Characterizing lignins from various sources and treatment processes after optimized sample preparation techniques and analysis via ESI-HRMS and custom mass defect software tools.

Sample preparation of complex, natural mixtures such as lignin prior to mass spectrometry analysis, however minimal, is a critical step in ensuring accurate and interference-free results. Modern shotgun-MS techniques, where samples are directly injected into a high-resolution mass spectrometer (HRMS) with no prior separation, usually still require basic sample pretreatment such as filtration and appropriate solvents for full dissolution and compatibility with atmospheric pressure ionization interfaces. In this study, sample preparation protocols have been established for a unique sample set consisting of a wide variety of degraded lignin samples from numerous sources and treatment processes. The samples were analyzed via electrospray (ESI)-HRMS in negative and positive ionization modes. The resulting information-rich HRMS datasets were then transformed into the mass defect space with custom R scripts as well as the open-source Constellation software as an effective way to visualize changes between the samples due to the sample preparation and ionization conditions as well as a starting point for comprehensive characterization of these varied sample sets. Optimized conditions for the four investigated lignins are proposed for ESI-HRMS analysis for the first time, giving an excellent starting point for future studies seeking to better characterize and understand these complex mixtures.

Combining Ultraviolet Photodissociation and Two-Dimensional Mass Spectrometry: A Contemporary Approach for Characterizing Singly Charged Agrochemicals.

Ultraviolet photodissociation (UVPD) has been shown to produce extensive structurally informative data for a variety of chemically diverse compounds. Herein, we demonstrate the performance of the 193 nm UVPD fragmentation technique on structural/moiety characterization of 14 singly charged agrochemicals. Two-dimensional mass spectrometry (2DMS) using infrared multiphoton dissociation (IRMPD) and electron-induced dissociation (EID) have previously been applied to a select range of singly charged pesticides. The ≥80% moiety coverage achieved for the majority of the species by the UVPD and 2D-UVPD methods was on par with and, in some cases, superior to the data obtained by other fragmentation techniques in previous studies, demonstrating that UVPD is viable for these types of species. A three-dimensional (3D) peak picking method was implemented to extract the data from the 2DMS spectrum, overcoming the limitations of the line extraction method used in previous studies, successfully separating precursor specific fragments with milli-Dalton accuracy. Whole spectrum internal calibration combined with 3D peak picking obtained sub-part-per-million (ppm) to part-per-billion (ppb) mass accuracies across the entire 2DMS spectrum.

Advantages of Two-Dimensional Electron-Induced Dissociation and Infrared Multiphoton Dissociation Mass Spectrometry for the Analysis of Agrochemicals.

Analysis of agrochemicals in an environmental matrix is challenging because these samples contain multiple agrochemicals, their metabolites, degradation products, and endogenous compounds. The analysis of such complex samples is achieved using chromatographic separation techniques coupled to mass spectrometry. Herein, we demonstrate a two-dimensional mass spectrometry (2DMS) technique on a 12 T Fourier transform ion cyclotron resonance mass spectrometer that can analyze a mixture of agrochemicals without using chromatography or quadrupole isolation in a single experiment. The resulting 2DMS contour plot contains abundant tandem MS information for each component in the sample and correlates product ions to their corresponding precursor ions. Two different fragmentation methods are employed, infrared multiphoton dissociation (IRMPD) and electron-induced dissociation (EID), with 2DMS to analyze the mixture of singly charged agrochemicals. The product ions of one of the agrochemicals, pirimiphos-methyl, present in the sample was used to internally calibrate the entire 2DMS spectrum, achieving sub part per million (ppm) to part per billion (ppb) mass accuracies for all species analyzed. The work described in this study will show the advantages of the 2DMS approach, by grouping species with common fragments/core structure and mutual functional groups, using precursor lines and neutral loss lines. In addition, the rich spectral information obtained from IRMPD and EID 2DMS contour plots can accurately identify and characterize agrochemicals.

Comparison of Fragmentation Techniques for the Structural Characterization of Singly Charged Agrochemicals.

Investigating the structure of active ingredients, such as agrochemicals and their associated metabolites, is a crucial requisite in the discovery and development of these molecules. In this study, structural characterization by electron-induced dissociation (EID) was compared to collisionally activated dissociation (CAD) on a series of agrochemicals. EID fragmentation produced a greater variety of fragment ions and complementary ion pairs leading to more complete functional group characterization compared to CAD. The results obtained displayed many more cross-ring fragmentation of the pyrimidine ring compared to the pyridine ring. Compounds that consisted of one aromatic heterocyclic moiety (azoxystrobin, fluazifop acid, fluazifop-p-butyl, and pirimiphos-methyl) displayed cross-ring fragmentation while compounds with only aromatic hydrocarbon rings (fenpropidin and S-metolachlor) displayed no cross-ring fragmentation. The advantages of high-resolution accurate mass spectrometry (HRAM MS) are shown with the majority of assignments at ppb range error values and the ability to differentiate ions with the same nominal mass but different elemental composition. This highlights the potential for HRAM MS and EID to be used as a tool for structural characterization of small molecules with a wide variety of functional groups and structural motifs.

Residual Complex I activity and amphidirectional Complex II operation support glutamate catabolism through mtSLP in anoxia.

Anoxia halts oxidative phosphorylation (OXPHOS) causing an accumulation of reduced compounds in the mitochondrial matrix which impedes dehydrogenases. By simultaneously measuring oxygen concentration, NADH autofluorescence, mitochondrial membrane potential and ubiquinone reduction extent in isolated mitochondria in real-time, we demonstrate that Complex I utilized endogenous quinones to oxidize NADH under acute anoxia. 13C metabolic tracing or untargeted analysis of metabolites extracted during anoxia in the presence or absence of site-specific inhibitors of the electron transfer system showed that NAD+ regenerated by Complex I is reduced by the 2-oxoglutarate dehydrogenase Complex yielding succinyl-CoA supporting mitochondrial substrate-level phosphorylation (mtSLP), releasing succinate. Complex II operated amphidirectionally during the anoxic event, providing quinones to Complex I and reducing fumarate to succinate. Our results highlight the importance of quinone provision to Complex I oxidizing NADH maintaining glutamate catabolism and mtSLP in the absence of OXPHOS.

Differentiation of Dihydroxylated Vitamin D3 Isomers Using Tandem Mass Spectrometry.

Vitamin D compounds are a group of secosteroids derived from cholesterol that are vital for maintaining bone health in humans. Recent studies have shown extraskeletal effects of vitamin D, involving vitamin D metabolites such as the dihydroxylated vitamin D3 compounds 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3. Differentiation and characterization of these isomers by mass spectrometry can be challenging due to the zero-mass difference and minor structural differences between them. The isomers usually require separation by liquid chromatography (LC) prior to mass spectrometry, which adds extra complexity to the analysis. Herein, we investigated and revisited the use of fragmentation methods such as collisional induced dissociation (CID), infrared multiphoton dissociation (IRMPD), electron induced dissociation (EID), and ultraviolet photodissociation (UVPD), available on a 12T Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) to generate characteristic fragments for the dihydroxylated vitamin D3 isomers that can be used to distinguish between them. Isomer-specific fragments were observed for the 1,25-dihydroxyvitamin D3, which were clearly absent in the 24,25-dihydroxyvitamin D3 MS/MS spectra using all fragmentation methods mentioned above. The fragments generated due to cleavage of the C-6/C-7 bond in the 1,25-dihydroxyvitamin D3 compound demonstrate that the fragile OH groups were retained during fragmentation, thus enabling differentiation between the two dihydroxylated vitamin D3 isomers without the need for prior chromatographic separation or derivatization.

Multimodal Tandem Mass Spectrometry Techniques for the Analysis of Phosphopeptides.

Collisionally activated dissociation (CAD), infrared multiphoton dissociation (IRMPD), ultraviolet photodissociation (UVPD), electron capture dissociation and electron detachment dissociation (EDD) experiments were conducted on a set of phosphopeptides, in a Fourier transform ion cyclotron resonance mass spectrometer. The fragmentation patterns were compared and varied according to the fragmentation mechanisms and the composition of the peptides. CAD and IRMPD produced similar fragmentation profiles of the phosphopeptides, while UVPD produced a large number of complementary fragments. Electron-based dissociation techniques displayed lower fragmentation efficiencies, despite retaining the labile phosphate group, and drastically different fragmentation profiles. EDD produced complex spectra whose interpretation proved challenging.

Stochasticity of poly(2-oxazoline) oligomer hydrolysis determined by tandem mass spectrometry.

Understanding modification of synthetic polymer structures is necessary for their accurate synthesis and potential applications. In this contribution, a series of partially hydrolyzed poly(2-oxazoline) species were produced forming poly[(2-polyoxazoline)-co-(ethylenimine)] (P(EtOx-co-EI)) copolymers; EI being the hydrolyzed product of Ox. Bulk mass spectrometry (MS) measurements accurately measured the EI content. Tandem mass spectrometry analysis of the EI content in the copolymer samples determined the distribution of each monomer within the copolymer and corresponded to a theoretically modelled random distribution. The EI distribution across the polymers was shown to be effected by the choice of terminus, with a permanent hydrolysis event observed at an OH terminus. A neighbouring group effect wasn't observed at the polymer length analysed (approximately 25-mer species), suggesting that previously observed neighbouring group effects require a larger polymer chain. Although clearly useful for random polymer distribution this approach may be applied to many systems containing non-specific modifications to determine if they are directed or random locations across peptides, proteins, polymers, and nucleic acids.

Two-Dimensional Mass Spectrometry Analysis of IgG1 Antibodies.

Two-dimensional mass spectrometry (2DMS) is a new, and theoretically ideal, data-independent analysis tool, which allows the characterization of a complex mixture and was used in the bottom-up analysis of IgG1 for the identification of post-translational modifications. The new peak picking algorithm allows the distinction between chimeric peaks in proteomics. In this application, the processing of 2DMS data correlates fragments to their corresponding precursors, with fragments from precursors which are <0.1 m/z at m/z 840 easily resolved, without the need for quadrupole or chromatographic separation.