra. This
study also helps in the discrimination of isomeric immonium ions
(e.g., leucine/isoleucine; lysine/glutamine) and screening of methyl-
ated peptides/proteins in post translation modification studies
using MS/MS of immonium ions. The methodology can also be
applied to determine the N-terminal amino acid of peptides.
Acknowledgements
The authors thank Dr M. Lakshmi Kantam, Director, CSIR-IICT for
facilities and encouragement. The authors acknowledge financial
support to the CSC-0110 (CMET) project by the Council of Scientific
and Industrial Research (CSIR), New Delhi. V. N. C and C. D.
thank CSIR, New Delhi for providing a senior research fellowship
and B. S. R. thank UGC, New Delhi for providing a junior research
fellowship.
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Supporting information
Additional supporting information may be found in the online
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