MEASUREMENT OF D-AMINOACIDS
Author: Amit Patel and
(University of Illinois at Urbana-Champaign)
SENSING AND DYNAMIC MEASUREMENT
Author: Elena Rosini
(Università degli Studi dell'Insubria)
|MEASUREMENT OF D-AMINOACIDS|
|The D-AAs are a class of signaling molecules which are largely understudied. The discovery and characterization of D-AAs in living systems necessitates the development, improvement and application of an assortment of analytical methods. Since the initial measurement of D-AAs in animals, advances in approaches for D-AA analysis have been numerous – including the development of capillary electrophoresis approaches with sufficient sensitivity to target sub-cellular samples, liquid chromatography approaches which simultaneously target many D-AAs and imaging approaches to uncover the localization of these molecules. These measurement approaches, when paired with appropriate experiment design and established animal models, have led to a variety of discoveries (including the characterization of novel enzymes, the establishment of two D-AAs as “classical” transmitters and a better understanding of many D-AAs in the nervous and endocrine systems of many animals).|
Capillary Electrophoresis (CE) is a separation technique which leverages the differences in ionic mobility of solvated ions in the presence of an applied electric field, where ionic mobility is dependent on the charge and stokes radius of the ions. CE has proven useful for the analysis of a variety biomolecules, ranging from single monosaccharides up to entire proteins. Relative to other separation techniques, such as liquid chromatography and gas chromatography, CE has a number of notable advantages — high efficiency, short separation times, and low sample volume requirements. The requirement for low sample volumes has allowed CE to characterize the DAAs in individual neurons.
In order to discriminate between the L- and D-forms of amino acids, chiral selectors are commonly employed. The differential interaction between the chiral forms of the analytes and the chiral selector result in their separation. Alternatively, CE without the use of such chiral selectors can also be used to separate and measure the amount of each enantiomer provided they are first reacted with an optically pure chiral reagent (resulting in the formation of diastereomers prior to analysis).
Capillary electrophoresis can be paired with a variety of detection modalities for D-AA analysis, where laser-induced fluorescence (LIF) and mass spectrometry (MS) are two of the more common means of detection. The employment of the aforementioned detection modalities provides sufficient sensitivity to enable the analysis of D-AAs in spite of limited sample volumes; yes, these analyses can even be carried out at the subcellular level!
resulting from the subcellular analysis of the processes and cell soma
of a single neuron from Aplysia.
Adapted from Miao, H., et al. J. Neurochem., 2006, 97(2), 595-606.
its advent, liquid chromatography (LC) has been a separation technique
choice for many analytical chemists. LC is well known as a robust
which separates different chemical species on the basis of different
times on a separation column as a consequence of differential
between the analytes, the liquid mobile phase and the stationary phase.
like the case described with CE, LC can discriminate on the basis of
by the employment of a chiral stationary phase, the incorporation of
which affect the retention time or by reacting the chiral species with
optically pure chiral reagent.
Many of the early
applications of LC for the analysis of D-AAs employed fluorescence as
detection modality. This was done in part to take advantage of the
afforded by fluorescence detection, which was useful since these
targeting what are often low abundance analytes. The use of
detection was also employed because the precolumn derivatization also
diasteroisomers. There are many examples of mass spectrometry being
used as the
detection modality for LC because of its sensitivity and selectivity.
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|In situ hybridization
ISH is an approach which enables the localization of a given sequence of nucleic acids (RNA/DNA) in tissue. This is generally accomplished through the application of a probe strand of nucleic acids which is complementary to the target strand, and labeled in some way to enable observations of the target localization in the tissue of interest. In this way, RNA or DNA corresponding to proteins connected to D-AAs, such as those related to known racemases or D-form amino acid oxidases, can be visualized.
ISH demonstrating the distribution of serine racemase expression in an adult mouse brain slice. Image acquired from the Allen Brain Atlas.
http://mouse.brain-map.org/experiment/show?id=74357621 (accessed Dec 29, 2016).
When interested in studying the localization of proteins associated with the D-AAs (such as a racemase responsible for the synthesis of a given D-AA), fluorescence imaging following sample treatment with antibodies is often employed. Where the ISH approach above targets strands of nucleic acids, this approach is generally used to measure proteins that have been translated - avoiding staining of cells/regions which contain the corresponding DNA/RNA but not the translated proteins. Shown below is a whole mount immunohistochemical stain of the D-aspartate racemase (DAR1) in Aplysia californica.
DAR1 immunoreactivity localization in the cerebral ganglion of Aplysia californica.
Image adapted from Wang, L., et al. J. Biol. Chem., 2011, 286(15), 13765–13774.
|Localization of Enzyme
Where ISH can localize specific strands of nucleic acids and IHC can be used to image translated proteins one can also treat tissue with carefully selected reagents to image on the basis of enzyme activity. This was eloquently demonstrated by Sasabe and coworkers with D-amino acid oxidase (DAO) in mouse tissue (shown below).
Visualization of DAO activity in the human cerebellum.
Note the higher levels of activity in molecular layer (Mol) relative to the granular layer (Gr) and minimal activity in the white matter (WM).
Image adapted from Sasabe, J. et al. Front Synaptic Neurosci., 2014, 6(14).
|SENSING AND DYNAMIC MEASUREMENTS||Go to the TOPICS LIST|
term “biosensor” is short for “biological sensor”, a chemical sensing
analytical device which converts a biological response into an
The device is made up of a transducer and a biological element (bioreceptor) that may be an enzyme, an antibody or a nucleic acid. The bioreceptor interacts with the analyte being tested and the biological response is converted by the transducer into an electrical, optical or thermal signal. The final result is a display depicting the presence of the target analyte (Turner et al., 1987).
key element of a biosensor is the transducer which makes use of a
physical change accompanying the reaction. This may be:
must possess the following beneficial features:
|The commercial application of biosensors had a significant impact in a number of areas. The market is comprised of different segments (namely: medical, environmental, and food), with medical applications being to dominant player (Hall, 1986). Biosensors represent a rapidly expanding field, with an estimated 60% annual growth rate; the major impetus coming from the health-care industry. The estimated world analytical market is about $ 12,000,000,000/year (30% is in the health care area): there is clearly a vast market expansion potential as less than 0.1% of this market is currently using biosensors. The demand for reliable, inexpensive and rapid methods for assessment of quality will increase.|
|Detection of selected D-amino acids (and derivatives)|
presence of D-amino acids in biological samples can be detected using
different analytical methods, such as capillary gas chromatography,
reversed-phase HPLC and capillary electrophoresis, which often are
time-consuming, expensive and not suitable for on-line application. A
rapid and selective determination of D-amino acids (and derivatives)
may have an important impact on life sciences at different levels. In
In this context, we set up two biosensors based on different, selected enzyme variants of the flavoenzyme glycine oxidase. The system is based on a simple fluorimetric assay, characterized by low cost and ease of use; the fluorescence intensity emission of a commercial dye transducer is proportional to the concentration of analyte (Rosini et al., 2014a). In real time the optical sensing system assays glycine or sarcosine in biological samples with a detection limit ≤ 0.5 µM. The glycine concentration detected in U87 human glioblastoma cell extracts is in good agreement with the value obtained by using the reference HPLC method (7.5 versus 6.7 µM, respectively); interestingly, the assay allowed to quantify the glycine concentration in human plasma, in good agreement with the values reported in the literature.
for "Measurement of D-AAs: Abundance":
Capillary Electrophoresis (CE)
for "Measurement of D-AAs: Localization":
for "Sensing and Dynamic Measurements":