Characterization of Phosphorylated Peptides by Electron-Activated and Ultraviolet Dissociation Mass Spectrometry: A Comparative Study with Collision-Induced Dissociation.

Mass-spectrometry-based methods have made significant progress in the characterization of post-translational modifications (PTMs) in peptides and proteins; however, room remains to improve fragmentation methods. Ideal MS/MS methods are expected to simultaneously provide extensive sequence information and localization of PTM sites and retain labile PTM groups. This collection of criteria is difficult to meet, and the various activation methods available today offer different capabilities. In order to examine the specific case of phosphorylation on peptides, we investigate electron transfer dissociation (ETD), electron-activated dissociation (EAD), and 193 nm ultraviolet photodissociation (UVPD) and compare all three methods with classical collision-induced dissociation (CID). EAD and UVPD show extensive backbone fragmentation, comparable in scope to that of CID. These methods provide diverse backbone fragmentation, producing a/x, b/y, and c/z ions with substantial sequence coverages. EAD displays a high retention efficiency of the phosphate modification, attributed to its electron-mediated fragmentation mechanisms, as observed in ETD. UVPD offers reasonable retention efficiency, also allowing localization of the PTM site. EAD experiments were also performed in an LC-MS/MS workflow by analyzing phosphopeptides spiked in human plasma, and spectra allow accurate identification of the modified sites and discrimination of isomers. Based on the overall performance, EAD and 193 nm UVPD offer alternative options to CID and ETD for phosphoproteomics.

Structural Elucidation and Relative Quantification of Sodium- and Potassium-Cationized Phosphatidylcholine Regioisomers Directly from Tissue Using Electron Induced Dissociation.

Comprehensive structural characterization of phosphatidylcholines (PCs) is essential to understanding their biological functions and roles in metabolism. Electron induced dissociation (EID) of protonated PCs directly generated from biological tissues has previously been shown to provide in-depth structural information on the lipid headgroup, regiosiomerism of fatty acyl tails and double bond positions. Although phosphatidylcholine ions formed via alkali metal cationization (i.e., [M + Na]+ and [M + K]+) are commonly generated during matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry experiments, the gas-phase ion chemistry behavior of EID on sodium- and potassium-cationized phosphatidylcholine ion types has not been studied for ions generated directly from tissue. Herein, we demonstrate EID on [M + Na]+ and [M + K]+ ion types in a MALDI imaging mass spectrometry workflow for lipid structural characterization. Briefly, near-complete structural information can be obtained upon EID of sodium- and potassium-cationized PCs, including diagnostic fragmentation of the lipid headgroup as well as identification of fatty acyl chain positions and double bond position. EID of cationized lipids generates sn-specific glycerol backbone cleavages as well as a favorable combined loss of sn-2 fatty acid with choline over sn-1, allowing for facile differentiation and relative quantification of PC regioisomers. Moreover, relative quantification of sn-positional isomers from biological tissue reveals that the relative percentages of sodium- and potassium-cationized sn-positional isomers varies significantly in different regions of rat brain tissue.

Improved metabolite characterization by liquid chromatography - Tandem mass spectrometry through electron impact type fragments from adduct ions.

Using a chimeric collision cell mounted on a quadrupole time-of-flight platform, collision induced dissociation (CID) and electron induced dissociation (EID) were investigated for the LC-MS analysis of low molecular weight compounds including drugs and endogenous metabolites. Compared to CID, EID fragmentation of the [M+H]+ species (10-20 eV) from standard compounds resulted in additional specific and informative fragments, mostly due to neutral losses and, in some cases due to ring openings. Some analytes, for example reserpine and vinpocetine, provided characteristic [M+H]•2+ species. For most analytes for sodium and potassium adducts and multimers a radical cation M•+ and electron impact type fragments were observed in the EID spectra, providing the opportunity to use EI libraries to support metabolite identification. EID opens the possibility to get structural information from adduct ions which is often not the case with CID. EID enabled the putative characterization of two metabolites in rat urine as glucuronides of 5,6-dihydroxyindole based on EID fragmentation of the potassium adducts.

TUTORIAL: ION ACTIVATION IN TANDEM MASS SPECTROMETRY USING ULTRA-HIGH RESOLUTION INSTRUMENTATION.

Tandem mass spectrometry involves isolation of specific precursor ions and their subsequent excitation through collision-, photon-, or electron-mediated activation techniques in order to induce unimolecular dissociation leading to formation of fragment ions. These powerful ion activation techniques, typically used in between mass selection and mass analysis steps for structural elucidation, have not only found a wide variety of analytical applications in chemistry and biology, but they have also been used to study the fundamental properties of ions in the gas phase. In this tutorial paper, a brief overview is presented of the theories that have been used to describe the activation of ions and their subsequent unimolecular dissociation. Acronyms of the presented techniques include CID, PQD, HCD, SORI, SID, BIRD, IRMPD, UVPD, EPD, ECD, EDD, ETD, and EID. The fundamental principles of these techniques are discussed in the context of their implementation on ultra-high resolution tandem mass spectrometers. © 2020 John Wiley & Sons Ltd. Mass Spec Rev.

Liquid Extraction Surface Analysis (LESA) Electron-Induced Dissociation and Collision-Induced Dissociation Mass Spectrometry of Small Molecule Drug Compounds.

Here, we present liquid extraction surface analysis (LESA) coupled with electron-induced dissociation (EID) mass spectrometry in a Fourier-transform ion cyclotron resonance mass spectrometer for the analysis of small organic pharmaceutical compounds directly from dosed tissue. First, the direct infusion electrospray ionisation EID and collision-induced dissociation (CID) behaviour of erlotinib, moxifloxacin, clozapine and olanzapine standards were compared. EID mass spectra were also compared with experimental or reference electron impact ionisation mass spectra. The results show that (with the exception of erlotinib) EID and CID result in complementary fragment ions. Subsequently, we performed LESA EID MS/MS and LESA CID MS/MS on singly charged ions of moxifloxacin and erlotinib extracted from a thin tissue section of rat kidney from a cassette-dosed animal. Both techniques provided structural information, with the majority of peaks observed for the drug standards also observed for the tissue-extracted species. Overall, these results demonstrate the feasibility of LESA EID MS/MS of drug compounds from dosed tissue and extend the number of molecular structures for which EID behaviour has been determined. Graphical Abstract ᅟ.

Quantitative structural multiclass lipidomics using differential mobility: electron impact excitation of ions from organics (EIEIO) mass spectrometry.

We report a method for comprehensive structural characterization of lipids in animal tissues using a combination of differential ion mobility spectrometry (DMS) with electron-impact excitation of ions from organics (EIEIO) mass spectrometry. Singly charged lipid ions in protonated or sodiated forms were dissociated by an electron beam having a kinetic energy of 10 eV in a branched radio-frequency ion trap. We established a comprehensive set of diagnostics to characterize the structures of glycerophospholipids, sphingolipids, and acylglycerols, including glycosylated, plasmalogen, and ester forms. This EIEIO mass spectrometer was combined with DMS as a separation tool to analyze complex lipid extracts. Deuterated quantitative standards, which were added during extraction, allowed for the quantitative analysis of the lipid molecular species in various lipid classes. We applied this technique to the total lipids extracted from porcine brain, and we structurally characterized over 300 lipids (with the exception of cis/trans double-bond isomerism in the acyl chains). The structural dataset of the lipidomes, whose regioisomers were distinguished, exhibit a uniquely defined distribution of acyl chains within each lipid class; that is, sn-1 and sn-2 in the cases of glycerophospholipids or sn-2 and (sn-1, sn-3) in the cases of triacylglycerols.

Structural identification of triacylglycerol isomers using electron impact excitation of ions from organics (EIEIO).

Electron-induced dissociation or electron impact excitation of ions from organics (EIEIO) was applied to triacylglycerols (TAGs) for in-depth molecular structure analysis using MS. In EIEIO, energetic electrons (∼10 eV) fragmented TAG ions to allow for regioisomeric assignment of identified acyl groups at the sn-2 or sn-1/3 positions of the glycerol backbone. In addition, carbon-carbon double bond locations within the acyl chains could also be assigned by EIEIO. Beyond the analysis of lipid standards, this technique was applied to edible oils and natural lipid extracts to demonstrate the power of this method to provide in-depth structural elucidation of TAG molecular species.

In-depth sphingomyelin characterization using electron impact excitation of ions from organics and mass spectrometry.

Electron impact excitation of ions from organics (EIEIO), also referred to as electron-induced dissociation, was applied to singly charged SM molecular species in the gas phase. Using ESI and a quadrupole TOF mass spectrometer equipped with an electron-ion reaction device, we found that SMs fragmented sufficiently to identify their lipid class, acyl group structure, and the location of double bond(s). Using this technique, nearly 200 SM molecular species were found in four natural lipid extracts: bovine milk, porcine brain, chicken egg yolk, and bovine heart. In addition to the most common backbone, d18:1, sphingosines with a range of carbon chain lengths, sphingadienes, and some sphinganine backbones were also detected. Modifications in natural SMs were also identified, including addition of iodine/methanol across a carbon-carbon double bond. This unparalleled new approach to SM analysis using EIEIO-MS shows promise as a unique and powerful tool for structural characterization.

Electron-induced dissociation (EID) for structure characterization of glycerophosphatidylcholine: determination of double-bond positions and localization of acyl chains.

Glycerophospholipids are a highly abundant and diverse collection of biologically relevant lipids, and distinction between isomeric and isobaric species is a fundamental aspect for confident identification. The ability to confidently assign a unique structure to a glycerophospholipid of interest is dependent on determining the number and location of the points of unsaturation and assignment of acyl chain position. The use of high-energy electrons (>20 eV) to induce gas-phase dissociation of intact precursor ions results in diagnostic product ions for localizing double-bond positions and determining acyl chain assignment. We describe a high-resolution, tandem mass spectrometry method for structure characterization of glycerophospholipids using electron-induced dissociation (EID). Furthermore, the inclusion of nomenclature to systematically assign bond cleavage sites with acyl chain position and double-bond location enables a uniform platform for lipid identification. The EID methodology detailed here combines novel application of an electron-based dissociation technique with high-resolution mass spectrometry that facilitates a new experimental approach for lipid biomarker discovery and validation.

Deciphering the structure of isomeric oligosaccharides in a complex mixture by tandem mass spectrometry: photon activation with vacuum ultra-violet brings unique information and enables definitive structure assignment.

Carbohydrates have a wide variety of structures whose complexity and heterogeneity challenge the field of analytical chemistry. Tandem mass spectrometry, with its remarkable sensitivity and high information content, provides key advantages to addressing the structural elucidation of polysaccharides. Yet, classical fragmentation by collision-activated dissociation (CAD) in many cases fails to reach a comprehensive structural determination, especially when isomers have to be differentiated. In this work, for the first time, vacuum ultra-violet (VUV) synchrotron radiation is used as the activation process in tandem mass spectrometry of large oligosaccharides. Compared to low energy CAD (LE-CAD), photon activated dissociation brought more straightforward and valuable structural information. The outstanding feature was that complete series of informative ions were produced, with only minor neutral losses. Moreover, systematic fragmentation rules could be drawn thus facilitating the definitive assignments of fragment identities. As a result, most of the structures present in a complex mixture of oligogalacturonans could be comprehensively resolved, including many isomers differing in the position of methyl groups along the galacturonic acid backbone.