Label-assisted laser desorption/ionization mass spectrometry (LA-LDI-MS): An emerging technique for rapid detection of ubiquitous cis-1,2-diol functionality

Article abstract of DOI:10.1039/c4ra07499h

The Label-Assisted Laser Desorption/Ionization (LA-LDI) technique has recently been applied to the detection of small molecules through a Time of Flight (TOF) mass spectrometric measurement. By excluding the external matrix, the mass spectrum becomes a lot cleaner being free of matrix related peaks and noise. In this paper we report a Label-Assisted Laser Desorption/Ionization Mass Spectrometry (LA-LDI-MS) based method for detection of various cis-1,2-diols, including the ones ubiquitous in biological samples. This journal is

Full text of DOI:10.1039/c4ra07499h

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Label-assisted laser desorption/ionization mass
spectrometry (LA-LDI-MS): an emerging technique
for rapid detection of ubiquitous cis-1,2-diol
functionality†
Cite this: RSC Adv., 2014, 4, 46555
*
Partha Sarathi Addy, Ahanjit Bhattacharya, Santi M. Mandal and Amit Basak
The Label-Assisted Laser Desorption/Ionization (LA-LDI) technique has recently been applied to the
detection of small molecules through a Time of Flight (TOF) mass spectrometric measurement. By
excluding the external matrix, the mass spectrum becomes a lot cleaner being free of matrix related
peaks and noise. In this paper we report a Label-Assisted Laser Desorption/Ionization Mass Spectrometry
(LA-LDI-MS) based method for detection of various cis-1,2-diols, including the ones ubiquitous in
biological samples.
Received 23rd July 2014
Accepted 3rd September 2014
DOI: 10.1039/c4ra07499h
www.rsc.org/advances
of this method over other available methods in the literature.
Although, so ionization based methods like Desorption Elec-
Introduction
trospray Ionization MS has been described for small molecules
by Cooks et al.,11 LA-LDI-MS offers the advantage of giving
highly intense molecular ion peaks, also being very selective at
the same time. Recently12 we have reported LA-LDI-MS method
for selective detection of Zn2+. In the present work, we have
applied the same method for detection of biologically impor-
tant cis-1,2-diols especially catecholamine neurotransmitters
dopamine and epinephrine thus demonstrating the scope of
this technique. 1,2-Diol functionality is important because of its
presence in various molecules of biological relevance.
Abnormal levels of dopamine and its metabolites in cerebro-
spinal uid (CSF) are implicated in several neurodegenerative
diseases, most notably Parkinson's disease.13 Also, other bio-
logically important molecules like ascorbic acid, citric acid and
simple sugars were studied.14
Commercially available MALDI-TOF-MS instruments are
equipped with UV lasers. On laser irradiation, the pyrene label
readily generates radical cation, which is detectable in MS. This
happens due to high molar absorptivity of pyrene or other
polyaromatic compounds. Based on the aforementioned facts,
we selected pyrene-1-boronic acid (PBA) as a selective probe for
identication of the cis-1,2-diol molecules (Fig. 1).
Identication of biologically important small molecules is an
important goal of bioanalytical chemistry1 having vast applica-
tions in medical diagnostics, microbiology, pharmacology2 and
environmental sciences. Traditionally, techniques like UV-Vis
Spectroscopy,3 Fluorescence Spectroscopy,4 Liquid Chromatog-
raphy-Mass Spectrometry5 and High Performance Liquid
Chromatography6 have been used for this purpose. Other
methods include surface plasmon resonance based optical
sensors7 and microuidics based sensors.8 Mass Spectrometry
can be a useful method for proling a mixture of molecules.
Recently in 2013, a novel method for reaction monitoring has
been established by Kozmin et al.9 using Label-Assisted Laser
Desorption/Ionization Time of Flight Mass Spectrometry
(LA-LDI-TOF-MS). In this technique, detection of small mole-
cules was carried out using Time of Flight Mass Spectrometer –
an instrument which is typically used for detection of macro-
molecules by MALDI.10 The most important merit of this
method is its high selectivity. Encouraged from this work we
envisioned that a pyrene based boronic acid could be useful for
the detection of various cis-1,2-diols. The selectivity of the
method lies in its working principle. It is only the polyaromatic
associated species which will undergo desorption/ionisation
and show their presence in the mass spectrum. That is why, cis-
1,2-diols, which form complexes with the pyrene based boronic
acid should be detected selectively in LA-LDI MS. Other
unreacted diols or unreacted contaminants will not interfere in
the mass spectrum. This selectivity issue is the main advantage
Results and discussion
Commercially available PBA was directly used as a probe for
cis-1,2-diols without further modication. Its solution was
prepared in phosphate buffer (0.1 M, pH 7): THF (1 : 1). The
mass spectrum recorded in positive mode showed an intense
molecular ion peak at m/z 246. Under the given conditions,
it also gives an intense fragmentation peak at m/z 218
IIT Kharagpur, Chemistry, Kharagpur, West Bengal, India
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ra07499h
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Fig. 1 Pyrene boronic acid and expected complexation behavior with
cis-1,2-diols.
Fig. 4 ‘Capture’ complexes of catechol and related molecules.
corresponding to pyren-1-ol, presumably generated by aerial
oxidation during spotting (Fig. 2).15
PBA. These include simple diols like ethylene glycol and tartaric
acid to biologically relevant molecules like dopamine,
epinephrine, L-DOPA and ascorbic acid. LDI mass spectra were
recorded in both positive and negative ion modes (Table 2, see
ESI†). An interesting observation was that while ethylene glycol
(see ESI† for spectrum), dopamine and epinephrine gave
molecular ion peaks for the respective boronate complexes in
The ability of PBA to complex with cis-1,2-diols and subse-
quent detection of the complex by LDI-MS was then proved. As
an initial attempt, catechol was chosen as the representative cis-
1,2-diol because of its well-known complexation behavior with
boronic acid. A solution of PBA (2.5 mM) in phosphate buffer
(pH 7.0)-THF mixture (1 : 1) was incubated (1 h) with a solution
of catechol in THF (1 mM). LDI-MS recorded on the mixture
showed a strong peak at m/z 320 (Fig. 3) corresponding to the
boronate complex A (in Fig. 4).
The experiment was repeated with 2,3-dihydroxynaph-
thalene and the peak at m/z 370 was found in LDI-MS (Table 1,
see ESI†) corresponding to the expected complex B (for spec-
trum see ESI†).
In order to extend the scope of the method, several other
compounds with cis-1,2-diol functionality were screened with
Fig. 5 ‘Capture’ complexes of tartaric acid, ascorbic acid and citric
acid.
Fig. 2 LDI mass spectrum of PBA (+ve mode).
Fig.
6 Structures of various possible species derived from PBA
Fig. 3 LDI mass spectrum (+ve mode) of PBA–catechol complex observed in different mass spectra. Sometimes the deprotonated form
(molecular ion peak at m/z ¼ 320).
46556 | RSC Adv., 2014, 4, 46555–46560
of the same species arrived in negative mode of the spectra.
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positive ion mode, the corresponding complexes E and F of
tartaric and ascorbic acid respectively (Fig. 5), could be detected
in the negative ion mode. One other point to note is the
appearance of peaks corresponding to free analytes in the mass
spectra recorded in the negative ion mode. For example, for
ascorbic acid, the peak at m/z 175 and for L-DOPA, the peak at
m/z 196 corresponding to respective molecular ions could be
observed. We believed that the unreacted PBA acted as a matrix
which in turn, helped the desorption cum ionization of the
analytes. The various molecular ions from different analytes
along with the proposed structures of the boronate complexes
are shown in Fig. 4–6.
Fig. 9 LDI mass spectrum (ꢁve mode) of PBA–L-DOPA complex.
As representative examples, LDI mass spectra of boronate
complexes of dopamine, epinephrine, L-DOPA and ascorbic acid
are shown in Fig. 7–10. Besides the molecular ion peak at m/z
363 (C), dopamine showed fragmentation peaks at m/z 347 (M)
+
and m/z 333 (N), possibly due to loss of NH2c and CH2]NH2 c
respectively (Scheme 1).
Mass spectrum of epinephrine did not show the molecular
ion peak at m/z 393. Instead it gave a prominent fragmentation
peak at m/z 376 (shown in inset in Fig. 8), due to the loss of
benzylic –OH. There are minor peaks at m/z 474 and m/z 684,
which can be attributed to formation of dimer and trimer
respectively due to self-condensation of PBA (structures I and J,
in Fig. 6). For ascorbic acid, the MS spectrum, recorded on a
negative mode, showed a peak at m/z 385 assigned for the
complex F. It also showed additional peaks at m/z 613 which is
attributed to the bis-complex G. We have also recorded the
complexation behaviour of citric acid, an a-hydroxy acid, with
Fig. 10 LDI mass spectrum (ꢁve mode) of PBA–ascorbate complex.
Scheme 1 Molecular ion and fragment species from dopamine.
PBA, which showed the molecular ion peak at m/z 401 corre-
sponding to the complex H (for spectrum, see ESI†).
Having been successful with the LDI-MS based detection of
small molecules including catecholamine neurotransmitters,
we turned our attention to sugars. Detection of simple sugars
and their derivatives is an important part of analytical
biochemistry, having applications in clinical diagnosis, eluci-
dation of biological pathways and reaction mechanisms. Two
representative examples, namely D-glucose and D-ribose were
chosen. Although stereochemically pure sugars were used for
analyses, they have complex solution chemistry, especially in
alkaline medium and exist as equilibrium of various cyclic and
acyclic forms. So, the exact nature of the captured complexes
was not determined. Mass spectra were recorded in both posi-
tive and negative ion modes. For ribose, ions were detected in
positive mode only at m/z 360 (100% intensity) and m/z 570
Fig. 7 LDI mass spectrum (+ve mode) of PBA–dopamine complex.
Fig. 8 LDI mass spectrum (+ve mode) of PBA–epinephrine complex. (ꢀ20% intensity) – corresponding to complexation with one and
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two molecules of PBA respectively (for spectrum see ESI†).
We have also checked the possible interference by glucose on
However, for glucose, the situation was more complex. In the the detection of dopamine whose complex with PBA showed up
positive ion mode, the spectrum showed a peak at m/z 600 while only in the positive ion mode. Solutions of dopamine of
in the negative ion mode, a peak appeared at m/z 599. With different strengths (100 mM, 50 mM, 10 mM) were kept incubated
time, the intensity of peak at m/z 600 decreased while that at m/z with PBA (2.5 mM) in presence of large excess of glucose
599 increased.16 The latter can arise through loss of one proton (10 mM). Peaks for the dopamine–PBA complex showed up in
from the doubly-complexed glucose molecule. However, the loss the positive ion mode LDI-MS even when glucose is present in
of a proton from the doubly complexed species appears to be 1000-fold excess by molar ratio. Thus presence of glucose has no
intriguing since there is apparently no signicantly free ionis- interference with the detection of dopamine. The peaks for the
able group in that species. This can be explained based on a glucose–PBA complex at m/z 599 were obtained in negative ion
similar observation reported by Norrild et al.,17 in which, all ve mode (all relevant spectra are included in ESI†).
–OH groups of a-glucofuranose are reported to be involved in
Sensitivity of detection of dopamine was also determined
complexation with overall loss of a proton. We believe that the thereaer using similar reaction conditions. In presence of
peak at m/z 600 arises due to bis-complexation with a-gluco- 2.5 mM pyrene-1-boronic acid, it was found that dopamine is
pyranose (structure “O”, Scheme 2), which in aqueous medium detectable in the concentration as low as 5 mM, which amounts
slowly converts to the thermodynamically more stable a-gluco- to 5 picomoles per 1 mL spotting (Fig. 12).18
furanose complex with m/z 599 via mutarotation followed by
One more point that needs to be mentioned here. Apart from
coordination of the C3–OH to the boron centre and concomitant selectivity, the LA-LDI MS has advantage over MALDI-MS for the
loss of a proton to generate structure “P”. The latter assumes a detection of small molecules in terms of quality of the spectra.
negative charge and the species shows a peak at m/z 599 This can be demonstrated by comparing the spectra of the same
recorded in the negative ion mode.
analyte, dopamine, using these two techniques separately. The
This was further veried by recording the LA-LDI-MS of LA-LDI mass spectrum (Fig. 13(i)) clearly shows the peaks for
aliquots taken out from the incubating mixture at different dopamine (complexed with PBA) at m/z 363, 347 and 333 along
time points in both positive and negative ion modes (Fig. 11). with pyren-1-ol (m/z 218) originating from PBA. The MALDI
There was a gradual decrease of peak intensity at m/z 600 (O) mass spectrum (Fig. 13(ii) and (iii)) shows the peaks from
with concomitant increase of the peak intensity at m/z 599 (P) dopamine at m/z 154 (dopamine + H+), 137 (from a-cleavage of
thus supporting the glucopyranose to glucofuranose NH2) and 123 (from benzylic cleavage); however, appearance of
conversion.
several other peaks arising from the matrix make the assign-
ment difficult. The analysis may become even more uncertain if
masses of the fragments arising from the matrix have same or
very close m/z values to those of the analyte. Between MALDI
and LA-LDI, MALDI is more sensitive as indicated by the
absolute intensity for same concentration of dopamine. This is
understandable as the matrix is used in large excess. However,
Scheme 2 Structures of two complexes of glucose.
Fig. 11 Time dependent variation of intensities of peaks at m/z 600
(+ve mode) (left panel) and m/z 599 (ꢁve mode) (right panel); time of
data recording: 30 min (red), 4 h (pink), 8 h (violet), 12 h (blue), 24 h Fig. 12 Concentration dependent variation of intensities of peaks for
(black).
dopamine at m/z 363, m/z 347 and m/z 334 (+ve mode).
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Fig. 15 LA-LDI (+ve mode) spectrum of the synthesized probe and
dopamine complex (intensity is much higher, and fragmentation is
much less).
Fig. 13 LDI MS (+ve mode) of dopamine (50 mM) taken in presence of
(i) PBA (ii) external matrix CHCA and (iii) external matrix + PBA (the
yellow shades show the relevant peaks (matrix : analyte ¼ 4000 : 1
(molar ratio).
LA-LDI has the following advantages: it gives much cleaner
spectrum; the spectrum mainly shows the peak for the complex
while in MALDI, the peaks from the analyte and from the matrix
may overlap as can be seen in case of dopamine (Fig. 13). We
believe that the sensitivity of LA-LDI can be increased further by
using a label with larger polyaromatic network. We are currently
checking other polyaromatic labels. We have already synthe-
sized phenyl ethynyl pyrene boronic acid (PEBA). Initial studies
showed that the spectra are much cleaner and ions due to dimer
or trimer are much less (Fig. 14 and 15).
Fig. 16 LDI MS of a mixture dopamine, epinephrine, L-DOPA, glycine
and L-alanine incubated with PBA and recorded on (i) positive ion
mode (ii) and negative ion mode.
mixture. Thus we have recorded LA-LDI mass spectrum on a
mixture of dopamine, epinephrine, L-DOPA, glycine and
L-alanine dissolved in a solution of THF and buffer (1 : 1, pH 8).
To this solution PBA was added keeping its concentration
2.5 mM and the nal concentration of each neurotransmitter
was 30 mM. The solution was incubated for 1 h and 1 mL was
spotted for LDI MS study. In the positive ion mode we have
found peaks corresponding to dopamine (at m/z 363, 347 and
334) and epinephrine (at m/z 376). In the negative ion mode we
have found peak corresponding to L-DOPA (at m/z 406) (Fig. 16).
Since a biological uid will contain all the catecholamine
neurotransmitters as well as other small analytes, we were
curious to know whether the method can detect them all in a
Conclusion
Thus we have developed a new label-assisted LDI mass spec-
trometry based technique for the detection of small molecules
with cis-1,2-diol functionality via selective capture.19 The
Fig. 14 LA-LDI (+ve mode) spectrum of the synthesized probe.
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Acknowledgements
DST is acknowledged for an SERC grant and for JC Bose
National Fellowship to AB which supported this research. PSA is
grateful to CSIR, Govt. of India for a Senior Research Fellow-
ship. ANB thanks DST, Govt. of India for INSPIRE Undergrad-
uate Scholarship.
15 (a) A. N. Cammidge, V. H. Goddard, C. P. Schubert,
H. Gopee, D. L. Hughes and D. Gonzalez-Lucas, Org. Lett.,
2011, 15, 6034; (b) Y.-Q. Zou1, J.-R. Chen, X.-P. Liu,
L.-Q. Lu1, R. L. Davis, K. A. Jørgensen and W.-J. Xiao,
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16 Aer 24 h, the intensities of peaks at m/z 600 and at m/z 599
are ꢀ5% and ꢀ16% respectively. This observation indicated
a slow conversion of complex “O” to complex “P”.
17 M. Bielecki, H. Eggert and J. C. Norrild, J. Chem. Soc., Perkin
Trans. 1, 1999, 2, 449.
18 All the experiments were carried out using Voyager-DE PRO
instrument. Linear mode; UV laser: N2 LASER, 337 nm
wavelength; LASER Intensity 3200; LASER Rep Rate 20.0
Hz. Dopamine and epinephrine were procured in
injectable form as dopamine hydrochloride and
epinephrine bitartrate respectively. Solutions containing
0.03 mM of dopamine and epinephrine were incubated
with 1 mM of pyrene-1-boronic acid in PBS (0.1 M, pH 7) +
THF (1 : 1) at room temperature for 1 h. The mass spectra
were recorded in positive ion mode.
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