Patent Application
20250137994
The present invention relates to a mass spectrometry method and to a mass spectrometry system.
The elucidation of biochemical and metabolic pathways is key for a deeper understanding of biological systems. Stable isotopes such as deuterium (2H) are powerful tools to achieve this endeavor.1,2 Deuterium (2H) has been deployed as a metabolic tracer in combination with different mass spectrometric methods such as hydrogen/deuterium exchange (HDX) mass spectrometry (MS) to explore dynamic protein structure or protein interactions and to study fatty acid/lipid biosynthesis.3-8
Recently, it has been suggested to monitor the incorporation of 2H into metabolites after ingestion of heavy water (2H2O). As an example, X. Fu et al., “Measurement of lipogenic flux by deuterium resolved mass spectrometry”, Nature Communications 2021, 12:3756, doi: 10.1038/s41467-021-23958-4 discloses investigations of de novo lipogenesis by monitoring deuterium incorporation into fatty acids following the administration of deuterated water to mice. Blood samples were taken from the mice, and the blood plasma was treated as follows: Plasma samples (5 μL) were treated with 2 mL (1:1, v/v) methanol/dichloromethane (DCM) to precipitate proteins and extract triglycerides. Samples were vortexed for 1 min and then centrifuged for 5 min at 1635×g. The DCM layer was transferred to a new tube and dried under N2. Samples were saponified with 1 mL 0.5 M KOH in methanol at 80° C. for 1 hr. Resulting FAs were extracted with 2 mL DCM/water (1:1, v/v) for 1 min, and the solvent was evaporated under N2. Dried lipid extract was resuspended in 50 μL of 1% triethylamine/acetone and reacted with 50 μL of 1% PFBBr/acetone for 30 min at room temperature. To this solution, 1 mL of isooctane was added before MS analysis. Additionally, liver extract was treated by a similar method. The treated plasma or liver extract was then analyzed by deuterium-resolved mass spectrometry. This method requires an invasive procedure for obtaining the blood samples or the liver abstract. Furthermore, sample preparation is very complex and time-consuming.
EP3559675A1 discloses methods of monitoring amino acid levels by investigating exhaled breath condensate using LC-MS or LC-MS/MS methods. Deuterated leucine was used as an internal standard.
US 2010255598A1 discloses the detection of 2H-labelled markers in exhaled breath by FTIR spectroscopy. In one embodiment, a parent therapeutic agent is labeled with a 2H-labelled marker. Upon metabolism (e.g., via enzymatic action) of the therapeutic agent, the marker becomes volatile or semi-volatile and is present in the breath. In another embodiment, the therapeutic agent is associated with a 2H-labelled taggant, which in turn will generate a marker in the breath that is easily measurable. 2H-labelled therapeutic agents are difficult to obtain and can be extremely expensive because they need to be specifically synthesized.
L. Shi et al., “Optical imaging of metabolic dynamics in animals”, Nature Communications, 2018, 9(1):2995 combined deuterium oxide (2H2O) probing with stimulated Raman scattering microscopy to image in situ metabolic activities.
S. Davies et al., “Rapid measurement of deuterium content of breath following oral ingestion to determine body water”, Physiol. Meas. 2001 November; 22(4):651-9, doi: 10.1088/0967-3334/22/4/301 discloses the use of flowing afterglow mass spectrometry to determine the deuterium content in exhaled breath water after oral ingestion of deuterated water. Only the deuterium content in the water signal is analyzed. This is also true for the following publications: P. Spanel et al., “Coordinated FA-MS and SIFT-MS analyses of breath following ingestion of D2O and ethanol: total body water, dispersal kinetics and ethanol metabolism”, Physiol. Meas. 2005 August; 26(4):447-57, doi: 10.1088/0967-3334/26/4/011; C. Chan et al., “A non-invasive, on-line deuterium dilution technique for the measurement of total body water in haemodialysis patients”, Nephrol. Dial. Transplant. 2008 June; 23(6):2064-70, doi: 10.1093/ndt/gfn045; D. Smith et al., “Comparative measurements of total body water in healthy volunteers by online breath deuterium measurement and other near-subject methods”, Am. J. Clin. Nutr. 2002 December; 76(6):1295-301, doi: 10.1093/ajcn/76.6.1295
It is an object of the present invention to provide a non-invasive method of investigating metabolic pathways in a subject, the method being capable to be carried out in real time and not requiring complex sample preparation protocols nor synthesis of labeled therapeutic agents.
This object is achieved by a method according to claim 1. Further embodiments of the invention are laid down in the dependent claims.
A mass spectrometry method is provided, comprising:
According to the present invention, a living human or animal subject ingests deuterated water. During or after a period of time of ingestion of the deuterated water, a gas sample is obtained from the subject, and a deuterium-resolved mass spectrum of the gas sample is determined. A deuterium-resolved mass spectrum is a mass spectrum that has sufficient resolution to distinguish between a deuterated compound and an isotopologue of the compound that has the same mass number but comprises no deuterons or a lower number of deuterons, instead comprising at least one heavy isotope of carbon (13C), nitrogen (15N) or oxygen (17O and 18O) in natural abundance. The deuterium-resolved mass spectrum is processed to determine a presence or amount of at least one deuterated metabolite in the gas sample. Specifically, the deuterium-resolved mass spectrum may be processed to identify at least one spectral feature associated with the presence of at least one deuterated metabolite in the gas sample, and an intensity of the spectral feature may be calculated to determine an indicator for the amount of the deuterated metabolite.
With this method, valuable insights into metabolic pathways in a subject can be gained. The method can readily be carried out in real time, i.e., the deuterium-resolved mass spectrum may be determined in real time immediately after the gas sample has been obtained from the subject, avoiding possible deterioration of the sample due to storage. The method does not require any complex sample preparation, as it would be the case with blood samples.
No isotopically labeled therapeutic agents are required. Therefore, the method can be carried out at relatively low cost.
In preferred embodiments, the gas sample comprises breath that has been exhaled by the subject. Breath samples can be particularly easily obtained from humans, and a breath sample can be readily transferred to an ion source in real time. In addition or in the alternative, the gas sample may comprise volatile metabolites that have been excreted through the skin.
Processing the deuterium-resolved mass spectrum may comprise a spectral separation step for separating a spectral feature associated with the deuterated metabolite from a spectral feature associated with an isotopologue of the metabolite that has identical mass number but comprises no deuterons or a lower number of deuterons. Such isotopologues will unavoidably be present due to the presence of heavy isotopes of carbon (13C), nitrogen (15N) and oxygen (17O and 18O) in natural abundance and should be distinguished from the deuterated metabolite of interest. The spectral separation step may comprise a binning procedure using a Kernel density function, as it is known in the art of high-resolution mass spectrometry per se. The Kernel density function may have a bandwidth that matches the resolution of the mass analyzer that was used for obtaining the mass spectrum.
Processing the deuterium-resolved mass spectrum may comprise determining an isotope ratio of the deuterated metabolite and a reference isotopologue of the metabolite in the gas sample. The reference isotopologue may be a fully protonated isotopologue, i.e., an isotopologue in which all deuterons of the deuterated metabolite are replaced by protons.
The steps of obtaining a gas sample may be repeated at a plurality of different times relative to ingestion of the deuterated water by the subject, and deuterium-resolved mass spectra may be determined for these gas samples to obtain a time series of the amount of the deuterated metabolite, in particular, of the aforementioned isotope ratio.
In another aspect, the present invention provides a mass spectrometry system that is configured for carrying out the method of the present invention. The mass spectrometry system comprises:
In advantageous embodiments, the ion source is a SESI source, and wherein the mass analyzer is an Orbitrap-type mass analyzer. However, other types of ion sources and high-resolution mass analyzers may be used.
In some embodiments, the mass spectrometry system may be specifically configured to process breath samples in real time. To this end, the system may comprise an interface for coupling a breathing mask or a mouthpiece to the mass spectrometry system. In this manner it becomes possible to sample air that has been exhaled by the subject, the exhaled air forming the gas sample and to transfer the gas sample to the ion source in real time. The mass spectrometry system may further comprise said breathing mask or mouthpiece. In other embodiments, the gas sample may first be stored before the mass spectrum of the gas sample is determined, i.e., the gas sample may be analyzed offline. For instance, the gas sample may be collected by having a subject exhale into a bag and may be stored in the bag for off-line analysis, as it is commonly done in the well-known urea breath test for identifying infections by Helicobacter pylori. The mass spectrometry system may accordingly comprise an interface configured to connect to a breath test bag.
The data processing system of the mass spectrometry system may be configured to carry out any of the data processing procedures discussed above. To this end, the data processing system may comprise a computer processor and program memory storing program data that cause the computer processor to carry out the data processing procedures. The data processing system may in particular be an appropriately programmed general-purpose computer.
Preferred embodiments of the invention are described in the following with reference to the drawings, which are for the purpose of illustrating the present preferred embodiments of the invention and not for the purpose of limiting the same. In the drawings,
Isotopologue: Isotopologues are molecules that differ only in their isotopic composition. They have the same chemical formula and bonding arrangement of atoms, but at least one atom has a different number of neutrons than the parent.
Deuterated water: The term “deuterated water” is to be understood to comprise both “heavy water” (2H2O) with two deuterium isotopes of hydrogen per molecules and “semi-heavy water” with one deuterium isotope per molecule (2H1HO) at an abundance that is higher than the natural abundance.
Metabolite: The term “metabolite” is to be understood in the usual manner as any intermediate or end-product of metabolism. The term is to be understood to include both primary and secondary metabolites. Water is not considered a metabolite.
Deuterated metabolite: A deuterated metabolite is a metabolite in which at least one proton has been replaced by a deuteron. Deuterated water is not considered a deuterated metabolite.
Mass spectrometer: A mass spectrometer comprises an ion source (ionizer) and a mass analyzer. The ion source converts a portion of the sample into ions. An extraction system removes ions from the sample, which are then targeted to the mass analyzer.
SESI: Secondary electrospray ionization (SESI) is a spray type, ambient ionization method where ions are produced by means of an electrospray. These ions then charge vapor molecules in the gas phase when colliding with them. SESI is an efficient ambient ionization method, that, when combined with high-resolution mass spectrometry (SESI-HRMS), enables capturing fine isotopic structures.
Orbitrap-type mass analyzer: An Orbitrap is an ion trap mass analyzer consisting of an outer barrel-like electrode and a coaxial inner spindle-like electrode that traps ions in an orbital motion around the spindle. The image current from the trapped ions is detected and converted to a mass spectrum using the Fourier transform of the frequency signal.
Deuterium-resolved mass spectrum: A deuterium-resolved mass spectrum is a mass spectrum with sufficient resolution to distinguish spectral features (contributions to the spectral intensity) originating from a deuterated molecule and an isotopologue of the same molecule that has the same total mass number (i.e., the same molecular mass in Daltons when rounded to the next integer), but comprises a lower number of deuterons while instead comprising a heavy isotope of carbon, nitrogen or oxygen, i.e., a nucleus selected from 13C, 15N, 17O or 18O. These heavy isotopes are typically present at low natural abundance in organic molecules. A deuterium-resolved mass spectrum may be obtained by using a mass analyzer with sufficiently high resolving power. Preferably the resolving power is at least 120,000 at m/z 200. Such high resolving power can be achieved, e.g., with commercially available Orbitrap-type mass analyzers and will lead to well-separated spectral lines for the different isotopologues. However, a separation of spectral features arising from different isotopologues with identical total mass number may also be possible at lower resolving power, using spectral separation methods such as kernel density estimation. It should be kept in mind that the resolving power of Orbitrap-type mass analyzers scales with 1/sqrt (m/Z). Therefore it is easier to separate features from different isotopologues with identical total mass number for lighter molecules than it is for heavy molecules.
Isotope ratio: The term “isotope ratio” or “isotopic ratio” is to understood to designate the ratio of the concentration of a particular isotopologue in a sample and the concentration of another isotopologue of the same metabolite in the same sample. The isotope ratio is well approximated by the ratio of the intensities of the associated spectral features (“peaks”) in the mass spectrum and may therefore be equated to this ratio.
Breathing mask: A breathing mask is a mask that covers the mouth, and usually other parts of the face or head, designed to direct the wearer's breath to a particular apparatus.
Two healthy female specific-pathogen-free (SPF) C57BL/6 mice weighing approximately 18-22 g were used in an example. Mice were purchased from Guangdong Medical Laboratory Animal Center (Foshan, China) and housed under SPF conditions at 22±2° C. on a 12 h light/dark cycle, and fed using standard mice chow and water ad libitum. The mice were acclimatized for a week before the experiment began. The animal studies were approved by the Laboratory Animal Ethics Committee of Jinan University, and conducted in uniformity with national guidelines of the care and use of laboratory animals.
The deuterium oxide (2H2O, 99.9 atom % 2H) was purchased from Merck (Darmstadt, Germany).
On day zero (D0) i.e. one day before the treatment with 2H2O, both mice were fed with standard drinking water (H2O) and a baseline measurement was acquired for both mice. Afterwards, mice were randomly divided into two groups. The treated group was fed with 2H2O (80%, v/v); the control group was fed with H2O. Volatile species emitted by the mice were measured at days 1, 2, 3, 10, 12 and 13 for 20 min by SESI-HRMS. SESI-HRMS analysis of mice's volatilome followed the procedure as previously reported13-16.
Pre-Processing: Data analysis was performed using MATLAB (version 2020b, MathWorks Inc., USA). Raw mass spectra files were converted to mzXML via msConvert (Proteowizard).17 Positive and negative scans per file were filtered out. We then computed the average spectrum of 100 scans per file and the spectra were recalibrated using a polynomial fitting. After calibration, the mass accuracy across the entire mass range was within 2 ppm for both polarities. Subsequently, the spectra were centroided and binned using a Kernel density function, selecting its bandwidth to match the instrument resolution at each m/z. The final feature list was obtained by centroiding the resulting Kernel density function. The signal intensity for each feature was computed by summing the intensity of the peaks within their full width at half maximum. As a result, a data matrix of 14 samples (seven time points for mouse drinking H2O and seven time points for mouse drinking 2H2O) ×3476 features in positive mode and ×1407 features in negative mode was obtained.
Post-Processing: In a first filtering step, the number of features was further reduced by considering only the cases were the signal intensity was greater than zero in at least four out of the seven data points measured for the mouse drinking 2H2O. Molecular formulae were assigned to the remaining 1350 positive and 1131 negative mode features based on the measured accurate mass within a tolerance of 2 ppm. Protonated (for positive mode features) and deprotonated species (for negative mode features) fulfilling “seven golden rules”18 with only C, H, N and O were considered. 2H/1H isotopologue pairs were searched by identifying mass spectral features with a mass difference of Δ=n*1.0063 u (±0.0005 u), where n=1,2, . . . 10. The ratio of the signal intensities 2H/1H was computed for both mice at all the time points. Of the resulting 2H/1H time traces, only those showing an increasing trend for the mouse drinking 2H2O vs. the control mouse were further considered (i.e. 60 isotopologue pairs). Moreover, to explore similarities across the different kinetics of such ratio time traces, we conducted hierarchical cluster analysis (average method; correlation distance). We then used the “lipids and non-lipids main chemical class” metabolite sets in MetaboAnalystR (version 3.0.3)19 to assign potential compound candidates to metabolites undergoing 2H-enrichment. Furthermore, compound hits were cross-referenced with HMDB and KEGG databases using “CrossReferencing” function of MetaboAnalystR.
We identified 60 isotopologue 2H/1H pairs (26 in positive and 34 in negative mode) fulfilling the mass difference and increasing intensity criteria.
Once we identified the species undergoing a relevant incorporation of 2H, we studied their evolution over time during the course of the experiment.
This example demonstrates that SESI-HRMS is a suitable tool to detect metabolic 2H-incorporation in vivo in real-time. The technique has the required sensitivity (i.e. part-per-trillion range22) to enable the detection of species emitted under physiological conditions without any sample preconcentration, and at the same time renders the required mass resolution to enable the separation of fine isotopic structures. This is illustrated in
In all examples shown in
In the latter, 2H2O forms N—2H, O—2H and S—2H bonds which occurs faster and more often because 2H/1H exchange is more prominent when the hydrogen is bound to a heteroatom as a consequence of the intrinsic electronegativity provided by the free electron pair whereas in an enzymatic exchange 2H2O forms C—2H bonds which is slower and dependent on the number of enzymes available and produced by the organism9, 25 26, 27. While all the molecular formulae identified in this study contain either O and/or N-atoms, it is unclear at this point whether the observed 2H-incorporations correspond to enzymatic, non-enzymatic or both types of reactions.
The cluster analysis revealed three major groups of patterns in these ratio time series. Cluster one contains pyruvic acid and lactic acid. The similarity in the temporal profiles with an increasing trend over the course of the days can be explained by anaerobic glycolysis in red blood cells, muscle cells or gut microbiome, whereby glucose produces pyruvate, which is then converted to lactate when supply of oxygen is limited28, 29. Cluster two contains lysine, which interestingly clusters together with m/z 161.1284, which fits with the molecular formula of methyl-lysine. In this cluster and cluster number three we found the three short chain fatty acids (i.e. acetic, propionic acid and butyric acid). These are the main products of gut microbiome activity and their importance modulating other metabolic, endocrine and immune functions is becoming increasingly evident.30
A method of monitoring in vivo 2H-incorporation using this setup is schematically illustrated in the flow diagram of
Data processing was done in a very similar as described above for Example 1. Data processing comprised, in particular, the spectral separation of spectral features associated with deuterated metabolites and isotopologues having the same nominal mass number but a different number of deuterons. To this end, binning was performed by employing a Kernel density function having a bandwidth that matched the instrument resolution at each m/z, as in Example 1.
Several dozens of deuterated metabolites were identified and quantified by determining the isotope ratio relative to their fully protonated isotopologues. Selected time series of the measured isotope ratios are shown in
SESI-HRMS allowed to monitor in vivo 2H-incorporation of metabolites in a non-invasive and real-time set-up, both for animals and humans, by sampling volatile compounds that are excreted via the skin or breath after oral ingestion of deuterated water. The obtained result show that the presently proposed method opens up new opportunities to use deuterium tracing to extend current metabolic studies, especially those with a focus on anaerobic glycolysis, lysine methylation and gut microbiome via monitoring of SCFAs.
In this study, secondary electrospray ionization was combined with high-resolution mass spectrometry (SESI-HRMS), using an Orbitrap-type mass analyzer. While this combination proved to be particularly well suited for the present purpose, the presently proposed method is not limited to a specific ionization method, and other types of ion sources may be employed, such as plasma ionization or atmospheric pressure chemical ionization. Neither is the method limited to a specific method of mass analysis, and other types of high-resolution mass analyzers may be used, such as FT-ICR mass analyzers.
The presently proposed method is not limited to a particular regime of administration of the deuterated water (e.g., single dose, multiple dose or continuous administration over a certain period of time) or to a particular scheme concerning the timing of when gas samples are obtained relative to the administration of the deuterated water. This is exemplified by the above Examples 1 and 2: In Example 1, the subject continuously ingested deuterated water over an extended period of time, and gas samples were obtained during this time. In Example 2, the subject ingested deuterated water only once over a very short period of time, and gas samples were obtained after the subject had stopped ingesting deuterated water. Many other schemes are conceivable, including schemes in which the ingestion of deuterated water follows a more complex pattern.
While in the above examples, mass analysis was carried out in real time, it is also possible to store the gas samples, e.g., in one or more bags, and to analyze them offline.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/075436 | 2/8/2022 | WO |
