Calibrants based on synthetic dendrimers have been recently proposed as a versatile alternative to peptides and proteins for both MALDI and ESI mass spectrometry calibration. Because of their modular synthetic platform, dendrimer calibrants are particularly amenable to tailoring for specific applications. Utilizing this versatility, a set of dendrimers has been designed as an internal calibrant with a tailored mass defect to differentiate them from the majority of natural peptide analytes. This was achieved by incorporating a tris-iodinated aromatic core as an initiator for the dendrimer synthesis, thereby affording multiple calibration points (m/z range 600–2300) with an optimized mass-defect offset relative to all peptides composed of the 20 most common proteinogenic amino acids.
Ion-molecule reactions of Me2S2 with a wide range of aliphatic carbanions differing by structure and proton affinity values have been studied in the gas phase using mass spectrometry techniques and DFT calculations. The analysis of the spectra shows a variety of product ions formed via different reaction mechanisms, depending on the structure and proton affinity of the carbanion. Product ions of thiophilic reaction (m/z 47), SN2 (m/z 79), and E2 elimination – addition sequence of reactions (m/z 93) can be observed. Primary products of thiophilic reaction can undergo subsequent SN2 and proton transfer reactions. Gibbs free energy profiles calculated for experimentally observed reactions using PBE0/6-311+G(2d,p) method show good agreement with experimental results.
Quadrupole mass filters using non-sinusoidal driving potentials present exciting opportunities for new functionality. Predicting figures of merit like resolving power and transmission efficiency helps characterize these emerging devices. To this end, matrix methods of solving the Hill equation of ion motion are employed to calculate stability diagrams and pseudopotential well depth maps in the a,q plane for arbitrary waveforms. The theoretical resolving power and well depth of digital, trapezoidal and sinusoidal mass filters are compared. Simplified expressions for digital mass filter operation are presented.
Dissociation of proteins and peptides by 193 nm ultraviolet photodissociation (UVPD) has gained momentum in proteomic studies because of the diversity of backbone fragments that are produced and subsequent unrivaled sequence coverage obtained by the approach. The pathways that form the basis for the production of particular ion types are not completely understood. In this study, a statistical approach is used to probe hydrogen atom elimination from a + 1 radical ions, and different extents of elimination are found to vary as a function of the identity of the C-terminal residue of the a product ions and the presence or absence of hydrogen bonds to the cleaved residue.
Phospholipids generally dominate in bacterial lipids. The negatively charged nature of phospholipids renders bacteria susceptible to cationic antibiotic peptides. In comparison with Gram-negative bacteria, Gram-positive bacteria in general have much less zwitterionic phosphatidylethanolamine. However, they are known for producing aminoacylated phosphatidylglycerol (PG), especially positively charged l-lysyl-PG, which is catalyzed by lysyl-PG synthase MprF, which appears to have a broad range of specificity for l-aminoacyl transfer RNAs. In addition, many Gram-positive bacteria also have a dlt-gene-coded d-alanylation pathway for lipoteichoic acids and wall teichoic acids covalently attached to a glycolipid or peptidoglycan. d-Alanylation also masks the dominant negative charge of the phosphate-rich polymers of teichoic acids. Using mass spectrometry, we have recently observed that precursor scans in negative mode for deprotonated amino acid fragments were most sensitive for ester-linked amino acids. Such a scan for precursors generating an m/z 145 lysyl anion revealed lysyl-PG as well as an additional species 100?m/z units greater than lysyl-PG. This unexpected species corresponded precisely to the expected mass of N-succinylated lysyl-PG. Tandem mass spectrometry revealed a precise match to the fragmentation pattern of this putative new species. PG, lysyl-PG, and N-succinyl-lysyl-PG may form a complete loop of charge reversal from -1 to +1 and then back to -1. Analogous charge reversal by N-succinylation of lysine residues in the bacterial as well as eukaryotic proteomes has been recently discovered as a major posttranslational modification. Such modification in bacterial lipids is possibly catalyzed by an enzyme homologous to the enzymes that modify lysine residues in proteins.
Brucellaceae are Gram-negative bacteria that cause brucellosis, one of the most distributed worldwide zoonosis, transmitted to humans by contact with either infected animals or their products. The lipopolysaccharide exposed on the cell surface has been intensively studied and is considered a major virulence factor of Brucella. In the last years, structural studies allowed the determination of new structures in the core oligosaccharide and the O-antigen of this lipopolysaccharide. In this work, we have reinvestigated the lipid A structure isolated from B. suis and B. abortus lipopolysaccharides. A detailed study by MALDI-TOF mass spectrometry in the positive and negative ion modes of the lipid A moieties purified from both species was performed. Interestingly, a new feature was detected: the presence of a pyrophosphorylethanolamine residue substituting the backbone. LID-MS/MS analysis of some of the detected ions allowed assurance that the Lipid A structure composed by the diGlcN3N disaccharide, mainly hexa-acylated and penta-acylated, bearing one phosphate and one pyrophosphorylethanolamine residue.
Ion isolation in a linear ion trap is demonstrated using dual resonance frequencies, which are applied simultaneously. One frequency is used to eject ions of a broad m/z range higher in m/z than the target ion, and the second frequency is set to eject a range of ions lower in m/z. The combination of the two thus results in ion isolation. Despite the simplicity of the method, even ions of low intensity may be isolated since signal attenuation is less than an order of magnitude in most cases. The performance of dual frequency isolation is demonstrated by isolating individual isotopes of brominated compounds.
Ion mobility spectrometry-mass spectrometry (IMS-MS) in combination with gas-phase hydrogen/deuterium exchange (HDX) and collision-induced dissociation (CID) is evaluated as an analytical method for small-molecule standard and mixture characterization. Experiments show that compound ions exhibit unique HDX reactivities that can be used to distinguish different species. Additionally, it is shown that gas-phase HDX kinetics can be exploited to provide even further distinguishing capabilities by using different partial pressures of reagent gas. The relative HDX reactivity of a wide variety of molecules is discussed in light of the various molecular structures. Additionally, hydrogen accessibility scoring (HAS) and HDX kinetics modeling of candidate (in silico) ion structures is utilized to estimate the relative ion conformer populations giving rise to specific HDX behavior. These data interpretation methods are discussed with a focus on developing predictive tools for HDX behavior. Finally, an example is provided in which ion mobility information is supplemented with HDX reactivity data to aid identification efforts of compounds in a metabolite extract.
We present a new two-plate linear ion trap mass spectrometer that overcomes both performance-based and miniaturization-related issues with prior designs. Borosilicate glass substrates are patterned with aluminum electrodes on one side and wire-bonded to printed circuit boards. Ions are trapped in the space between two such plates. Tapered ejection slits in each glass plate eliminate issues with charge build-up within the ejection slit and with blocking of ions that are ejected at off-nominal angles. The tapered slit allows miniaturization of the trap features (electrode size, slit width) needed for further reduction of trap size while allowing the use of substrates that are still thick enough to provide ruggedness during handling, assembly, and in-field applications. Plate spacing was optimized during operation using a motorized translation stage. A scan rate of 2300 Th/s with a sample mixture of toluene and deuterated toluene (D8) and xylenes (a mixture of o-, m-, p-) showed narrowest peak widths of 0.33 Th (FWHM).
Hydrogen/deuterium exchange coupled with high-resolution mass spectrometry was successfully applied for the identification of A-type tetrameric, pentameric, and hexameric procyanidins in peanut skin. This extended a previous study on isomeric cyclic B-type unconventional tetramer, pentamer, and hexamer procyanidins found in wine and cranberries. Not only had the method successfully identified the procyanidins with a single A-linkage (e.g., tetrameric m/z 1153.2608) by means of distinguishing them from their isomeric cyclic B-type analogues, but this method also worked for procyanidins with two or more A-linkages (such as the tetrameric m/z 1151.2452). As a further consequence, B-type cyclic pentamers and hexamers in wine have been elucidated with hydrogen/deuterium exchange (HDX) for the first time.
Hydration reactions of deprotonated nucleobases (uracil, thymine, 5-fluorouracil,2-thiouracil, cytosine, adenine, and hypoxanthine) produced by electrospray have been experimentally studied in the gas phase at 10 mbar using a pulsed ion-beam high-pressure mass spectrometer. The thermochemical data, ΔHo, ΔSo, and ΔGo, for the monohydrated systems were determined. The hydration enthalpies were found to be similar for all studied systems and varied between 39.4 and 44.8 kJ/mol. A linear correlation was found between water binding energies in the hydrated complexes and the corresponding acidities of the most acidic site of nucleobases. The structural and energetic aspects of the precursors for the hydrated complexes are discussed in conjunction with available literature data.
Characterization of the cysteine content of proteins is a key aspect of proteomics. By defining both the total number of cysteines and their bound/unbound state, the number of candidate proteins considered in database searches is significantly constrained. Herein we present a methodology that utilizes 266 nm UVPD to count the number of free and bound cysteines in intact proteins. In order to attain this goal, proteins were derivatized with N-(phenylseleno)phthalimide (NPSP) to install a selectively cleavable Se–S bond upon 266 UVPD. The number of Se–S bonds cleaved upon UVPD, a process that releases SePh moieties, corresponds to the number of cysteine residues per protein.
Mixtures of pollen grains of three different species (Corylus avellana, Alnus cordata, and Pinus sylvestris) were investigated by matrix-assisted laser desorption/ionization time-of-flight imaging mass spectrometry (MALDI-TOF imaging MS). The amount of pollen grains was reduced stepwise from >?10 to single pollen grains. For sample pretreatment, we modified a previously applied approach, where any additional extraction steps were omitted. Our results show that characteristic pollen MALDI mass spectra can be obtained from a single pollen grain, which is the prerequisite for a reliable pollen classification in practical applications. MALDI imaging of laterally resolved pollen grains provides additional information by reducing the complexity of the MS spectra of mixtures, where frequently peak discrimination is observed. Combined with multivariate statistical analyses, such as principal component analysis (PCA), our approach offers the chance for a fast and reliable identification of individual pollen grains by mass spectrometry.
Radical activation methods, such as electron transfer dissociation (ETD), produce structural information complementary to collision-induced dissociation. Herein, electron transfer dissociation of 3-fold protonated DNA hexamers was studied to gain insight into the fragmentation mechanism. The fragmentation patterns of a large set of DNA hexamers confirm cytosine as the primary target of electron transfer. The reported data reveal backbone cleavage by internal electron transfer from the nucleobase to the phosphate linker leading either to a?/w or d/z? ion pairs. This reaction pathway contrasts with previous findings on the dissociation processes after electron capture by DNA cations, suggesting multiple, parallel dissociation channels. However, all these channels merely result in partial fragmentation of the precursor ion because the charge-reduced DNA radical cations are quite stable. Two hypotheses are put forward to explain the low dissociation yield of DNA radical cations: it is either attributed to non-covalent interactions between complementary fragments or to the stabilization of the unpaired electron in stacked nucleobases. MS3 experiments suggest that the charge-reduced species is the intact oligonucleotide. Moreover, introducing abasic sites significantly increases the dissociation yield of DNA cations. Consequently, the stabilization of the unpaired electron by π–π-stacking provides an appropriate rationale for the high intensity of DNA radical cations after electron transfer.
Paper spray tandem mass spectrometry is used to identify and quantify eight individual amphetamines in whole blood in 1.3 min. The method has been optimized and fully validated according to forensic toxicology guidelines, for the quantification of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxy-N-methylamphetamine (MDMA), 3,4-methylenedioxy-N-ethylamphetamine (MDEA), para-methoxyamphetamine (PMA), para-methoxymethamphetamine (PMMA), and 4-fluoroamphetamine (4-FA). Additionally, a new concept of intrinsic and application-based selectivity is discussed, featuring increased confidence in the power to discriminate the amphetamines from other chemically similar compounds when applying an ambient mass spectrometric method without chromatographic separation. Accuracy was within ±15% and average precision was better than 15%, and better than 20% at the LLOQ. Detection limits between 15 and 50 ng/mL were obtained using only 12 μL of whole blood.
Tissue imaging using matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a well-established technique that, in recent years, has seen wider adoption and novel application. Applications such imaging mass spectrometry (IMS) and biotyping are beginning to gain greater exposure and use; however, with limitations in optimization methods, producing the best result often relies on the ability to customize the physical characteristics of the instrumentation, a task that is challenging for most mass spectrometry laboratories. With this in mind, we have described the effect of making simple adjustments to the laser optics at the final collimating lens area, to adjust the laser beam size and shape in order to allow greater customization of the instrument for improving techniques such as IMS. We have therefore been able to demonstrate that improvements can be made without requiring the help of an electrical engineer or external funding in a way that only costs a small amount of time.
Sample throughput in electrospray ionization mass spectrometry (ESI-MS) is limited by the need for frequent ion path cleaning to remove accumulated debris that can lead to charging and general performance degradation. Contamination of ion optics within the vacuum system is particularly problematic as routine cleaning requires additional time for cycling the vacuum pumps. Differential mobility spectrometry (DMS) can select targeted ion species for transmission, thereby reducing the total number of charged particles entering the vacuum system. In this work, we characterize the nature of instrument contamination, describe efforts to improve mass spectrometer robustness by applying DMS prefiltering to reduce contamination of the vacuum ion optics, and demonstrate the capability of DMS to extend the interval between mass spectrometer cleaning. In addition, we introduce a new approach to effectively detect large charged particles formed during the electrospray ionization (ESI) process.
Mass spectroscopic investigations on tetrahydrofuran (THF, C4H8O), a common model molecule of the DNA-backbone, have been carried out. We irradiated isolated THF and (hydrated) THF clusters with low energy electrons (electron energy ~70 eV) in order to study electron ionization and ionic fragmentation. For elucidation of fragmentation pathways, deuterated TDF (C4D8O) was investigated as well. One major observation is that the cluster environment shows overall a protective behavior on THF. However, also new fragmentation channels open in the cluster. In this context, we were able to solve a discrepancy in the literature about the fragment ion peak at mass 55 u in the electron ionization mass spectrum of THF. We ascribe this ion yield to the fragmentation of ionized THF clusters.
Over the last several decades, mass spectrometry has become one of the principle methods for compound identification and quantification. While for analytical purposes, fragments which are not fully characterized in terms of origin and intensity as a function of experimental conditions have been used, understanding the nature of those species is very important. Herein we discuss such issues relative to triacetone triperoxide (TATP) and its frequently observed fragment at m/z 89. This “fragment” has been identified as the gas-phase reaction product of TATP with one or two methanol molecules/ions. Additionally, the origin and conditions of other fragments at m/z 91, 75, and 74 associated with TATP will be addressed. Similar analytical issues associated with other multi-peroxide organic compounds [hexamethylene triperoxide diamine (HMTD), methyl ethyl ketone peroxides (MEKP)] will also be discussed. Solution storage conditions for TATP, HMTD, and tetramethylene diperoxide diamine dialdehyde have been determined.
We report on the ultraviolet photodissociation (UVPD) chemistry of protonated tyrosine, iodotyrosine, and diiodotyrosine. Distonic loss of the iodine creates a high-energy radical at the aromatic ring that engages in hydrogen/proton rearrangement chemistry. Based on UVPD kinetics measurements, the appearance of this radical is coincident with the UV irradiation pulse (8 ns). Conversely, sequential UVPD product ions exhibit metastable decay on ca. 100 ns timescales. Infrared ion spectroscopy is capable of confirming putative structures of the rearrangement products as proton transfers from the imine and β-carbon hydrogens. Potential energy surfaces for the various reaction pathways indicate that the rearrangement chemistry is highly complex, compatible with a cascade of rearrangements, and that there is no preferred rearrangement pathway even in small molecular systems like these.
