Chiral recognition plays an important role in many fundamental interactions of living systems. Different spectroscopic methods such as fluorescence spectroscopy, mass spectrometry, and NMR spectroscopy, have been applied to achieve the analysis of chiral enantiomeric compound10,66,67.
In this study, targeted analysis of amino acids by LC-MS as well as NMR spectroscopy were employed. Since both techniques are achiral methods, chiral enantiomeric discrimination requires the formation of diastereoisomeric entities, which can then be distinguished.
Accumulation of D-Glutamate in starved E. coli revealed by LC-MS analysis
Targeted LC-MS analysis is a widely used technique in biological studies. It is based on the separation of components in a complex mixture by liquid chromatography, and the detection of their mass-to-charge ratio by mass-spectrometry. However, direct chromatography of enantiomers that have identical molecular weight will also have the same retention time, making simple LC-MS ineffective. This challenge can be overcome using an LC column with a chiral stationary phase68. However, this method requires a precise selection of specific columns, multiple optimizations for specific compounds and still is not widely accessible for biological samples where the difference between concentrations of L- and D-amino acids can be orders of magnitude. In this study, a chemical derivatization followed by a relatively simple reverse-phase LC-MS technique that demonstrated a better alternative was used.
To directly distinguish between the chiral isomers, derivatization of the L- and D-glutamic acid with a chiral reagent (S)-NIFE69,70 that allows separation of derivatized isomers chromatographically was done. The identity of the targets was confirmed by the exact mass-to-charge ratio recorded by a high-resolution mass spectrometer that also allows monitoring isotopic composition, further supporting the identification. The results of the analysis are presented in Fig. 2. The data reveals the presence of L- and D-glutamate in the samples under stress conditions, while the control samples had L-glutamate only (Fig. 2E-G). Remarkably, 10-hour-long stress showed a visibly stronger accumulation of D-glutamate compared to 24 hours of stress. Considering the large difference in abundance between the two forms of glutamate in the biological samples, it was impossible to precisely quantify them using LC-MS. However, relative quantification of metabolites was performed by comparative analysis, calculating the peak's areas. The results support the visual observation of higher D-glutamate content under 10 hours-long stress compared to 24 hours-long- stress. Thus, the ratio between L- and D-glutamate after 10 h stress was 6:1 while after 24 h it was 12:1 only. These results were consistent among the analysis of three sets of samples from three independent preparations, containing each two duplicates of unstressed E. coli (control) of 10-h stressed cells, and of 24-h stressed cells.
D-Glutamate detection in E. coli cells by NMR Spectroscopy
NMR Spectroscopy is also an achiral technique that requires the creation of diastereoisomeric entities, whereby spectral differences between an antipodal pair can be recognized. For enantiomeric discrimination, an optically pure chiral reagent (chiral auxiliary) is required to convert the mixture of enantiomers into a diastereomeric mixture through in-situ formation of nonequivalent diastereomeric complexes with substrate enantiomers. NMR resonances of diastereomers are anisochronous and, therefore, can often be distinguished in the NMR spectrum. Over many years, a variety of chiral NMR auxiliaries have been introduced, e.g. tartaric acid, which is a bidentate ligand with two chiral centers forming a seven-membered chelate ring, as well as different shift reagents. In this study, D-tartarate was applied to the chiral sample and the complex was prepared in situ.
In an attempt to differentiate and increase chemical shift differences between D- and L-glutamate, several chiral auxiliary reagents were tested, under different experimental conditions. Aizawa and coworkers71 used Somarium Lantanide shift reagent in the presence of chiral Tartarate, pH 8. Unfortunately, under these conditions, our biological samples had precipitated, and no NMR signal was detected. Therefore, it was decided to use D-tartaric acid solely, testing different pH conditions. Best results were obtained using a 3-fold excess of D-tartaric acid and pH 7, conditions that were eventually used in this study.
One-dimensional (1D) 1H NMR spectra, as well as 2D 1H-13C Heteronuclear Single Quantum Correlation (HSQC) NMR spectra were used to characterize and identify the presence of glutamate in stressed cell extract. A comparison of the 1H and 13C chemical shifts between standard samples of D- and L-glutamate reveals slight differences between the NMR signals of the two glutamate enantiomers. Superposition of the 1D 1H NMR spectra of D-glutamate (red), L-glutamate (blue), and a mixture of D- & L-glutamate, 1:15 (green), is displayed in Fig. 3. (This 1:15 ratio was chosen based on results obtained by LC-MS). The largest chemical shift difference is observed for the Hβ signals (~ 2.36 ppm; see Fig. 3 insert/enlargement). Chemical shifts of the 1:15 D: L-glutamate mixture (Fig. 3) further support the chemical exchange between the two forms, revealing weighted average chemical shifts of D- and L-glutamate.
13 C chemical shift differences between D- and L-glutamate followed the trend observed for the 1H shift differences. To simultaneously track 1H and 13C shift differences, a 2D 1H-13C Heteronuclear Single Quantum Correlation (HSQC) NMR spectrum was recorded. Figure 4 presents the aliphatic region of the 2D 1H-13C correlation spectrum of stressed cell extract (red), superimposed on the corresponding spectrum of a 1:15 mixture of D- & L-glutamate, (blue). For clarity, the 1D 1H NMR spectrum of the latter mixture sample is shown as well (green). The chemical shifts of the 1:15 mixture signals nicely fit the signals of the stressed cell extract, clearly supporting and identifying the presence of glutamate in the cell extract sample.
Table 1
D-amino acid accumulation, as signaling agents, under various stress or disease conditions- a universal phenomenon.
Entry No.
|
Type of stress/
disease
|
Organism
|
The D-amino acid accumulated
|
Ref.
|
Prokaryotic
|
1
|
Density
|
Bacteria
Bacillus anthracis (germination of fresh spore is inhibited in a density-dependent manner by D-ala)
|
D-alanine
|
72
|
2
|
Hyperthermal stress
|
Archaea
Pyrobaculum islandicum
Methanosarcina barkeri Halobacterium salinarium
|
D-alanine
|
73
|
3
|
Vibrio cholerae mrcA mutant
|
Bacteria
a mutant in Vibrio cholerae mrcA, which encodes a PBP1A homolog
and Bacillus subtilisgenerated
|
D-Met and
D-Leu
D-Tyr and
D-Phe
|
74
|
Eukaryotic
|
4
|
Osmotic stress
|
Parasitic protozoan
Leishmania amazonensis
|
D-alanine
|
75
|
5
|
Hypersalinity acclimation
|
Crustaceans Aquatic invertebrates
Penaeus japonicus
Procambarus clarkia
Juasus lalandi
Chionoecetes opili
Eriocheir japonicus
|
D-alanine
|
76
|
6
|
Changes in external salinity
|
A brackish-water mollusc,
Corbicula japonica
|
D-alanine
|
77
|
7
|
Hypersalinity acclimation
|
Mollusks Aquatic invertebrates
Scapharca broughtonii
Crassostrea gigas
Patinopecten yessoensis
Meretrix lusoria
Ruditapes philippinarum
Pseudocardium sachalinensis
Tresus keenae
|
D-alanine
|
76
|
8
|
Hypertonic or Hypotonic stress
|
Mollusks aquatic invertebrates
Lucinoma aequizonata
|
D-alanine
|
78
|
9
|
Herbicides
|
Plant
Nicotiana tabacum
|
D-alanine
|
79
|
10
|
Ultraviolet
radiation
|
Duckweed plants
Landoltia punctata
|
D-alanine
|
10
|
11
|
Amino acid deprivation
|
Plant
Arabidopsis thaliana
|
D-alanine
|
80
|
12
|
Tidal freshwater marshes
|
Plant
Phragmites australis
|
D-alanine
|
81
|
13
|
Most exposed to chronic mild stress (CMS), also some of them with Alzheier's disease (AD)
|
Male Wistar Rats
mammalian tissues
frontal cortex
|
D-glutamate
|
82
|
14
|
Mutant lacing D-amino acid oxidase
|
Mouse
mammalian tissues
|
D- amino acids:
D- serine ;
D-alanine;
D- proline
|
83
84 85 86
|
15
|
Treated with vehicle or drugs employed for therapy of mood/anxiety and subjected to food shock stress
|
Rats
mammalian tissues
|
D-glutamate
|
87
|
16
|
Adult male -aging
|
Rat
mammalian tissues
salivary glands
|
D-aspartic acid
|
88
|
17
|
Adult male-aging
|
Rat
mammalian tissues
CNS anterior pituitary gland
and in the pancreas
|
D-alanine
|
89
|
18
|
Mutant lacing D-amino acid oxidase
|
Mouse
mammalian tissues
|
D- praline;
D- leucine
|
90
|
19
|
Mutant ddY/DAO− mice lacking D-amino-acid oxidase
|
Mouse
mammalian tissues
in the pituitary and pineal glands
|
five D-amino acids
(D-Asp;
D-Ser;
D-Ala;
D-Leu and
D-Pro)
|
91
|
20
|
Adult male- aging
|
Rat
mammalian tissues
Islets of Langerhans of rat pancreas
|
D-alanine
|
92
|
21
|
Renal - kidney disease-
|
Human
Homo sapiens
mammalian tissues
|
D- amino acids:
D- serine ;
D-alanine;
D- proline
|
85
|
22
|
Hunger
|
Human
Homo sapiens
mammalian tissues
produced in salivary glands
|
D-alanine;
D- proline;
D-aspartate
|
37
|
23
|
Alzheimer’s disease (AD)
|
Human
|
D-aspartate
|
93
|
24
|
Motor Neuron Disease(MND)/Amyotrophic Lateral Sclerosis(ALS)
|
Human
|
D- serine;
D-glutamate;
D-aspartate
|
94
|
25
|
Alzheimer’s disease (AD)
|
Human
|
D-glutamate
|
40
|
Mitochondrial dysfunction is one of the earliest pathophysiological events in ALS5 The mitochondrial ultrastructure is a useful tool for assessing mitochondrial quality95. Aggregated mitochondria, with a swollen and vacuolated appearance, are one of the first changes96–98. The activity of the complexes involved in the electron transport chain is decreased in ALS. This results in decreased ATP generation, and increased generation of ROS leading to oxidative damage to DNA, RNA and mRNA5. In synaptic pruning, microglia derived C1q may play an essential source of excessive synapse removal leading to pathological conditions46,49,99.