Functional dihydro-1H-imidazole derivatives for MALDI signal enhancement of a lysine-specific chemical modification

Article abstract of DOI:10.1002/jccs.200900144

The enhancement of signal sensitivity and quantification of low-abundance proteins is of great importance in proteomic analysis. The preparation of 2-methoxy-4,5-dihydro-1H-imidazole (MeO-DHIM) derivatives was accomplished by one-step synthesis of O-methyl-isourea. The signal enhancement induced by these attached moieties increases in an order of compound 1 ≈ 4 < 2 < 5 ≈ 6 < 3 in MALDI mass spectrometry. Peptide-compound 3 adduct was approximately 20 times signal enhancement of the unmodified peptide and of 9 times of the peptide-compound 1 adduct. This result demonstrates that the rational designed organic molecules are capable of providing a sensitive tool in the detection of low-abundance proteins in proteomics.

Full text of DOI:10.1002/jccs.200900144

Journal of the Chinese Chemical Society, 2009, 56, 987-994
987
Functional Dihydro-1H-imidazole Derivatives for MALDI Signal
Enhancement of a Lysine-Specific Chemical Modification
Ming-Yi Hoa (
), Yu-Tse Shieha (
), Chung-Lin Liaob (
),
Yau-Hung Chenc (
) and Chien-Chung Chengd* (
)
aDepartment of Chemistry, Tamkang University, Tamsui 25137, Taiwan, R.O.C.
bGenomics Research Center, Academia Sinica, Taipei 11529, Taiwan, R.O.C.
cGraduate Institute of Life Sciences, Tamkang University, Tamsui, Taipei 25137, Taiwan, R.O.C.
dDepartment of Applied Chemistry, National Chia-Yi University, Chia-Yi City 60004, Taiwan, R.O.C.
The enhancement of signal sensitivity and quantification of low-abundance proteins is of great impor-
tance in proteomic analysis. The preparation of 2-methoxy-4,5-dihydro-1H-imidazole (MeO-DHIM) de-
rivatives was accomplished by one-step synthesis of O-methyl-isourea. The signal enhancement induced
by these attached moieties increases in an order of compound 1 » 4 < 2 < 5 » 6 < 3 in MALDI mass spec-
trometry. Peptide-compound 3 adduct was approximately 20 times signal enhancement of the unmodified
peptide and of 9 times of the peptide-compound 1 adduct. This result demonstrates that the rational de-
signed organic molecules are capable of providing a sensitive tool in the detection of low-abundance pro-
teins in proteomics.
Keywords: MALDI; Dihydroimidazole; Lysine-specific; Guanidinium; Signal enhancement;
Mass spectrometry.
The enhancement of detection sensitivity and quanti-
fication of low-abundance proteins are of great importance
in proteomic analysis.1-3 Peptide mapping experiments
usually employ mass spectrometry;4-6 for example, peptide
mass fingerprinting with MALDI-TOF or ESI-MS/MS tan-
dem mass spectrometry, in combination with other separa-
tion technologies such as CE and HPLC, is routinely per-
formed. Although many sophisticated methods, such as 2D
gel electrophoresis and multi-dimensional protein identifi-
cation technology (MudPIT), attempt to resolve the thou-
sands of proteins of living organisms, only some hundreds
of proteins can be identified because of large disparities in
the concentrations of expressed proteins. Low-abundance
proteins are sometimes important biomarkers in disease di-
agnosis, as well as in the monitoring of disease progression
and drug responsiveness; e.g., prostate-specific antigen
(PSA), a marker of prostate cancer.7 Therefore, the detec-
tion and analysis of low-abundance proteins is a challeng-
ing but vital topic in proteomics.8,9
Aside from techniques for the enrichment of target
proteins and effective cell-fractionation steps prior to such
enrichment, low-abundance proteins may be more readily
detected after attachment of a charged species. For in-
stance, an arginine-containing peptide, attached to peptide
fragments obtained by trypsin digestion, was found to in-
crease detection sensitivity of MALDI-TOF mass spec-
trometry by 4-8-folds.10-14 Recently, significant enhance-
ments in signal sensitivities were achieved using a cyclic
guanidinium group as a charge carrier (Fig. 1). In addition,
the isotopes were incorporated into the framework of the
Fig. 1. General concept of signal enhancement in proteomic analysis.
* Corresponding author. Tel: +886-5-276-6084; Fax: +886-5-271-7901; E-mail: cccheng@mail.ncyu.edu.tw

988 J. Chin. Chem. Soc., Vol. 56, No. 5, 2009
Ho et al.
cyclic guanidinium group to act as internal standards for
protein quantification. The preparation of 2-methoxy-4,5-
dihydro-1H-imidazole (MeO-DHIM) was accomplished
via the formation of O-methyl-isourea.14-18 The modified
guanidinium species coupled to lysine-containing residues
in tryptic peptides of horse myoglobin; however, no further
details on chemical structures, nor signal enhancements of
peptide fragments after coupling, were reported.
ence of glacial acetic acid, refluxing at 40 °C for 5 h. The
cyclization was quenched with water, followed by extrac-
tion with CH2Cl2, drying with MgSO4, and evaporation of
solvent to obtain desired compounds in reasonable yields
(60-85%), as shown in Scheme I. In MALDI mass spec-
trometry, analyzes are believed to receive a proton from the
chemical matrix such as aCHCA to become a positive-
charged molecule. Therefore, signal enhancement of a pep-
Here, we report a one-step ring-closing synthesis for
the preparation of a variety of 2-ethoxy-4,5-dihydro-1H-
imidazoles (EtO-DHIMs), all with functional groups, in
reasonable yields. Ethoxy-dihydroimidazole derivatives
are capable of substituting a suitable amino group to form a
cyclic guanidinium group. Moreover, we show that signal
enhancements of tryptic digestion peptides are related to
the chemical properties of the enhancers, including basicity
and the potential for stabilization of the protonated species.
Therefore, a functionalized guanidinium group, used as a
conjugated marker, may increase the number of low-abun-
dance proteins that can be detected in and characterized by
proteomic techniques.
One-step synthesis of 2-ethyl-1,3-dial-
kylisourea derivatives with various func-
tional groups
Scheme I
A typical procedure for the one-step synthesis of
EtO-DHIM derivatives was followed. This involved con-
densation of three equivalents of tetraethyl orthocarbo-
nate19 and one equivalent of diamine in CH2Cl2 in the pres-
Fig. 2. Observed mass of peptide-DHIM adducts, by MALDI-TOF mass spectrometry, to confirm the formation of cyclic
guanidinium moieties. The model peptide with m/z 1271.66 was isolated from a tryptic digest of horse myoglobin.

Modified-imidazoles Enhances the Signal in MALDI
J. Chin. Chem. Soc., Vol. 56, No. 5, 2009 989
tide fragment in mass spectrometry can be achieved if the
analyte were to carry a stable charge or if the analyte showed
a high basicity favoring protonation.20 To this end, meth-
ylation of the amino group of Compound 2 was carried out
to increase the basicity of DHIM. In addition, six-mem-
ber-ring analogs, Compounds 3 and 4, were prepared to
offer improved resonance forms after protonation. Com-
pounds 5 and 6 were synthesized to increase hydrophobic
characteristics and to introduce chirality, to permit us to ex-
plore the influence of chirality on reactivities with peptide
fragments. The more hydrophobic Compound 718 was eas-
ily prepared by a ring-closing reaction, but had extremely
low solubility in water and low reactivity in coupling with a
peptide fragment. A typical structural analysis of DHIM
derivatives revealed that the ethoxy group appeared at ca.
4.1 ppm as a quartet-splitting peak and at ca. 1.5 ppm as a

990 J. Chin. Chem. Soc., Vol. 56, No. 5, 2009
Ho et al.
Fig. 3. Fragment analysis of peptide-DHIM adducts in MALDI-TOF/TOF mass spectrometry, to confirm the formation of
y-ions and lysine-specific attachment.

Modified-imidazoles Enhances the Signal in MALDI
J. Chin. Chem. Soc., Vol. 56, No. 5, 2009 991
triplet-splitting peak in NMR spectra.
nant y ion series through a C-terminal lysine derivatization.
Through the chemical modification of lysine residue, it
seems to change ion fragmentation pattern in MALDI-TOF/
To explore possible signal enhancements by these
compounds in MALDI-TOF mass spectroscopy, a model
peptide with 11 amino acid residues (LFTGHPETLEK),
purified from a horse myoglobin tryptic digest,6 was al-
lowed to react with different DHIM derivatives as shown in
Fig. 2. Because trypsin specifically23 cleaves at the C-ter-
mini of lysine and araginine, the model peptide with a
lysine at one end shows up as a protonated molecular ion,
[M+H]+, with a mass-charge ratio (m/z) of 1271.66, as a
reference peak in MALDI-MS experiments.12 This model
peptide has been well characterized by proteomic analysis
after reaction of Compound 1 with trypsin-digested pep-
tides of myoglobin.11 A peptide-DHIM adduct was pre-
pared by reacting an EtO-DHIM derivative in an aqueous
2.5 M NH4OHin 50% v/v MeOH solution at 40 °C for 16 h,
and then purified on a C18 reverse-phase HPLC column to
give a yield of 5-77%. The target peptide was chemically
modified at the side-chain amino group of lysine using a se-
ries of y-ions, as confirmed by MALDI-TOF/TOF21 (see
Fig. 3). Compound 111 was found to attach to both the ly-
sine residue and the N-terminal amino group of the peptide
fragment; however, Compounds 2-5 reacted with the pep-
tide fragment to yield only lysine-specific adducts without
modification of the N-terminus. From MALDI tandem
spectra (see Fig. 3), we observed fragment ions form domi-
TOF experiments. Asynthesis peptide (NH2YKELGFQGamide
)
exhibited the same proclivity for near exclusive formation
of a ions and especial y7 and b2-NH3 ions while modified
lysine residue was near c-terminal of peptide (data not
shown). It’s found all observed fragment ions contain mod-
ified lysine residue. This evidence supports all modified
lysine moiety were strong proton carrier, indirectly ex-
plained MALDI signal enhancement through proton affin-
ity increased. It also simplified tandem mass spectra, make
peptide denovo sequencing more easily.
The above results suggest that the introduction of
alkyl groups increases steric effects and results in regio-se-
lectivity of the coupling reaction. The chiral sense change
between Compounds 5 and 6 did not influence the yields or
reactivities of peptide-DHIM adducts. The observed masses
of the adducts are shown in Fig. 2. The retention times of
the peptide-DHIM adducts of Compounds 1-5, monitored
at 214 nm in HPLC, were 12.02, 12.22, 12.89, 11.39, and
14.36 min (see Fig. 4). It is interesting to note that an at-
tached small (68-122 Da) chemical moiety is capable of
dominating the polarity of a large (1270 Da) peptide.
A comparison of signal enhancements by different
chemicals requires that the amounts of protein-adducts
Fig. 4. Reversed-phase HPLC profile of model peptide tethered to different cyclic guanidino moieties.

992 J. Chin. Chem. Soc., Vol. 56, No. 5, 2009
Ho et al.
Fig. 5. (A) Relative signal intensities of model peptide tethered to different cyclic guanidinium moieties in MALDI-TOF
mass spectrometry. (B) Relative signal intensities in a bar-graph format. These experiments were repeated at least
three times.

Modified-imidazoles Enhances the Signal in MALDI
J. Chin. Chem. Soc., Vol. 56, No. 5, 2009 993
must be normalized. Because yields after lysine condensa-
tion varied, peptide-DHIM adducts were further isolated
and purified by HPLC on a reverse-phase C18 column. To
quantify the concentration of each isolated adduct, capil-
lary LC with a fixed absorption wavelength at 214 nm was
used to assay each sample. Peptide-DHIM adducts were
thoroughly mixed at similar concentrations and then sub-
jected, as a mixture, to MALDI-TOF analysis. The proce-
dure was repeated at least three times. Signal enhancements
induced by the attached moieties increased in the order of
Compound 4 < 1 < 2 » 5 » 6 < 3, as shown in Fig. 5. The
more hydrophobic peptide-DHIM adducts, as indicated by
polarity data from HPLC, displayed better signal enhance-
ments. The opposing chiralities of Compounds 5 and 622
did not significantly influence yields from coupling reac-
tions or signal enhancements. The simplicity with which
charged species may be formed is of fundamental impor-
tance in attempts to increase signal intensities in MALDI-
TOF analysis. The proton source is usually supplied by the
matrix, such as a-cyano-4-hydroxycinnamic acid (aCHCA)
in MALDI experiments. The methylated Compound 2 was
synthesized to provide an electron-donating group favor-
ing protonation. As a result, Compound 2, containing a cy-
clic guanidinium group, showed better signal enhancement
(by 1.5-fold) than did Compound 1. Thus, increasing basic-
ity favors protonation resulting in signal enhancement.
The signal enhancement of the peptide-Compound 3
ration of a linker to be immobilized on a solid support,
where tethering could be monitored using a fluorophore.
Such work is ongoing in our laboratory.
In conclusion, signal responses in MALDI mass
spectrometry are intrinsic properties of fragment pep-
tides. Low-abundance protein characterization can be pep-
tide-dependent; however, chemical conjugation of a pep-
tide provides a strategy whereby a peptide fragment can
gain a charge, permitting mass spectrometry signals to be
amplified. Such modifications of peptides may eliminate
tedious sample accumulation processes, conventionally re-
quired before analysis of low-abundance proteins could
commence. In this paper, a simple synthetic route for the
preparation of functionalized cyclic diamine compounds is
presented. Furthermore, guanidinium species generated by
condensation of our compounds with lysine residues of a
peptide fragment can enhance signal intensities in MALDI-
TOF tests. Modifications of peptides, to increase basicity
or to stabilize desirable resonance forms, are important to
improve signal intensities in MALDI mass spectrometry
experiments. The synthetic method we describe is a simple
route for the introduction of other functional groups24 such
as ICAT25 and ITRAQ,26 for increasingly sensitive quanti-
fication of peptide fragments.4,5,27
ACKNOWLEDGMENTS
The authors thank Prof. Hsiu-Fu Hsu at Tamkang
University for kindly reviewing the manuscript. We grate-
fully acknowledge the financial support from National
Chia-Yi University, Tamkang University, and the National
Science Council of Taiwan.
adduct was approximately 15.1 times that of the unmodi-
fied peptide and 1.6 times that of the peptide-Compound 1
adduct (Fig. 5). The significant enhancement afforded by
Compound 3 compared with Compound 1 is ascribed to
lower ring strain and better resonance after protonation, to
stabilize the positive charge. This enhancement may allow
more proteins of low abundance to be detected in peptide
mapping experiments. Compound 4 was observed to en-
hance signals to a level comparable to that seen with use of
Compound 1. The hydroxyl group in the six-membered
ring increases the polarity of a peptide-DHIM adduct, as
demonstrated by the earlier retention time from HPLC;
however, a hydroxyl group is known to interfere with pro-
tonation in mass spectrometry, resulting in suppression of
signal intensity. This result suggests that signal suppres-
sion by a hydroxyl group can be overcome by suitable
structural modification. Therefore, modification of the
hydroxyl group of Compound 4 may well provide a variety
of functional analogs that do not cause signal suppression.
For example, O-alkylation is a possible strategy for prepa-
Received December 18, 2008.
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