Gas-phase stabilization of Cu(II) reveals phosphatidylcholine isomeric features by ESI and MALDI-MS

Glycerophospholipids (GPLs) are amphiphilic metabolites that make up most of the lipid bilayer in mammalian cells. Literature has pointed out the significant implications of double bond (DB) positions and linkage positions on the glycerol moiety in the chemical, biochemical, and biophysical roles of lipids.1 Currently, derivatization or instrumental modifications are requested to highlight these isomeric features in GPLs.2 Copper-adducted fatty acid dissociation spectra proved to be efficient in locating DB in aliphatic chains. However, the driving force of the diagnostic fragmentation mechanism was attributed to the presence of the free carboxylic acid function that is lacking in GPLs. We have recently demonstrated that radical-driven fragmentations could be obtained from quaternary copper complexes of GPLs. The CID MS/MS fragmentation of such species yields both DB and stereo-numbering (sn) locations in a single experiment.In this work, we will present our mechanistic hypothesis for phosphatidylcholine (PC) analogs, based on MS/MS and MSn experiments. Different isomers and classes of GPLs were used to confirm the unambiguous location of stereoisomers and the role of the polar head. In addition, this method was applied to generate Cu(II) quaternary PC complexes by MALDI-MS.We tried different MALDI matrices such as 1,5 DAN, 9-AA, DCTB, DHB, and THAP ionize and characterize the quaternary Cu(II)-based complex, and it turned out that DCTB was the only matrix that allowed the +II oxidation state of Cu to be preserved, suggesting high complex stability.3 MALDI-TOF/TOF experiments were performed on these complexes and allowed locating the double bonds.[1] X. Ma and Y. Xia, Angew. Chem. Int. Ed. Engl., 2014, 53, 2592–2596. [2] A. Bednařík, et al. Anal. Chem. 2022, 94, 4889–4900.[3] J. Zhang, et al. J. Am. Soc. Mass Spectrom. 2003, 14, 42–50

Gas-phase stabilization of Cu(II) reveals phosphatidylcholine isomeric features by ESI and MALDI-MS

International audienceGlycerophospholipids (GPLs) are amphiphilic metabolites that make up most of the lipidbilayer in mammalian cells. literature has pointed out the significant implications of doublebond (DB) positions and linkage positions on the glycerol moiety in the chemical, biochemical,and biophysical roles of lipids.1 Currently, derivatization or instrumental modifications are re-quested to highlight these isomeric features in GPLs.Copper-adducted fatty acid proved to be efficient in localizing DB in aliphatic chains. However,the driving force of this fragmentation mechanism was attributed to the presence of the freecarboxylic acid function that is lacking in GPLs. We recently demonstrated that radical-drivenfragmentation could be obtained from quaternary copper complexes of GPLs. The CID MS/MSfragmentation of such species yields both DB and stereo-numbering (sn) locations in a singleexperiment.In this work, we will present our mechanistic hypothesis for phosphatidylcholine analogs basedon MS/MS and MSn experiments. Different isomers and classes of GPLs were used to confirmthe unambiguous location of stereoisomers and the role of the polar head. In addition, thismethod was applied to generate Cu(II) quaternary PC complexes by MALDI-MS.We tried different MALDI matrixes to characterize the quaternary Cu(II)-based complex. Forthis purpose, 1,5 DAN, 9-AA, DCTB, DHB, and THAP were tested to ionize this complex.DCTB was the only matrix that allowed to keep the +II oxidation state of Cu, suggesting highcomplex stability.3 MALDI-TOF/TOF experiments were performed on these complexes and al-lowed to locate the double bonds

Surface Plasmon Resonance coupled to Mass Spectrometry to studylectin-sugar interactions

International audienceBiomolecular interactions are at the heart of the functioning of all living systems. The study ofbiointeractions is essential for understanding the global organization of the cellular machinery and theirrole in physiological processes. They constitute a significant challenge in analytical chemistry,diagnostics, and therapeutic research.Lectins are proteins that specifically recognize edible sugars. Our project aims to develop a couplingbetween Surface Plasmon Resonance Imaging and Mass Spectrometry (SPRi -MS) to analyze protein-sugar interactions. The aim is to create a multiplex SPR biochip with immobilized lectins and then to usethis biochip in coupling with MALDI-TOF-MS. This coupling allows the kinetics and thermodynamics ofthe interaction to be studied in real-time, together with the structural identification of the sugarscaptured from a complex mixture.By SPRi, this work confirmed significant interactions, between the lectin WGA and the neoglycosylatedBSA gratied carrying the sugars N-acetylgalactosamine and N-acetylglucosamine, and between the lectinAIA and neoglycosylated BSA carrying galactose and its N-acetylated form. We atempted the MSdetection of captured glycosylated BSA directly from the biochip surface; however, the lack of sensitivityin MS detection hindered the development of the coupling. The sensitivity depends, on the amount ofligands retained on biochip surface, which itself depends, among other factors, on the chemicalfunctionalization of the biochip surface, the nature of the receptors and their immobilization on thesurface [1]. Modifications of the MALDI-TOF-MS analysis conditions are being carried out with the useof MALDI imaging and the use of alternative receptors in order to evaluate their impact on the sensitivityof detection by SPRi-MS

2D Sequence-Coded Oligourethane Barcodes for Plastic Materials Labeling

International audienceMixtures of uniform sequence-defined oligourethanes were evaluated as 2D molecular barcodes for labeling three different commodity polymers, namely polystyrene, polyvinylchloride and polyethylene terephthalate. Six different oligourethanes were synthesized by solid-phase iterative synthesis and were coded using a binary monomer alphabet.High-resolution mass spectrometrystudies indicated that all oligomers are uniform and sequence-defined. However, instead of using them as individual codedchains, oligomers with different chain-length mass and sequence were mixed into intentionally polydisperse libraries. In particular, a three-component and a four-component library were created to encode a 2-bytes model binary sequence. These 2D-coded libraries were incorporated in all commodity plastics via a simple solvent casting procedure. Furthermore, in all cases, the oligomer mixtures could be extracted from the host polymer films and deciphered by mass spectrometry, thus opening interesting avenues for anti-counterfeiting and traceability applications.-

Structural characterization of phosphatidylcholine using VUVPD and ETD MS

Lipids present a challenging analytical task, due to the number of known or potential isomers in biological samples. Currently, many analogs remain undifferentiated, and despite the many technical and methodological advances made in MS for lipid analysis, their characterization at the isomeric level still remains an analytical challenge.1 Structural characterization of glycerophospholipids (GPLs) shows particular interest since literature has pointed out the significant implications of isomeric variations in their chemical, biochemical, and biophysical roles.2 Since CID MS/MS fragmentation of protonated or sodiated GPLs does not yield structural information about the double bond (DB) or stereo-numbering (sn) locations, we will present alternative activation methods to CID to characterize phosphatidylcholines at this isomeric level. For this purpose, electron transfer dissociation (ETD) and ultraviolet photodissociation (UVPD) will be used to locate DB position, and to distinguish the aliphatic chain positions on the glycerol moiety, respectively. In this way, we have demonstrated that radical fragmentations enabling DB localization can be obtained by ETD from lithiated species. In addition, the generation of radical cations along the aliphatic chain was improved by avoiding adduction at the polar head. While photodissociation in the hundreds of nm leads to low-intensity diagnostic fragment ions of the double bond,3 UV photo-fragmentation at shorter wavelengths generates products ions specific of the aliphatic chain position on the glycerol, and more particularly intense diagnostic product ions with exclusively odd-electron sn-1 ester cleavage, and even-electron sn-2 ester cleavage ions.[1] A. Bednařík, et al., Anal. Chem. 2022, 94, 4889–4900.[2] R. Bandu, et al., Mass Spectrom Rev, 2018, 37, 107–138. [3] D. Klein and J. Brodbelt, Anal. Chem. 2017, 89, 1516–1522

Structural identification of acetogenins by MS/MS of copper-adducted ions

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Coupling of Surface Plasmon Resonance with Mass Spectrometry to study protein glycosylation

International audienceLectins are widely appreciated, in the fields of hematology and immunology, due to their specificbinding to carbohydrate structures. Lectins are commonly present on the cell surface or elsewhere; they arealso known as specific markers for membrane glycoprotein structures. This study is focused on the use ofJacalin lectin (Artocarpus integrifolia) (AIA), which belongs to the family of galactose-binding lectins and isused here as a model of lectin. The lack of sensitivity in MS detection hinders the development of approachesusing Surface Plasmon Resonance (SPR) coupled with Mass Spectrometry (MS). Among the factors affectingsensitivity, the amount of ligands retained on the biochip surface is crucial [1]. Thanks to their smaller size andmultimerism, lectins offer an attractive receptor alternative to usual antibodies for increasing ligand capture,which is particularly interesting in glycoconjugates analysis.Our project aims to develop a multiplex SPR biochip with immobilized lectins to afford the couplingbetween SPR Imaging and MS (SPRi-MS) to determine protein glycosylation and further study of unknownlectins and their interactions with N-glycans. This coupling allows the real-time monitoring of interactions andthe determination of their kinetics and thermodynamics parameters, together with the structuralidentification of the glycoconjugates captured from a complex mixture [1], [2], [3].Fetuin is a plasma glycoprotein containing N-acetylglucosamine (GlcNac) and N-acetylgalactosamine(GalNac) in its structure, which is commonly used as a model for N- and O-glycans analysis. Our SPRi analysisconfirmed significant interactions between AIA and fetuin. The sensorgrams showed considerable variationsin reflectivity accompanied by the lighting of the spots upon the injection of increasing concentrations offetuin. Detection of the captured fetuin was successfully achieved by MALDI-TOF MS directly on the lectinbiochip.Moreover, a protocol for the on-chip spraying of the MALDI matrix is currently in development in thelaboratory. This mode of matrix deposit will be combined with a direct on-chip MALDI-TOF MS imaging (MSi)experiment to evaluate the impact on both the sensitivity and the selectivity of detection of fetuin by a SPRi-MSi approach

Convenient Graphical Visualization of Messages Encoded in Sequence-Defined Synthetic Polymers Using Kendrick Mass Defect Analysis of their MS/MS Data

WOS:000442218000008Kendrick mass defect (KMD) analysis is shown here to be a convenient method to read binary messages encoded in the structure of two types of sequence-defined synthetic polymers, namely, polyurethanes and poly(alkoxyamine phosphodiester)s. KMD analysis allows graphical ranking of mass data obtained for species containing repeating units. This is performed on MS/MS data in which distribution of fragments reveals the comonomer sequence of digital macromolecules. Choosing one coding monomer as the base unit, KMD computation of MS/MS data leads to stair-like plots where flat steps correspond to that monomer selected as the base unit while oblique steps reveal the other monomer. To correct for any point misalignments resulting from slight inaccuracy of fragment mass measurement, fractional base units are used to perform resolution-enhanced KMD (RE-KMD) analysis. As the length of the chain increased, a procedure aiming at correct aliased points is also implemented to achieve continuous, more convenient, stair-like plots

Kinetic study of azobenzene E/Z isomerization using ion mobility-mass spectrometry

International audienceIntroductionAzobenzene molecular switches can interconvert both photochemically and thermally. Due to their important development, their fast identification has become a challenge. Usually identified using UV-VIS spectrophotometry, we propose different coupling to MS such as ion mobility spectrometry and liquid chromatography to study the thermal Z to E back relaxation kinetic of isomers.MethodsDifferent synthetized isomerically pure azobenzenes were analysed in MS/MS using different cation adducts. LC experiments were performed using a 1260 series Infinity system packed with a Kinetex XB C-18 column coupled to an ion trap mass analyser (AmaZon speed ETD). Ion mobility-mass spectrometry experiments were carried out on a hybrid instrument (Synapt G2-Si HDMS) equipped with an ESI source. ResultsAnalyses of isomers by tandem mass spectrometry were carried out on different azobenzene pairs and an azobenzene sample consisting of three isomers. Differentiation of isomers was achieved for isomer couples using MS/MS. An upstream separation dimension was necessary to characterise the sample with three isomers. For these compounds, IMS-MS and LC-(UV)-MS were evaluated in parallel to study the kinetic of thermal Z to E isomerisation. Baseline separation of two isomers was achieved in IMS-MS from [M+Ag]+, while the third isomer was differentiated from [2M+Ag]+. In addition, LC separation of the three isomers was achieved in less than 10 minutes. These methodologies can be applied to different systems in order to determine their thermal back relaxation kinetic. IMS-MS and MS/MS methods will be preferred for fast systems as they allowed differentiation in the millisecond scale.Novel AspectFor the first time, the thermal back relaxation kinetic of azobenzene isomers is studied using hyphenated techniques (IMS and LC) to MS and MS/MS

Kinetic study of azobenzene E/Z isomerization using ion mobility-mass spectrometry

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