In-Electrospray source Hydrogen/Deuterium exchange coupled to multistage fragmentation for the investigation of the protonation and fragmentation pathways of gas phase ions.

Identification of molecules in complex natural matrices relies on matching the fragmentation spectra of ions under investigation and the spectra acquired for the corresponding analytical standards. Currently, there are many databases of experimentally measured tandem mass spectrometry spectra (such as NIST, MzCloud, and Metlin), and considerable progress has been made in the development of software for predicting tandem mass spectrometry fragments in silico using combinatorial, machine learning, and quantum chemistry approaches (such as MetFrag, CFM-ID, and QCxMS). However, the electrospray ionization molecules can be ionized at different sites (protonated or deprotonated), and the fragmentation spectra of such ions are different. Here, we are using the combination of the in-ESI source hydrogen/deuterium exchange reaction and MSn fragmentation for the investigation of the fragmentation pathways for different protomers of organic molecules. It is shown that the distribution of the deuterium in the fragment ions reflects the presence of different protomers. For several molecules, the distribution of deuterium was traced up to the MS5 level of fragmentation revealing many unusual and unexpected effects. For example, we investigated the loss of HF from the ciprofloxacin and norfloxacin ions and observed that for ions protonated at -COOH group, the eliminating hydrogen always comes from -NH group. When ions are protonated at another site, the elimination of hydrogen with a probability of 30% occurs from the -NH group, and with a probability of 70%, it originates from other sites on the molecule. Such effects were not described previously. Quantum chemical simulation was used for the verification of the protonated structures and simulation of the corresponding fragmentation spectra.

Investigating the Metabolism of Plants Germinated in Heavy Water, D2O, and H218O-Enriched Media Using High-Resolution Mass Spectrometry.

Mass spectrometry has been an essential technique for the investigation of the metabolic pathways of living organisms since its appearance at the beginning of the 20th century. Due to its capability to resolve isotopically labeled species, it can be applied together with stable isotope tracers to reveal the transformation of particular biologically relevant molecules. However, low-resolution techniques, which were used for decades, had limited capabilities for untargeted metabolomics, especially when a large number of compounds are labelled simultaneously. Such untargeted studies may provide new information about metabolism and can be performed with high-resolution mass spectrometry. Here, we demonstrate the capabilities of high-resolution mass spectrometry to obtain insights on the metabolism of a model plant, Lepidium sativum, germinated in D2O and H218O-enriched media. In particular, we demonstrated that in vivo labeling with heavy water helps to identify if a compound is being synthesized at a particular stage of germination or if it originates from seed content, and tandem mass spectrometry allows us to highlight the substructures with incorporated isotope labels. Additionally, we found in vivo labeling useful to distinguish between isomeric compounds with identical fragmentation patterns due to the differences in their formation rates that can be compared by the extent of heavy atom incorporation.

Untargeted Lipidomics after D2O Administration Reveals the Turnover Rate of Individual Lipids in Various Organs of Living Organisms.

The administration of low doses of D2O to living organisms was used for decades for the investigation of metabolic pathways and for the measurement of the turnover rate for specific compounds. Usually, the investigation of the deuterium uptake in lipids is performed by measuring the deuteration level of the palmitic acid residue using GC-MS instruments, and to our knowledge, the application of the modern untargeted LC-MS/MS lipidomics approaches was only reported a few times. Here, we investigated the deuterium uptake for >500 lipids for 13 organs and body liquids of mice (brain, lung, heart, liver, kidney, spleen, plasma, urine, etc.) after 4 days of 100% D2O administration. The maximum deuteration level was observed in the liver, plasma, and lung, while in the brain and heart, the deuteration level was lower. Using MS/MS, we demonstrated the incorporation of deuterium in palmitic and stearic fragments in lipids (PC, PE, TAG, PG, etc.) but not in the corresponding free forms. Our results were analyzed based on the metabolic pathways of lipids.

Simple In Vitro 18O Labeling for Improved Mass Spectrometry-Based Drug Metabolites Identification: Deep Drug Metabolism Study.

The identification of drug metabolites formed with different in vitro systems by HPLC-MS is a standard step in preclinical research. In vitro systems allow modeling of real metabolic pathways of a drug candidate. Despite the emergence of various software and databases, identification of compounds is still a complex task. Measurement of the accurate mass, correlation of chromatographic retention times and fragmentation spectra are often insufficient for identification of compounds especially in the absence of reference materials. Metabolites can "slip under the nose", since it is often not possible to reliably confirm that a signal belongs to a metabolite and not to other compounds in complex systems. Isotope labeling has proved to be a tool that aids in small molecule identification. The introduction of heavy isotopes is done with isotope exchange reactions or with complicated synthetic schemes. Here, we present an approach based on the biocatalytic insertion of oxygen-18 isotope under the action of liver microsomes enzymes in the presence of 18O2. Using the local anesthetic bupivacaine as an example, more than 20 previously unknown metabolites were reliably discovered and annotated in the absence of the reference materials. In combination with high-resolution mass spectrometry and modern methods of mass spectrometric metabolism data processing, we demonstrated the ability of the proposed approach to increase the degree of confidence in interpretating metabolism data.

Analysis of 16O/18O and H/D Exchange Reactions between Carbohydrates and Heavy Water Using High-Resolution Mass Spectrometry.

Mono- and polysaccharides are an essential part of every biological system. Identifying underivatized carbohydrates using mass spectrometry is still a challenge because carbohydrates have a low capacity for ionization. Normally, the intensities of protonated carbohydrates are relatively low, and in order to increase the corresponding peak height, researchers add Na+, K+, or NH4+to the solution. However, the fragmentation spectra of the corresponding ions are very poor. Based on this, reliably identifying carbohydrates in complex natural and biological objects can benefit frommeasuring additional molecular descriptors, especially those directly connected to the molecular structure. Previously, we reported that the application of the isotope exchange approach (H/D and 16O/18O) to high-resolution mass spectrometry can increase the reliability of identifying drug-like compounds. Carbohydrates possess many -OH and -COOH groups, making it reasonable to expect that the isotope exchange approach would have considerable potential for detecting carbohydrates. Here, we used a collection of standard carbohydrates to investigate the isotope exchange reaction (H/D and 16O/18O) in carbohydrates and estimate its analytical applications.

PyFragMS-A Web Tool for the Investigation of the Collision-Induced Fragmentation Pathways.

Dissociation induced by the accumulation of internal energy via collisions of ions with neutral molecules is one of the most important fragmentation techniques in mass spectrometry (MS), and the identification of small singly charged molecules is based mainly on the consideration of the fragmentation spectrum. Many research studies have been dedicated to the creation of databases of experimentally measured tandem mass spectrometry (MS/MS) spectra (such as MzCloud, Metlin, etc.) and developing software for predicting MS/MS fragments in silico from the molecular structure (such as MetFrag, CFM-ID, CSI:FingerID, etc.). However, the fragmentation mechanisms and pathways are still not fully understood. One of the limiting obstacles is that protomers (positive ions protonated at different sites) produce different fragmentation spectra, and these spectra overlap in the case of the presence of different protomers. Here, we are proposing to use a combination of two powerful approaches: computing fragmentation trees that carry information of all consecutive fragmentations and consideration of the MS/MS data of isotopically labeled compounds. We have created PyFragMS-a web tool consisting of a database of annotated MS/MS spectra of isotopically labeled molecules (after H/D and/or 16O/18O exchange) and a collection of instruments for computing fragmentation trees for an arbitrary molecule. Using PyFragMS, we investigated how the site of protonation influences the fragmentation pathway for small molecules. Also, PyFragMS offers capabilities for performing database search when MS/MS data of the isotopically labeled compounds are taken into account.