Location Of Double Bonds Research Articles

A deep learning-guided automated workflow in LipidOz for detailed characterization of fungal fatty acid unsaturation by ozonolysis.

Understanding fungal lipid biology and metabolism is critical for antifungal target discovery as lipids play central roles in cellular processes. Nuances in lipid structural differences can significantly impact their functions, making it necessary to characterize lipids in detail to understand their roles in these complex systems. In particular, lipid double bond (DB) locations are an important component of lipid structure that can only be determined using a few specialized analytical techniques. Ozone-induced dissociation mass spectrometry (OzID-MS) is one such technique that uses ozone to break lipid DBs, producing pairs of characteristic fragments that allow the determination of DB positions. In this work, we apply OzID-MS and LipidOz software to analyze the complex lipids of Saccharomyces cerevisiae yeast strains transformed with different fatty acid desaturases from Histoplasma capsulatum to determine the specific unsaturated lipids produced. The automated data analysis in LipidOz made the determination of DB positions from this large dataset more practical, but manual verification for all targets was still time-consuming. The DL model reduces manual involvement in data analysis, but since it was trained using mammalian lipid extracts, the prediction accuracy on yeast-derived data was reduced. We addressed both shortcomings by retraining the DL model to act as a pre-filter to prioritize targets for automated analysis, providing confident manually verified results but requiring less computational time and manual effort. Our workflow resulted in the determination of detailed DB positions and enzymatic specificity.

Comprehensive analysis of phospholipids in pumpkin (Cucurbita maxima) seeds: Comparison with cashew nuts and almonds

Pumpkin (Cucurbita maxima) seeds have gained increasing attention due to their high nutraceutical value; however, data regarding the phospholipid (PL) profile of this oily seed are scarce. Through the combination of RPLC-MS/MS, HILIC-MS, and Paternò-Büchi photochemical reaction, we comprehensively characterized PLs in three accessions of pumpkin seeds. We identified a total of 183 PL molecular species across eight classes. Phosphatidylcholine (PC) represents the most abundant class, followed by phosphatidylinositol (PI), phosphatidylglycerol (PG), and phosphatidic acid (PA). The total PL content is comparable to that of almonds and cashew nuts. In terms of PL structures, pumpkin seeds feature a higher abundance of C18:2-containing PL isomers, a preferential esterification of C18:1 and C18:2 at the sn-2 position as well as a predominant Δ9/Δ9,12 double bond locations for C18:1 and C18:2 respectively. This is the first systematic study on PLs of pumpkin seeds to our knowledge. Our findings add further evidence on the potential value of pumpkin seeds as a functional food or as a source material for making plant-based drink.

Cross-Validation of Lipid Structure Assignment Using Orthogonal Ion Activation Modalities on the Same Mass Spectrometer.

The onset and progression of cancer is associated with changes in the composition of the lipidome. Therefore, better understanding of the molecular mechanisms of these disease states requires detailed structural characterization of the individual lipids within the complex cellular milieu. Recently, changes in the unsaturation profile of membrane lipids have been observed in cancer cells and tissues, but assigning the position(s) of carbon-carbon double bonds in fatty acyl chains carried by membrane phospholipids, including the resolution of lipid regioisomers, has proven analytically challenging. Conventional tandem mass spectrometry approaches based on collision-induced dissociation of ionized glycerophospholipids do not yield spectra that are indicative of the location(s) of carbon-carbon double bonds. Ozone-induced dissociation (OzID) and ultraviolet photodissociation (UVPD) have emerged as alternative ion activation modalities wherein diagnostic product ions can enable de novo assignment of position(s) of unsaturation based on predictable fragmentation behaviors. Here, for the first time, OzID and UVPD (193 nm) mass spectra are acquired on the same mass spectrometer to evaluate the relative performance of the two modalities for lipid identification and to interrogate the respective fragmentation pathways under comparable conditions. Based on investigations of lipid standards, fragmentation rules for each technique are expanded to increase confidence in structural assignments and exclude potential false positives. Parallel application of both methods to unsaturated phosphatidylcholines extracted from isogenic colorectal cancer cell lines provides high confidence in the assignment of multiple double bond isomers in these samples and cross-validates relative changes in isomer abundance.

Consequential Effect of Intake of Fatty Acid through Milk and Butterfat (Ghee)

Milk fat is observed to have high nutritional properties and other health benefits. The fat in milk is the most complicated natural fat since it comprises over 400 distinct fatty acids. The saturated fatty acids present in milk account for approximately 70% by weight (5). The most important fatty acid from a quantitative viewpoint is palmitic acid (16:0), which accounts for approximately 30% by weight of the total fatty acids. Myristic acid (14:0) and stearic acid (18:0) make up 11 and 12% by weight, respectively. Oleic acid is the major unsaturated fatty acid present in milk. The fatty acid composition of milk fat determines its nutritional value. The fatty acids found in milk vary in terms of their structure, cis or trans isomers, amount and location of double bonds, and chain length. The chemical makeup and content of the fatty acids present determine the physical and nutritional characteristics of the fat. The fatty acids in milk also have nutritional value and offer certain health advantages. Eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and other polyunsaturated fatty acids are considered to help prevent coronary artery disease (CAD) and other age-related neurodegenerative illnesses. Many of these therapeutic properties are unknown even today. The impact of fatty acids on human health is a topic that is always under dispute. Here we discuss how fatty acids act in terms of human health. Regulations have been implemented to ensure reduced risk due to the intake of fatty acids like trans fatty acids. Trans fats can increase the risk of CHD.

Optimized quantification of fatty acids in complex media using advanced analytical techniques.

Various medium formulations contain essential fatty acids at concentrations ranging from 10 to 100 mg/L. Accurate and precise lipid measurement in media is crucial for monitoring media quality and conducting studies on lipids in the context of cell culture. This study employed two-dimensional gas chromatography (GC × GC) analyses to offer enhanced resolution, sensitivity, and separation performance compared to GC. Quantification of fatty acid methyl esters (FAMEs) in a medium was conducted using GC × GC combined with a high-resolution mass spectrometer and flame ionization detector, considering potential interference from nonionic surfactant Tween 80, which was precipitated and removed by optimizing the concentration of cobalt thiocyanate (CTA) solution during pretreatment. This advanced analytical approach enabled identification of cis and trans isomers of identical molecular weights and determination of the location and number of double bonds in the same carbon number structure. Our analysis identified 36 FAMEs within the C6-C24 region, and a 5% CTA solution was optimal for efficient removal of Tween 80 during lipid extraction. Additionally, this advanced method minimized FAME contamination and loss during pretreatment, thereby significantly reducing the sample volume required to detect trace levels of FAMEs. This improvement led to a fatty acid recovery rate of 106% while maintaining the average relative standard deviation for the target FAMEs of about 3%. Our research paves the way for future investigation into medium quality control and the role of fatty acids in cell culture. This offers the possibility for economical and effective trace quantification of fatty acids in complex media.

Triboelectric Nanogenerators for the Masses: A Low-Cost Do-It-Yourself Pulsed Ion Source for Sample-Limited Applications.

Triboelectric nanogenerators (TENG) are useful devices for converting mechanical motion into electric current using readily available materials. Though the applications for these devices span across many fields, TENG can be leveraged for mass spectrometry (MS) as inexpensive and effective power supplies for pulsed nanoelectrospray ionization (nESI). The inherently discontinuous spray provided by TENG is particularly useful in scenarios where high sample economy is imperative, as in the case of ultraprecious samples. Previous work has shown the utility of TENG MS as a highly sensitive technique capable of yielding quality spectra from only a few microliters of sample at low micromolar concentrations. As the field of miniaturized, fieldable mass spectrometers grows, it remains critical to develop advanced ion sources with similarly small power requirements and footprints. Here, we present a redesigned TENG ion source with a sub-1000 USD material cost, lower power consumption, reduced footprint, and improved capabilities. We validate the performance of this new device for a diverse set of applications, including lipid double bond localization and native protein analysis.

Sample preparation for fatty acid analysis in biological samples with mass spectrometry-based strategies.

Fatty acids (FAs) have attracted many interests for their pivotal roles in many biological processes. Imbalance of FAs is related to a variety of diseases, which makes the measurement of them important in biological samples. Over the past two decades, mass spectrometry (MS) has become an indispensable technique for the analysis of FAs owing to its high sensitivity and precision. Due to complex matrix effect of biological samples and inherent poor ionization efficiency of FAs in MS, sample preparation including extraction and chemical derivatization prior to analysis are often employed. Here, we describe an updated overview of FA extraction techniques, as well as representative derivatization methods utilized in different MS platforms including gas chromatography-MS, liquid chromatography-MS, and mass spectrometry imaging based on different chain lengths of FAs. Derivatization strategies for the identification of double bond location in unsaturated FAs are also summarized and highlighted. The advantages, disadvantages, and prospects of these methods are compared and discussed. This review provides the development and valuable information for sample pretreatment approaches and qualitative and quantitative analysis of interested FAs using different MS-based platforms in complex biological matrices. Finally, the challenges of FA analysis are summarized and the future perspectives are prospected.

Paternò-Büchi Reaction Mass Spectrometry Enables Positional Assignment of Polymethylene-Interrupted Double Bonds in Food-Derived Lipids.

Fatty acids (FAs) containing polymethylene-interrupted (PMI) double bonds are a component of human foods; however, they present a significant analytical challenge for de novo identification. Covalent adduct chemical ionization and ozone-induced dissociation mass spectrometry (MS) methods enable unambiguous assignment of PMI-FA double bond positions, however, no method has been reported with electrospray ionization (ESI) platform using off-the-shelf systems. In the current work, we studied the Paternò-Büchi (PB) fragmentation patterns of PMI-FA and triacylglycerol (TG) by analyzing several known food sources. PB-MS/MS and MS3 enabled complete double bond location assignments, including the isolated double bond in PMI-FA and triacylglycerols. Sea urchin ("uni"), oyster, pine nut, and ginkgo nut were characterized for their signature PMI-FA, 20:2(5Z,11Z), 22:2(7Z, 15Z), 18:3(5Z,9Z,12Z), and 20:3(5Z,11Z,14Z), respectively. Quantitative analyses of the relative abundance of these PMI-FA led to results similar to reference methods. 18:3(5Z,9Z,12Z) was enriched at the sn-1/sn-3 position in pine nut major TG.

Location of Double or Triple Bonds in Lipids Using Mass Spectrometry, Part II

This text is the second part of a review article on advances in mass spectrometry for characterizing double and triple bonds in lipid acyl chains by LC-MS and direct ionization of liquid samples at atmospheric pressure. It is devoted to epoxidation, forming adducts from acetonitrile during atmospheric pressure chemical ionization, photodissociation, and electron-ion reaction-based dissociations.

Structural Elucidation and Relative Quantification of Fatty Acid Double Bond Positional Isomers in Biological Tissues Enabled by Gas-Phase Charge Inversion Ion/Ion Reactions.

Fatty acids (FAs) contain a vast amount of structural diversity, and differences in fatty acid structure have been associated with various disease states. Accurate identification and characterization of fatty acids is critical to fully understand the biochemical roles these compounds play in disease progression. Conventional tandem mass spectrometry (MS/MS) workflows do not provide sufficient structural information, necessitating alternative dissociation methods. Gas-phase charge inversion ion/ion reactions can be used to alter the ion type subjected to activation to provide improved or complementary structural information. Herein, we have used an ion/ion reaction between fatty acid (FA) anions and magnesium tris-phenanthroline [Mg(Phen)3] dications to promote charge remote fragmentation of carbon-carbon bonds along the fatty acid chain, allowing for localization of carbon-carbon double bond (C=C) positions to successfully differentiate monounsaturated fatty acid isomers. Relative quantification was also performed to obtain the relative abundance of fatty acid isomers in different biological tissues. For example, the relative abundance of FA 18:1 (9) was determined to vary across regions of rat brain, rat kidney, and mouse pancreas, and FA 16:1 (9) was found to have a higher relative abundance in the dermis layer compared to the sebaceous glands in human skin tissue.

Location of Double or Triple Bonds in Lipids Using Mass Spectrometry, Part I

Structural analysis of lipids is one of the classical topics of organic mass spectrometry. In recent years, we have witnessed a rapid development of new methods that allow the characterization of lipid structures more accurately, more rapidly, and in more complex samples. These methods are mostly based on derivatization reactions or newly available fragmentation techniques. Basic mass spectrometric analysis of lipids, i.e., determination of lipid class, acyl chain length, and degree of unsaturation, is usually relatively easy but the detailed structural description is more challenging. In this two-part review article, we present advances in mass spectrometry for characterizing double and triple bonds in lipid acyl chains by LC-MS and direct ionization of liquid samples at atmospheric pressure. The first paper discusses charge-switch derivatizations, ozonolysis, and the Paternò-Büchi reaction.

Revolutions in Lipid Isomer Resolution: Application of Ultrahigh-Resolution Ion Mobility to Reveal Lipid Diversity.

Many families of lipid isomers remain unresolved by contemporary liquid chromatography-mass spectrometry approaches, leading to a significant underestimation of the structural diversity within the lipidome. While ion mobility coupled to mass spectrometry has provided an additional dimension of lipid isomer resolution, some isomers require a resolving power beyond the capabilities of conventional platforms. Here, we present the application of high-resolution traveling-wave ion mobility for the separation of lipid isomers that differ in (i) the location of a single carbon-carbon double bond, (ii) the stereochemistry of the double bond (cis or trans), or, for glycerolipids, (iii) the relative substitution of acyl chains on the glycerol backbone (sn-position). Collisional activation following mobility separation allowed identification of the carbon-carbon double-bond position and sn-position, enabling confident interpretation of variations in mobility peak abundance. To demonstrate the applicability of this method, double-bond and sn-position isomers of an abundant phosphatidylcholine composition were resolved in extracts from a prostate cancer cell line and identified by comparison to pure isomer reference standards, revealing the presence of up to six isomers. These findings suggest that ultrahigh-resolution ion mobility has broad potential for isomer-resolved lipidomics and is attractive to consider for future integration with other modes of ion activation, thereby bringing together advanced orthogonal separations and structure elucidation to provide a more complete picture of the lipidome.

Deep-profiling of phospholipidome via rapid orthogonal separations and isomer-resolved mass spectrometry

A lipidome comprises thousands of lipid species, many of which are isomers and isobars. Liquid chromatography-tandem mass spectrometry (LC-MS/MS), although widely used for lipidomic profiling, faces challenges in differentiating lipid isomers. Herein, we address this issue by leveraging the orthogonal separation capabilities of hydrophilic interaction liquid chromatography (HILIC) and trapped ion mobility spectrometry (TIMS). We further integrate isomer-resolved MS/MS methods onto HILIC-TIMS, which enable pinpointing double bond locations in phospholipids and sn-positions in phosphatidylcholine. This system profiles phospholipids at multiple structural levels with short analysis time (<10 min per LC run), high sensitivity (nM detection limit), and wide coverage, while data analysis is streamlined using a home-developed software, LipidNovelist. Notably, compared to our previous report, the system doubles the coverage of phospholipids in bovine liver and reveals uncanonical desaturation pathways in RAW 264.7 macrophages. Relative quantitation of the double bond location isomers of phospholipids and the sn-position isomers of phosphatidylcholine enables the phenotyping of human bladder cancer tissue relative to normal control, which would be otherwise indistinguishable by traditional profiling methods. Our research offers a comprehensive solution for lipidomic profiling and highlights the critical role of isomer analysis in studying lipid metabolism in both healthy and diseased states.

Characterization of Oxidized Glycerophosphoethanolamines via Radical-Directed Dissociation Tandem Mass Spectrometry and the Paternò-Büchi Derivatization.

Oxidized glycerophosphoethanolamines (oxPEs) represent a subclass of bioactive lipids that have intricate roles in various physiological and pathological events. Conventional mass spectrometric methods cannot provide unambiguous information to locate the OH group and the sites of unsaturation. Herein, we report a combined strategy for in-depth structural characterization of oxPEs, including radical-directed dissociation tandem mass spectrometry (RDD-MS/MS) for localizing the OH group and the Paternò-Büchi derivatization coupled with tandem mass spectrometry for pinpointing carbon-carbon double-bond locations. The RDD-MS/MS method has been integrated on a reversed-phase liquid chromatography-mass spectrometry workflow. It enables the profiling of 24 distinct oxPE molecules with unequivocal assignment of the OH sites at nM sensitivity in bovine liver lipid extract treated by soybean 15-lipoxygenase. These findings showcase that the developed method has a good potential in analyzing biological systems where oxPEs may play important roles.

Rapidly identifying and quantifying of unsaturated lipids with carbon‐carbon double bond isomers by photoepoxidation

Unsaturated lipids play an essential role in life activities. Identifying and quantifying their carbon-carbon double bond (CC) isomers have become a hot topic in recent years. In lipidomics, the analysis of unsaturated lipids in complex biological samples usually requires high-throughput methods, which puts forward the requirements of rapid response and simple operation for identification. In this paper, we proposed a photoepoxidation strategy, which uses benzoin to open the double bonds of unsaturated lipids to form epoxides under ultraviolet light and aerobic conditions. Photoepoxidation is controlled by light and has a fast response. After 5 min, the derivatization yield can reach 80% with no side reaction products. Besides, the method has the advantages of high quantitation accuracy and a high yield of diagnostic ions. It was successfully applied to rapidly identify the double bond locations of various unsaturated lipids in both positive and negative ion modes, and to rapidly identify and quantitatively analyze the various isomers of unsaturated lipids in mouse tissue extract. So the method has the potential for large-scale analysis of unsaturated lipids in complex biological samples.

Identification of Carbon-Carbon Double Bond Stereochemistry in Unsaturated Fatty Acids by Charge-Remote Fragmentation of Fixed-Charge Derivatives.

Separation and identification of fatty acid (FA) isomers in biological samples represents a challenging problem for lipid chemists. Notably, FA regio- and stereo-isomers differing in the location or (cis/trans) geometry of carbon-carbon double bonds are often incompletely separated and ambiguously assigned in conventional chromatography-mass spectrometry analyses. To address this challenge, FAs have been derivatized with the charge-switch derivatization reagents N-methyl-pyridinium-3-methanamine and N-(4-aminomethylphenyl)pyridinium and subjected to reversed-phase liquid chromatography-tandem mass spectrometry. Charge-remote fragmentation of the fixed-charge derivatives leads to characteristic product ions arising from dissociation at allylic positions that enable assignment of position(s) of unsaturation, while a newly discovered dihydrogen neutral loss was found to be dominant for double bonds with cis-stereochemistry. The structure of the [M - 2]+ product ions was probed by gas-phase ozonolysis revealing the presence of two new carbon-carbon bonds on either side of the initial position of unsaturation consistent with an electrocyclic mechanism of 1,4-dihydrogen elimination. Charge-remote fragmentation pathways diagnostic of double bond position and stereochemistry were found to be generalized for FAs of different carbon-chain lengths, double bond positions, and degrees of unsaturation and were effective in the unequivocal assignment of the FA structure in complex mixtures of FA isomers, including bovine milk powder.

Quantitative Analysis and Structural Elucidation of Fatty Acids by Isobaric Multiplex Labeling Reagents for Carbonyl-Containing Compound (SUGAR) Tags and m-CPBA Epoxidation.

In this study, a novel analytical method was developed to investigate fatty acids (FAs) for relative quantification, carbon-carbon double-bond localization, and cis-/trans-geometry differentiation by isobaric multiplex labeling reagents for carbonyl-containing compound (SUGAR) tag conjugation and meta-chloroperoxybenzoic acid (m-CPBA) epoxidation. FAs are essential components of cells and have diverse functions in energy storage and as complex lipid constituents. It has been reported that FAs play different roles in various biological processes such as the functional development of the brain. The comprehensive characterization and quantification of FAs are crucial to further elucidate their biological roles. However, it is challenging to perform relative quantification and structural elucidation of FAs using integrated mass spectrometry (MS)-based methods. Recently, our group developed isobaric multiplex SUGAR tags for quantitative glycomics. Besides aldehyde/ketone groups on glycans, hydrazide groups also possess reactivity toward carboxylic acids on FAs. In this study, we extended SUGAR tag labeling with FAs for the quantitative analysis by liquid chromatography (LC)-MS/MS in the positive ion mode and applied this strategy for the comparative analysis of FAs hydrolyzed from oil samples. In addition, to comprehensively elucidate the structures of unsaturated FAs, epoxidation by m-CPBA was performed before SUGAR tag labeling to enable carbon-carbon double-bond localization. Moreover, the cis- and trans-geometries of carbon-carbon double bonds in multiple pairs of monounsaturated FAs could also be differentiated in higher-energy collisional dissociation (HCD)-MS/MS. This study developed a high-throughput comprehensive FA analysis platform, which could be widely applied and utilized in biological and clinical studies.

Liquid Chromatography-Mass Spectrometry (LC-MS) Derivatization-Based Methods for the Determination of Fatty Acids in Biological Samples.

Fatty acids (FAs) play pleiotropic roles in living organisms, acting as signaling molecules and gene regulators. They are present in plants and foods and may affect human health by food ingestion. As a consequence, analytical methods for their determination in biological fluids, plants and foods have attracted high interest. Undoubtedly, mass spectrometry (MS) has become an indispensable technique for the analysis of FAs. Due to the inherent poor ionization efficiency of FAs, their chemical derivatization prior to analysis is often employed. Usually, the derivatization of the FA carboxyl group aims to charge reversal, allowing detection and quantification in positive ion mode, thus, resulting in an increase in sensitivity in determination. Another approach is the derivatization of the double bond of unsaturated FAs, which aims to identify the double bond location. The present review summarizes the various classes of reagents developed for FA derivatization and discusses their applications in the liquid chromatography-MS (LC-MS) analysis of FAs in various matrices, including plasma and feces. In addition, applications for the determination of eicosanoids and fatty acid esters of hydroxy fatty acids (FAHFAs) are discussed.

Effect of double bond on electronic and optical properties of coelenteramide: A time-dependent density functional theory investigation

Coelenteramide has got attention for the great latent application in biologically inspired organic light-emitting devices. However, only blue light was emitted by coelenteramide. In order to obtain a wide distribution of light, the effect of double bond on electronic and optical properties of coelenteramide were studied by CAM-B3LYP/6-31+G(d) method. The results showed that the location of double bond was very important. For 1a, 2a, 2b and 3a, the double bond was inserted between amidopyrazine core and phenol group, which had strong π-conjugation effect, effectively improved the HOMO and LUMO energy levels, the capabilities of holes and electrons injection. The absorption and emission wavelengths generate a large bathochromic shift compared with native coelenteramide. In contrast, for 1b, 1c, 2c, double bond between amidopyrazine core and p-cresol, or between amidopyrazine core and benzyl group have little impact on HOMO and LUMO energy levels, energy gap, ionization potentials, electron affinities and absorption spectra.

A derivatization strategy for comprehensive identification of 2- and 3-hydroxyl fatty acids by LC-MS

2/3-Hydroxy fatty acids (2/3-OHFAs) are a class of important biological molecules closely related to many diseases, of which the unsaturated ones (2/3-OHUFAs) obtain special recognition for their bioactivities. However, the comprehensive identification of 2/3-OHFAs has been a daunting task due to the similarity of isomeric structures and lack of authentic standards. Herein, we report a strategy for the 2/3-OHUFA identification by using an in-house synthesized derivatization reagent, 4-amino-1,1-dimethylpiperidin-1-ium iodide hydrochloride (ADMI). Through ADMI derivatization, the diagnostic ion m/z 155.1 or 171.1 were produced by liquid chromatography-mass spectrometry (LC-MS) analysis, which could distinguish the 2-OH or 3-OH group, respectively. Then the meta-chloroperoxybenzoic acid (mcpba) was used to resolve the locations of double bonds in 2/3-OHUFAs by the characteristic cleavage of the newly formed epoxides. Thus, the identification of 2/3-OHUFAs was enabled by all these characteristics. Moreover, in order to simplify the whole analysis, an isotope-labeled ADMI was designed to quickly pick out the low-abundant 2/3-OHFAs from complex biological matrices. Finally, a combined derivatization strategy was established to analyze mouse lung tissues with melanoma metastasis. Different long-chain 2-OHFAs and 3-OHFAs were identified, including FA 12:0-3OH, FA 12:1(Δ9)-3OH, FA 14:0-3OH, FA 14:1(Δ5)-3OH, FA 16:0-2/3OH, and FA 18:0-2/3OH. The results showed that the contents of most identified 2/3-OHFAs were lower in the cancer group than in the control group, suggesting the plausible role of 2/3-OHFAs in cancer development. In summary, the new derivatization method provides a powerful analysis strategy for 2/3-OHUFAs, which sheds light on a better understanding of the biological functions of 2/3-OHUFAs.