Charge Enhancement of Single-Stranded DNA in Negative Electrospray Ionization Using the Supercharging Reagent Meta-nitrobenzyl Alcohol

Charge enhancement of single-stranded oligonucleotide ions in negative ESI mode is investigated. The employed reagent, meta-nitrobenzyl alcohol (m-NBA), was found to improve total signal intensity (I_tot), increase the highest observed charge states (z_high), and raise the average charge states (z_avg) of all tested oligonucleotides analyzed in negative ESI. To quantify these increases, signal enhancement ratios (SER_1%) and charge enhancement coefficients (CEC_1%) were introduced. The SER_1%, (defined as the quotient of total oligonucleotide ion abundances with 1 % m-NBA divided by total oligonucleotide abundance without m-NBA) was found to be greater than unity for every oligonucleotide tested. The CEC_1% values (defined as the average charge state in the presence of 1 % m-NBA minus the average charge state in the absence of m-NBA) were found to be uniformly positive. Upon close inspection, the degree of charge enhancement for longer oligonucleotides was found to be dependent upon thymine density (i.e., the number and the location of phospho-thymidine units). A correlation between the charge enhancement induced by the presence of m-NBA and the apparent gas-phase acidity (largely determined by the sequence of thymine units but also by the presence of protons on other nucleobases) of multiply deprotonated oligonucleotide species, was thus established. Ammonium cations appeared to be directly involved in the m-NBA supercharging mechanism, and their role seems to be consistent with previously postulated ESI mechanisms describing desorption/ionization of single-stranded DNA into the gas phase.

Improved MALDI-TOF Microbial Mass Spectrometry Imaging by Application of a Dispersed Solid Matrix

The key step in high quality microbial matrix-assisted laser desorption/ionization mass spectrometry imaging (microbial MALDI MSI) is the fabrication of a homogeneous matrix coating showing a fine-grained morphology. This application note addresses a novel method to apply solid MALDI matrices onto microbial cultures grown on thin agar media. A suspension of a mixture of 2,5-DHB and α-CHCA is sprayed onto the agar sample surface to form highly homogeneous matrix coatings. As a result, the signal intensities of metabolites secreted by the fungus Aspergillus fumigatus were found to be clearly enhanced.

Using Gas Modifiers to Significantly Improve Sensitivity and Selectivity in a Cylindrical FAIMS Device

Recent reports describing enhanced performance when using gas additives in a DMS device (planar electrodes) have indicated that comparable benefits are not attainable using FAIMS (cylindrical electrodes), owing to the non-homogeneous electric fields within the analyzer region. In this study, a FAIMS system (having cylindrical electrodes) was modified to allow for controlled delivery of gas additives. An experiment was carried out that illustrates the important distinction between gas modifiers present as unregulated contaminants and modifiers added in a controlled manner. The effect of contamination was simulated by adjusting the ESI needle position to promote incomplete desolvation, thereby permitting ESI solvent vapor into the FAIMS analyzer region, causing signal instability and irreproducible CV values. However, by actively controlling the delivery of the gas modifier, reproducible CV spectra were obtained. The effects of adding different gas modifiers were examined using 15 positive ions having mass-to-charge (m/z) values between 90 and 734. Significant improvements in peak capacity and increases in ion transmission were readily attained by adding acetonitrile vapor, even at trace levels (≤0.1%). Increases in signal intensity were greatest for the low m/z ions; for the six lowest molecular weight species, signal intensities increased by ～10- to over 100-fold compared with using nitrogen without gas additives, resulting in equivalent or better signal intensities compared with ESI without FAIMS. These results confirm that analytical benefits derived from the addition of gas modifiers reported with a uniform electric field (DMS) also are observed using a non-homogenous electric field (FAIMS) in the analyser region.

Impact of homogeneous and filamentary discharge modes on the efficiency of dielectric barrier discharge ionization mass spectrometry

The present study contributes to the evaluation of dielectric barrier discharge-based ambient ionization for mass spectrometric analysis (DBDI-MS) by providing a further step towards an understanding of underlying ionization processes. This examination highlights the effect of physical discharge modes on the ionization efficiency of the DBDI source. A distinction is made between the homogeneous and filamentary discharge mode due to different plasma gases in barrier configurations. Therefore, we first report on discharge modes of DBDI by demonstrating a universally applicable method to classify the predominant modes. Then, the ionization efficiency of these two modes is evaluated by a laser desorption-DBDI-MS with different molecular analytes. Here, the laser desorption is used to deliver neutral analytes which will be ionized by the plasma jet applied as dielectric barrier discharge ionization. With a clear increase of signal intensities in the homogeneous mode in contrast to the filamentary one, the present study indicates a pronounced dependence of the ionization efficiency on the discharge mode allowing further insight into the mechanisms of the ionization process.

Structural Distinction of Diacyl-, Alkylacyl,and Alk-1-Enylacyl Glycerophosphocholines as [M – 15]– Ions by Multiple-Stage Linear Ion-Trap Mass Spectrometry with Electrospray Ionization

We describe a linear ion-trap (LIT) multiple-stage (MSⁿ) mass spectrometric approach towards differentiation of alkylacyl, alk-1-enylacyl- and diacyl-glycerophoscholines (PCs) as the [M – 15]^– ions desorbed by electrospray ionization (ESI) in the negative-ion mode. The MS⁴ mass spectra of the [M – 15 – R₂′CH = CO]^– ions originated from the three PC subfamilies are readily distinguishable, resulting in unambiguous distinction of the lipid classes. This method is applied to two alkyl ether rich PC mixtures isolated from murine bone marrow neutrophils and kidney, respectively, to explore its utility in the characterization of complex PC mixture of biological origin, resulting in the realization of the detailed structures of the PC species, including various classes and many minor isobaric isomers.

Study of the ESI and APCI interfaces for the UPLC–MS/MS analysis of pesticides in traditional Chinese herbal medicine

In this work, 53 selected pesticides of different chemical groups were extracted from Chinese herbal medicines and determined by ultra-high-performance liquid chromatography (UHPLC)–tandem mass spectrometry (MS/MS) using both electrospray ionization (ESI) and atmospheric-pressure chemical ionization (APCI). Extracts were obtained using the acetonitrile-based quick, easy, cheap, effective, rugged, and safe (QuEChERS) sample preparation technique. Cleanup was performed by dispersive solid-phase extraction using primary secondary amine, graphitized carbon black, and octadecylsilane. Two atmospheric-pressure interfaces, ESI and APCI, were checked and compared. The validation study, including detection limits, linearity, and matrix effects, was conducted on fritillaria, radix ginseng, folium isatidis, semen persicae, and flos lonicerae in multiple reaction monitoring mode. These matrices represent a variety of plants used in traditional Chinese medicine. Fritillaria and radix ginseng were chosen as representatives for roots, folium isatidis was chosen as a representative for leaves, semen persicae was chosen as a representative for seeds, and flos lonicerae was chosen as a representative for flowers. The limits of detection for pesticides were lower in the UHPLC–ESI-MS/MS method than in the UHPLC–APCI-MS/MS method. Matrix effects on the two ionizations were evaluated for the five matrices. Soft signal enhancement in UHPLC–APCI-MS/MS and signal suppression in UHPLC–ESI-MS/MS were observed.

Amino and Acetamide Functional Group Effects on the Ionization and Fragmentation of Sugar Chains in Positive-Ion Mass Spectrometry

To elucidate the influence of amino (-NH₂) and acetamide (-NHCOCH₃, -NAc) groups in sugar chains on their ionization and fragmentation, cycloamyloses (cyclodextrins, CyDs) and lacto-oligosaccharide are analyzed by MALDI TOF/TOF and ESI Q-TOF mass spectrometry. CyD derivatives substituted by amino or acetamide groups are ideal analytes to extract the function group effects, which are amino-CyD with one hexosamine (HexNH₂) and acetamide-CyD with one N-acetyl hexosamine (HexNAc). Interestingly, the relative ion intensities and isotope-like patterns in their product ion spectra depend on the functional groups and ion forms of sugar chains. Consequently, the results indicate that a proton (H⁺) localizes on the amino group of the amino sugar, and that the proton (H⁺) induces their fragmentation. Sodium cation (Na⁺) attachment is independent from amino group and exerts no influence on their fragmentation patterns in amino group except for mono- and disaccharide fragment ions because there is the possibility of the reducing end effect. In contrast, a sodium cation localizes much more frequently on the acetamide group in acetamide-CyDs because the chemical species with HexNAc are stable. Thus, their ions with HexNAc are abundant. These results are consistent with the fragmentation of lacto-neo-N-tetraose and maltotetraose, suggesting that a sodium cation generally localizes much more frequently on the acetamide group in sugar chains.

In-Line Desalting of Proteins from Buffer and Synthetic Urine Solution Prior to ESI-MS Analysis via a Capillary-Channeled Polymer Fiber Microcolumn

Presented here is a novel in-line solid phase extraction (SPE) method utilizing a capillary-channeled polymer (C-CP) fiber microcolumn prior to introduction to an electrospray ionization (ESI) source. The high permeability of the microcolumn allows for operation under syringe pump or HPLC driven flow, ultimately providing greater mass spectral clarity and accurate molecular weight determinations for different protein/buffer combinations. Studies presented here focus on the desalting of several target proteins from a standard phosphate buffered saline (PBS) matrix and a synthetic urine solution prior to ESI-MS determinations. In every case, responses for μM-level proteins in PBS improve from the situation of not permitting molecular weight determinations to values that are precise to better than ±10 Da, without internal standards, with relative improvements in the signal-to-background ratios (S/B) on the order of 3,000×. De-salting of a myoglobin-spiked (12 μM) synthetic urine results in equally-improved spectral quality.      

ESI Mass Spectrometric Exploration of Selective Recognition of G-Quadruplex in c-myb Oncogene Promoter Using a Novel Flexible Cyclic Polyamide

In this research, electrospray ionization mass spectrometry (ESI-MS) was used to probe the binding selectivity of a flexible cyclic polyamide (cβ) to G-quadruplexes from the long G-rich sequences in the c-myb oncogene promoter. The results show that three G-rich sequences, including d[(GGA)₃GGTCAC(GGA)₄], d[(GGA)₄GAA(GGA)₄], and d[(GGA)₃GGTCAC(GGA)₄GAA(GGA)₄] species in the c-myb promoter can form parallel G-quadruplexes, and cβ selectively binds towards these G-quadruplexes over both several other G-quadruplexes and the duplex DNA. These properties of cβ have profound implications on future studies of the regulation of c-myb oncogene expression.

Plasma-Spray Ionization (PLASI): A Multimodal Atmospheric Pressure Ion Source for Liquid Stream Analysis

A new ion generation method, named plasma-spray ionization (PLASI) for direct analysis of liquid streams, such as in continuous infusion experiments or liquid chromatography (LC), is reported. PLASI addresses many of the analytical limitations of electrospray ionization (ESI) and has potential for real time process stream analysis and reaction monitoring under atmospheric conditions in non-ESI friendly scenarios. In PLASI-mass spectrometry (MS), the liquid stream is pneumatically nebulized and partially charged at low voltages; the resultant aerosol is thus entrained with a gaseous plasma plume from a distal glow discharge prior to MS detection. PLASI-MS not only overcomes ESI-MS limitations but also generates simpler mass spectra with minimal adduct and cluster formation. PLASI utilizes the atomization capabilities of an ESI sprayer operated below the ESI threshold to generate gas-phase aerosols that are then ionized by the plasma stream. When operated at or above the ESI threshold, ionization by traditional ESI mechanisms is achieved. The multimodal nature of the technique enables readily switching between plasma and ESI operation. It is expected that PLASI will enable analyzing a wide range of analytes in complex matrices and less-restricted solvent systems, providing more flexibility than that achievable by ESI alone. Figure

?     

Laser-Induced Azomethine Ylide Formation and Its Covalent Entrapment by Fulleropyrrolidine Derivatives During MALDI Analysis

Two novel monofunctionalized fulleropyrrolidine derivatives (Prato adducts) were prepared and characterized by matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). MALDI experiments conducted in the positive-ion mode on pure and mixed samples of both monofunctionalized fullerene derivatives revealed the efficient formation of bisadducts (in the case of the pure samples) and mixed bisadducts (in the case of a mixed sample). Bisadducts were not observed in the ESI experiments and thus not present in the sample. A mechanism for the MALDI formation of these bisadduct ions is proposed in which an azomethine ylide fragment is formed in situ from the monofunctionalized fulleropyrrolidine species upon laser irradiation. This fragment, which can survive as an intact moiety in the gas phase in the special environment provided by the MALDI experiment, is then able to attach to a fulleropyrrolidine monoadduct which acts as a dipolarophile, thus leading to the formation of a bisadduct fullerene derivative. The unprecedented re-attachment of the azomethine ylide implies that the establishment of the ligand attainment of Prato adducts based on MALDI analysis alone can lead to wrong assignments.

Plasma-Assisted Reaction Chemical Ionization for Elemental Mass Spectrometry of Organohalogens

We present plasma-assisted reaction chemical ionization (PARCI) for elemental analysis of halogens in organic compounds. Organohalogens are broken down to simple halogen-containing molecules (e.g., HBr) in a helium microwave-induced plasma followed by negative mode chemical ionization (CI) in the afterglow region. The reagent ions for CI originate from penning ionization of gases (e.g., N₂) introduced into the afterglow region. The performance of PARCI-mass spectrometry (MS) is evaluated using flow injection analyses of organobromines, demonstrating 5–8 times better sensitivities compared with inductively coupled plasma MS. We show that compound-dependent sensitivities in PARCI-MS mainly arise from sample introduction biases.

Using Metal Complex Ion-Molecule Reactions in a Miniature Rectilinear Ion Trap Mass Spectrometer to Detect Chemical Warfare Agents

The gas-phase reactions of a series of coordinatively unsaturated [Ni(L)_n]^y+ complexes, where L is a nitrogen-containing ligand, with chemical warfare agent (CWA) simulants in a miniature rectilinear ion trap mass spectrometer were investigated as part of a new approach to detect CWAs. Results show that upon entering the vacuum system via a poly(dimethylsiloxane) (PDMS) membrane introduction, low concentrations of several CWA simulants, including dipropyl sulfide (simulant for mustard gas), acetonitrile (simulant for the nerve agent tabun), and diethyl phosphite (simulant for nerve agents sarin, soman, tabun, and VX), can react with metal complex ions generated by electrospray ionization (ESI), thereby providing a sensitive means of detecting these compounds. The [Ni(L)_n]²⁺ complexes are found to be particularly reactive with the simulants of mustard gas and tabun, allowing their detection at low parts-per-billion (ppb) levels. These detection limits are well below reported exposure limits for these CWAs, which indicates the applicability of this new approach, and are about two orders of magnitude lower than electron ionization detection limits on the same mass spectrometer. The use of coordinatively unsaturated metal complexes as reagent ions offers the possibility of further tuning the ion-molecule chemistry so that desired compounds can be detected selectively or at even lower concentrations.

Pharmaceutical Analysis by Solid-Substrate Electrospray Ionization Mass Spectrometry with Wooden Tips

Electrospray ionization (ESI) using wooden tips as solid substrates allows direct ionization of various samples and their simple and efficient analyses by mass spectrometry (MS). In this study, wooden-tip ESI-MS was used for pharmaceutical analysis. A wide variety of active components present in pharmaceuticals with forms of tablets, capsules, granules, dry suspensions, suspensions, drops, and oral liquids, etc., were all successfully ionized directly for mass spectrometric analysis. Trace degradation products were also sensitively detected using wooden-tip ESI-MS. This strategy was extended to construct chemical fingerprints of herbal products containing complex and unknown components, and the fingerprints provided valuable information for their quality assessment and origin tracing. Our experimental data demonstrated that wooden-tip ESI-MS is a useful tool for rapid pharmaceutical analysis, with high sensitivity and wide applicability, showing promising perspectives for quality assessment and control, authentication, and origin tracing of pharmaceuticals.

Mass spectrometry and inflammation—MS methods to study oxidation and enzyme-induced changes of phospholipids

Many diseases such as arthritis or atherosclerosis are accompanied by inflammatory processes. Inflammation is characterized by the infiltration of cells such as neutrophilic granulocytes and (a) the release of phospholipases [particularly phospholipase A₂ (PLA₂)] and (b) the generation of reactive oxygen as well as nitrogen species (ROS and RNS). While PLA₂ leads to defined lyso products (lacking one acyl residue), lipid oxidation is characterized by much more complex product patterns, including lipid peroxides, aldehydes (by double bond cleavage), and many others. Nevertheless, oxidation processes are highly important under in vivo conditions because molecules with regulatory functions are generated by the oxidation of lipids and/or free fatty acids. Therefore, lipid oxidation products as well as lysolipids are increasingly assumed to represent important disease (bio)markers. Consequently, there is also increasing interest in methods to characterize these products qualitatively and quantitatively. Mass spectrometry (MS) seems to be the method of choice to study (phospho)lipids changed under inflammatory conditions: nowadays, soft ionization MS methods are regularly used to study oxidative lipid modifications because of their high sensitivities and the tremendous mass resolutions that are achievable by using modern mass spectrometers. However, experimental care is required to be able to detect all relevant products. Although electrospray ionization (ESI) MS is so far most popular, applications of matrix-assisted laser desorption and ionization (MALDI) MS are continuously increasing. This review aims to summarize the so far available data on MS analyses of oxidized lipids as well as lysolipids. In addition to model systems, special attention will be paid to the monitoring of oxidized lipids and lysolipids under in vivo conditions. It is the aim of this review to provide a critical survey of the advantages and drawbacks of the different MS methods, with the focus on MALDI and ESI.

High Throughput Screening of High-Affinity Ligands for Proteins with Anion-Binding Sites using Desorption Electrospray Ionization (DESI) Mass Spectrometry

A high throughput screening system involving a linear ion trap (LTQ) analyzer, a house-made platform and a desorption electrospray ionization (DESI) source was established to screen ligands with a high affinity for proteins with anion-binding sites. The complexes were analyzed after incubation, ultrafiltration, washing, and displacement. A new anionic region inhibited dissociation (ARID) mechanism that was suitable for a protein with anion-binding site was proposed. We utilized the differences in detectable dissociation of protein–ligand complexes, combined with displacement experiments, to distinguish free ligands displaced from anion-binding sites from liberated ligands dissociated from nonspecific interactions. The method was validated by α₁-acid glycoprotein (AGP) and (R), (S)-amlodipine. Site-specific enantioselectivity shown in our experiments was consistent with earlier studies. Obtaining all of the qualitative information of 15*3 samples in 2.3 min indicates that the analysis process is no longer the time-limiting step in the initial stage of drug discovery. Quantitative information verified that our method was at least a semiquantitative method.

Differentiation of Diastereoisomers of Protected 1,2-Diaminoalkylphosphonic Acids by EI Mass Spectrometry and Density Functional Theory

Electron ionization mass spectrometry and density functional theory (DFT) calculations have been used to study the fragmentation of diastereoisomers of protected 1,2-diaminoalkylphosphonic acids. The loss of a diethoxyphosphoryl group and the elimination of diethyl phosphonate were found to be competitive fragmentation processes, which can be used to differentiate both stereoisomers. Selective deuterated analogs and product- and precursor-ion mass spectra allowed the elucidation of the fragmentation mechanisms. The structures of the transition states and product ions were optimized using the density functional theory (DFT), and free energy calculations confirmed the observed differences in the formation and relative intensities of specific fragment ions.

Using electrospray laser desorption ionization mass spectrometry to rapidly examine the integrity of proteins stored in various solutions

Electrospray laser desorption ionization mass spectrometry (ELDI/MS) allows the rapid desorption and ionization of proteins from solutions under ambient conditions. In this study, we have demonstrated the use of ELDI/MS to efficiently examine the integrity of the proteins stored in various solutions before they were further used for other biochemical tests. The protein standards were prepared in the solutions containing buffers, organic salts, inorganic salts, strong acid, strong base, and organic solvents, respectively, to simulate those collected from solvent extraction, filtration, dialysis, or chromatographic separation. Other than the deposit of a drop of the sample solution on the metallic sample plate in an ELDI source, no additional sample pretreatment is needed. The sample drop was then irradiated with a pulsed laser; this led to desorption of the analyte molecules, which subsequently entered the ESI plume to undergo post-ionization. Because adjustment of the composition of the sample solution is unnecessary, this technique appears to be useful for rapidly evaluating the integrity of proteins after storage or prior to further biochemical treatment. In addition, when using acid-free and low-organic-solvent ESI solutions for ELDI/MS analysis, the native conformations of the proteins in solution could be detected.

Isolation and sequence analysis of peptides from the skin secretion of the Middle East tree frog Hyla savignyi

Novel peptides were identified in the skin secretion of the tree frog Hyla savignyi. Skin secretions were collected by mild electrical stimulation. Peptides were separated by reversed-phase high-performance liquid chromatography. Mass spectra were acquired by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), and fragment ion spectra were obtained after collision-induced dissociation and electron capture dissociation. Peptides were analyzed by manual de novo sequencing and composition-based sequencing (CBS). Sequence analyses of three so far undescribed, structurally unrelated peptides are presented in this paper, having the sequences DDSEEEEVE-OH, P*EEVEEERJK-OH, and GJJDPJTGJVGGJJ-NH₂. The glutamate-rich sequences are assumed to be acidic spacer peptides of the prepropeptide. One of these peptides contains the modified amino acid hydroxyproline, as identified and localized by high-accuracy FTICR-MS. Combination of CBS and of experience-based manual sequence analysis as complementary and database-independent sequencing strategies resulted in peptide identification with high reliability.

Why do the Abundances of Ions Generated by MALDI Look Thermally Determined?

In a previous study (J. Mass Spectrom. 48, 299–305, 2013), we observed that the abundance of each ion in a matrix-assisted laser desorption ionization (MALDI) spectrum looked thermally determined. To find out the explanation for the phenomenon, we estimated the ionization efficiency and the reaction quotient (Q_A) for the autoprotolysis of matrix, M + M → [M + H]⁺ + [M ? H]^?, from the temperature-controlled laser desorption ionization spectra of α-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB). We also evaluated the equilibrium constants (K_A) for the autoprotolysis at various temperatures by quantum chemical calculation. Primary ion formation via various thermal models followed by autoprotolysis-recombination was compatible with the observations. The upper limit of the effective temperature of the plume where autoprotolysis-recombination occurs was estimated by equating Q_A with the calculated equilibrium constant.