MALDI Ion Research Articles

MALDI-2 Mass Spectrometry for Synthetic Polymer Analysis.

Synthetic polymers are ubiquitous in daily life, and their properties offer diverse benefits in numerous applications. However, synthetic polymers also present an increasing environmental burden through their improper disposal and subsequent degradation into secondary micro- and nanoparticles (MNPs). These MNPs accumulate in soil and water environments and can ultimately end up in the food chain, resulting in potential health risks. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) has the potential to study localized biological or toxicological changes in organisms exposed to MNPs. Here, we investigate whether MALDI-2 postionization can provide a sensitivity enhancement in polymer analysis that could contribute to the study of MNPs. We evaluated the effect of MALDI-2 by comparing MALDI and MALDI-2 ion yields from polyethyleneglycol (PEG), polypropylene glycol (PPG), polytetrahydrofuran (PTHF), nylon-6, and polystyrene (PS). MALDI-2 caused a signal enhancement of the protonated species for PEG, PPG, PTHF, and nylon-6. PS, by contrast, preferentially formed radical ions, which we attribute to direct resonance-enhanced multiphoton ionization (REMPI). REMPI of PS led to an improvement in sensitivity by several orders of magnitude, even without cationizing salts. The improved sensitivity demonstrated by MALDI-2 for all polymers tested highlights its potential for studying the distribution of certain classes of polymers in biological systems.

Spatial visualization of comprehensive brain neurotransmitter systems and neuroactive substances by selective in situ chemical derivatization mass spectrometry imaging.

Molecule-specific techniques such as MALDI and desorption electrospray ionization mass spectrometry imaging enable direct and simultaneous mapping of biomolecules in tissue sections in a single experiment. However, neurotransmitter imaging in the complex environment of biological samples remains challenging. Our covalent charge-tagging approach using on-tissue chemical derivatization of primary and secondary amines and phenolic hydroxyls enables comprehensive mapping of neurotransmitter networks. Here, we present robust and easy-to-use chemical derivatization protocols that facilitate quantitative and simultaneous molecular imaging of complete neurotransmitter systems and drugs in diverse biological tissue sections with high lateral resolution. This is currently not possible with any other imaging technique. The protocol, using fluoromethylpyridinium and pyrylium reagents, describes all steps from tissue preparation (~1 h), chemical derivatization (1-2 h), data collection (timing depends on the number of samples and lateral resolution) and data analysis and interpretation. The specificity of the chemical reaction can also help users identify unknown chemical identities. Our protocol can reveal the cellular locations in which signaling molecules act and thus shed light on the complex responses that occur after the administration of drugs or during the course of a disease.

Competing Deprotonation and Electron Capture Dissociation in MALDI Mass Spectrometry.

A protonation/deprotonation mechanism has been established for the interpretation of ions in MALDI. We show herein that negative ions can be generated in different ways. Molecules with different electron affinities have been spotted on surfaces of TiO2, ZnO, and a stainless steel plate for the investigation of electron capture dissociation in comparison with photo- or thermal-induced deprotonation upon irradiation of the third harmonic of Nd3+:YAG (355 nm) laser pulses. Detection of C60•- and Fe (II) (porph•-) radical anions unambiguously demonstrates the electron-transfer process and the exothermic capture of electrons. Radical anions of fatty acids were difficult to observe because of electron-directed ultrafast homolytic cleavage of O-H bonds unless there is a conjugated system as that in C60 and porphyrin for the delocalization and stabilization of acquired changes. The surface basicity of substrate materials was found to determine the competition of the electron-capture dissociation with deprotonation processes. Multiple electron transfers to pyrrole, -COOH, and Fe2+ of the heme were observed on TiO2 and the stainless steel plate but not on ZnO. When the heme was deprotonated by proton sponge 1,8-bis(dimethylamino)naphthalene, the occurrence of electron transfer on TiO2 was also not observed. It is proposed that negative charges of deprotonated ions prevent electron transfer due to the repulsive force. When both deprotonation and electron transfer are inhibited, adsorbed fatty acids on TiO2 undergo dehydration reactions to form titanium esters. In contrast, ZnO generates gaseous micelles composed of positive metal ions and negative fatty acid ions through either deprotonation or electron-capture dissociation.

Protein ion yield enhancement in matrix-assisted laser desorption/ionization mass spectrometry after sample and matrix low-pressure glow discharge plasma irradiation.

Plasma-assisted ionization is widely used in mass spectrometry; in this study, a low-pressure glow discharge is introduced as a new method to improve the detection of large proteins, and bovine serum albumin (BSA) is used as a protein model. The treatment of analyte, matrix, and the matrix/analyte mixture is evaluated under optimal conditions. Low-pressure radio-frequency capacitively coupled plasma (RF-CCP) treatment is utilized in the sample preparation step of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to enhance the protein MALDI ion signal. Plasma treatment can be an effective tool for enhancing the non-covalent binding of the analyte with the matrix, incorporation of the analyte into the matrix, production of matrix/analyte crystals, and analyte protonation through plasma activation, resulting in an improved MALDI ion signal. Fourier-transform infrared (FTIR) spectroscopy allows us to distinguish between the functional groups of plasma-treated and control samples. In addition, optical emission spectroscopy (OES) determines the plasma species, and zeta potential analysis characterizes the potential difference between plasma-treated and control samples before MALDI-TOF MS analysis. Plasma-treated BSA can provide a five-times enhancement of ion intensity. The combination of the plasma-treated analyte with the plasma-treated matrix leads to an increase in the ion intensity by a factor of 14. Low-pressure glow discharge plasma treatment greatly enhances MALDI ion signals, with a noticeable increase in incorporation, co-crystallization, protonation, and the concentration of the sample functional groups.

Drosophila melanogaster chemical ecology revisited: 2-D distribution maps of sex pheromones on whole virgin and mated flies by mass spectrometry imaging

BackgroundSexual behavior in Drosophila melanogaster flies is greatly influenced by chemical cues. In this study, a spatial distribution of female and male sex pheromones was investigated on the surface of virgin and mated six-day-old fruit flies. Surface analysis was performed using the technique of mass spectrometry imaging (MSI) matrix-assisted laser desorption/ionization – time of flight (MALDI-TOF) and confirmed by gas chromatography – mass spectrometry (GC-MS) analysis of hexane extracts prepared from dissected flies.ResultsMALDI-TOF MSI experiments focused on female pheromones (7Z,11Z)-heptacosa-7,11-diene (7,11-HD) and (7Z,11Z)-nonacosa-7,11-diene (7,11-ND) were enhanced by using lithium 2,5-dihydroxybenzoate (LiDHB) matrix to improve the ionization and quality of MS spectra. Oxygenated compounds represented by male anti-attractants 11-cis-vaccenyl acetate (cVA) and 3-O-acetyl-1,3-dihydroxy-octacosa-11,19-diene (CH503) were successfully ionized without MALDI matrix in the form of potassium adducts in laser desorption ionization (LDI-TOF MSI) mode. A similar pattern of distribution for 7,11-HD and 7,11-ND was observed on the surface of both the left and the right female wing, with the strongest signals at the base of the wing. 7,11-HD and 7,11-ND were additionally detected on female legs but not on the body. The distribution of both male pheromones, cVA and CH503, was localized in virgin male flies solely on the abdominal tip and anogenital region. In addition, results from MSI experiments with mated flies showed the distribution of cVA and CH503 also on the female abdomen and thorax, demonstrating that anti-attractants were transferred from males to females during copulation. Results from LDI/MALDI-TOF MSI were supported by GC-MS analysis of hexane extracts prepared from different parts of virgin male or female Drosophila flies. Similar amounts of 7,11-HD and 7,11-ND were present on the legs, body and wings (127 ± 5 ng and 170 ± 8 ng, respectively). cVA was detected only on the male body. All acquired MSI datasets were affected by mass shift (predominantly between ±0.2 Da to ±0.4 Da), which was reduced using a mass recalibration approach.ConclusionsThe LDI/MALDI-TOF MSI technique makes it possible to study the distribution of female and male sexual pheromones on D. melanogaster flies. Moreover, the technique enables the transfer of male sex pheromones to females during copulation to be visualized. However, imaging experiments of 3-D biological samples performed on a single TOF-MS instrument equipped with a MALDI ion source and UV nitrogen laser evinced a photo-electric charging/discharging, a phenomenon that often leads to unpredictable mass shifts and poor mass accuracy.

Mouse single oocyte imaging by MALDI-TOF MS for lipidomics

Reproductive cells are a very special kind of material for the analysis. Depending on the species, their dimensions allow for the application of mass spectrometry imaging-based techniques to receive a reasonable data for interpretation of their condition without any additional sample preparation steps, except for typical sample preparation characteristic for IMS protocols. A comparison between lipid profiles of oocytes could answer the question of the overall quality of the cells in the function of time or conditions of storage. Even tiny differences in the lipid profiles, but still detectable by bioinformatic analysis, could be crucial for the estimation of the conditions of the cells in various stages of development or aging. In our study, MALDI-TOF/TOF MSI was used to analyze and visualize the single oocytes. We deposited the cells on the transparent indium-tin-oxide (ITO) glass and marked their positions, which allowed for the fast localization of the cells and precise laser targeting in the ion source. We also optimized the usage of different MALDI matrices and different approaches. The proposed way of measurement allows analyzing quite a significant quantity of oocytes in a reasonably short time. During the analysis, the lipid composition of the single cell was successfully estimated in a conventional usage of the MALDI ion source, and the localization of lipids was confirmed by imaging mass spectrometry (IMS) analysis. The observed quantity of the lipids allowed for the application of the LIFT™ technique to obtain MS/MS spectra sufficient for lipids’ unambiguous identification. We hope that our idea of the oocyte analysis will help to elucidate chemical changes that accompany different processes in which oocytes are involved. There could be such fascinating phenomena as the oocyte maturation, changes in the lipid components during their storage, and much more.

Matrix-assisted laser desorption/ionization behavior of neat linear and cyclic poly(L-lactide)s and their blends.

RationaleNumerous new tin catalysts that enable the synthesis of cyclic polylactides with broad variation in their molecular mass were recently developed. The abundance of cyclics in matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectra is, however, frequently reported to greatly exceed that of linears. Thus, the MALDI ionization behavior of various end‐capped linear poly(L‐lactide)s and one cyclic poly(L‐lactide) was investigated and compared.MethodsNeat compounds and various blends of cyclic and linear species were prepared and studied under identical conditions with regard to sample preparation and instrumental condition, except for the laser power. For this purpose, two different MALDI‐TOF mass spectrometers were applied.ResultsOur results reveal that cyclics indeed show a slightly better ionization in MALDI, although their ionization as a neat compound seems to be less effective than that of linear polylactides. The ionization of most linear polylactides investigated does not depend on the end group structure. However, linear polylactides containing 12‐bromododecyl end groups reveal an unexpected saturation effect that is not caused by fragmentation of the polymer or the end group, or by electronic saturation of the detector digitizer. Furthermore, polylactides with a 2‐bromoethyl end group did not show such a saturation effect.ConclusionsAn overestimation of cyclic species in MALDI‐TOF mass spectra of poly(L‐lactide)s must be considered, but the commonly assumed peak suppression of linear polymers in mixtures of both structures can be excluded.

Electroporation and Mass Spectrometry: A New Paradigm for In Situ Analysis of Intact Proteins Direct from Living Yeast Colonies.

Yeasts constitute an oft-neglected class of pathogens among which the resistance to first-line treatments, attributed in part to mutations in efflux pumps, is rapidly emerging. Their thick, chitin-reinforced cell walls render cell lysis difficult, complicating their analysis and identification by methods routinely used for bacteria, including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Liquid extraction surface analysis mass spectrometry (LESA-MS) has previously been applied to the analysis of intact proteins from Gram-positive and Gram-negative bacterial colonies sampled directly on solid nutrient media. To date, a similar analysis of yeast colonies has not proved possible. Here we demonstrate the rapid release of intact yeast proteins for LESA-MS by electroporation using a home-built high-voltage device designed to lyse cells grown in colonies on agar media. Detection and identification of previously inaccessible proteins from baker’s yeast Saccharomyces cerevisiae, as well as two clinically relevant yeast species (Candida glabrata and Cryptococcus neoformans), is shown. The electroporation approach also has the potential to be translated to other mass spectrometric analysis techniques, including MALDI and various ambient ionization methods.

Poly-l-lysine-based tissue embedding compatible with matrix-assisted laser desorption ionization-mass spectrometry imaging analysis of dry and fragile aristolochia plants

The integrity of tissue is crucial for a high-quality analysis of matrix-assisted laser desorption ionization-mass spectrometry image (MALDI-MSI). Various embedding media utilized in traditional tissue-sectioning techniques are generally not recommended for MALDI-MSI of dry and fragile plant tissues because of the ion suppression effect in MALDI ionization in the low-mass region as well as the undesirable structural deformation during the sample preparation. In this work, a novel poly-L-lysine (PLL)-based tissue embedding method was developed for MALDI-MSI analysis of dry and fragile aristolochia plant (AP) tissues. The practical application in fixation, embedding, cryosectioning, and mounting of the dry and fragile AP tissues demonstrated that the PLL-based embedding technique could provide good rigidity to the plant tissues analysis compared to that without embedding and gelatin embedding. With the assistance of the PLL embedding medium, high spatial resolution molecular ion maps of main compounds, including aristolochic acids I (AAI) and aristolochic acids II (AAII) in AP root tissue, could be achieved by MALDI-MSI with enhanced signal intensities and no obvious background interference. This work provides an alternative approach for embedding the dry and fragile plant tissues comparable with MALDI-MSI analysis.

A reflection on the role of ICP-MS in proteomics: Update and future perspective

Mass spectrometry is the technique of reference for the identification and quantification of proteins. Whereas ESI and MALDI ionization sources are inherently not quantitative being highly influenced by the chemical nature of the analyte and the matrix, ICP-MS uses a hard ionization source that destroys proteins into its atoms and measures the elemental signal, which is independent of its chemical environment. As a consequence, ICP-MS turns up as an excellent technique for the screening, mapping and quantification of peptides and proteins in a sample through elemental detection (any element but C, H, N, or O) once they have been previously separated by chromatography. In this time, great efforts have been put in developing instrumentation and new methodologies that enable a better, more efficient, and more useful analysis of proteins with ICP-MS. Moreover, quantitative capabilities but lack of molecular information of ICP has led to a synergic relationship both with identifying capabilities of ESI-MS, or the use of protein-specific antibodies carrying an elemental label. Journal significanceWe are delighted to participate in this special issue and have the chance to congratulate Journal of Proteomics for its 10th Anniversary, and wish for many further successful anniversaries. During this last decade, Journal of Proteomics has been a clear promotor of works integrating ICP-MS for proteomics analysis. In fact, already in 2009, a review was published by invitation of the editor in chief focused on the established and potential role of ICP-MS in different areas of the proteomics analysis at the time: “The emerging role of ICP-MS in proteomics” [1]. Even though ICP-MS is not fully known or acknowledged in the proteomics world yet, its impact was significant as demonstrated by the really high interest in such publication (over 150 citations). Since then, several excellent papers relating to ICP-MS applications in proteomics have been published in this journal. Following the trend, we expect through this personal view of the current standing of ICP-MS in proteomics to enlighten the readers of Journal of Proteomics with a vision of the full present and future potential of ICP-MS in proteomics.

Remote Atmospheric Pressure Infrared Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry (Remote IR-MALDI MS) of Proteins

Remote Infrared Matrix-Assisted Laser Desorption/Ionization (Remote IR-MALDI) system using tissue endogenous water as matrix was shown to enable in vivo real-time mass spectrometry analysis with minimal invasiveness. Initially the system was used to detect metabolites and lipids. Here, we demonstrate its capability to detect and analyze peptides and proteins. Very interestingly, the corresponding mass spectra show ESI-like charge state distribution, opening many applications for structural elucidation to be performed in real-time by Top-Down strategy. The charge states show no dependence toward laser wavelength or length of the transfer tube. Indeed, remote analysis can be performed 5 m away from the mass spectrometer without modification of spectra. On the contrary, addition of glycerol to water shift the charge state distributions toward even higher charge states. The desorption/ionization process is very soft, allowing to maintain protein conformation as in ESI. Observation of proteins and similar spectral features on tissue, when protein standards are deposited on raw tissue pieces, could potentially open the way to their direct analysis from biological samples. This also brings interesting features that could contribute to the understanding of IR MALDI ionization mechanism.

Development of a Visualization Method for Imidacloprid in Drosophila melanogaster via Imaging Mass Spectrometry.

Imidacloprid is widely used for exterminating harmful insects; however, information regarding its distribution in insects is limited. Herein, we developed a visualization method for imidacloprid in Drosophila melanogaster, by using matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS). IMS requires sample cryosections; however, certain challenges prevail in retaining fly morphology in sections owing to their small size and heterogeneous components. Therefore, the section preparation method was optimized first, followed by imidacloprid distribution visualized using MALDI-IMS. Using 10% gelatin as an embedding material and 70% ethanol for pretreatment, the gaps between embedding material and D. melanogaster body surface were reduced. The tight adhesion between embedding media and D. melanogaster retains fly morphology in sections. Furthermore, the imidacloprid standard was analyzed separately via MALDI and electrospray ionization (ESI), and imidacloprid was converted to guanidine-imidacloprid via laser irradiation. Consequently, the imidacloprid distribution in D. melanogaster was successfully visualized using guanidine-imidacloprid as the target peak.

Soft-ionization mass spectrometric observation of isomeric effects occurring in the synthesis of bis-sulfonium salts from thiacyclanes/xylylene dibromides and collision induced dissociation of bis-sulfonium cations

Micro-synthesis of bis-sulfonium salts from ortho-, meta- and para-xylylene dibromides and mono- and bicyclic sulfides was performed for their further investigation by tandem electrospray/ionization mass spectrometry (ESI-MS) with the use of collision-induced dissociation (CID). Reaction products were detected by ESI-MS (without chromatographic separation), combined high-performance liquid chromatography/ESI-MS and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). It was shown that only the reaction of ortho- and para-xylylene dibromides with cyclic sulfides gave rise to corresponding bis-sulfonium salts yielding products of elimination of protonated sulfide from doubly charged ions during ESI and MALDI ionization. Fragmentation pathways of bis-sulfonium even-electron dications generated in ESI conditions were examined for the first time. The mass spectra revealed peaks for only single charged fragments included thiacyclane hydrocarbon residues, benzene- and sulfur-containing ions. Drastic quantitative and qualitative differences between ortho- and meta(para)-isomers allowing their easy differentiation were observed and explained. They were manifested in the character of eliminated thiacyclane residue and in the peak intensity ratios of m/z 104 and 91 benzene-containing ions. Isomeric di-cations with regio-isomeric structures of monocyclic sulfides demonstrated strong differences in CID spectra.

MALDI Mass Spectrometry of Fullero[C60]tetrahydropyridines

Mass spectra of positive and negative MALDI ions of the series of fullero[C60] tetrahydropyridines with different substituents in a heterocycle are systematically studied for the first time. All mass spectra contain C60 fullerene peaks as a result of the reduction of fullero[C60]tetrahydropyridines. The intensities of the protonated molecular ions’ [M + H]+ peaks are highest in the mass spectra of positive ions of the studied compounds, while molecular radical ion [M]+ is less intense. The intensities of the peaks of molecular radical ion [M]– are highest in the mass spectra of the negative ions. The [C60C2H5] ions formed during the decay of the molecular ions with the detachment of neutral nitrile molecules is characteristic of all compounds. Using DFT quantum–chemical calculations (PBE/3z), the energies of the highest (HOMO) and lowest unoccupied molecular orbitals (LUMO) are determined for fullero[C60]tetrahydropyridines with substituents in the heterocycle.

Generation of multiply charged peptides and proteins from glycerol-based matrices using lasers with ultraviolet, visible and near-infrared wavelengths and an atmospheric pressure ion source

Conventional matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is characterized by the predominant generation of singly charged analyte ions. Recent studies demonstrated that high charge states of peptides and small proteins can be produced with an atmospheric pressure (AP) MALDI ion source if liquid glycerol-based matrix systems are utilized and the AP-vacuum transfer is realized via a heated ion inlet tube [R. Cramer, A. Pirkl, F. Hillenkamp, K. Dreisewerd, Angew. Chem. Int. Ed. 52 (2013) 2364–2367]. Here, we used this AP ion source and employed an optical parametric oscillator (OPO) laser to study the wavelength dependence of the ion generation between 260 and 1080 nm. Three matrix systems with different optical absorption characteristics were investigated: non-absorbing glycerol mixed with trifluoroacetic acid (TFA) and glycerol mixed with either 2,4- or 2,5-dihydroxybenzoic acids as two classical UV-MALDI matrices. The highest ion yields of multiply charged peptides were consistently obtained at laser wavelengths for which the chromophore-containing matrix systems exhibited the lowest optical absorption. Best sensitivities were even achieved with the transparent glycerol/TFA mixture and by use of a Nd:YAG-laser operated at 1064nm (or with the OPO laser tuned to a similar wavelength). Experiments with absorbing and transparent sample substrates demonstrated the involvement of substrate absorption. In contrast to the results obtained with the AP ion source, using the same matrix systems in combination with a standard oMALDI2™ ion source, operated at a fine vacuum of ∼1mbar and without an inlet tube, the inverse wavelength dependence was observed. Or in other words, like in standard UV-MALDI under these conditions the highest analyte ion yields were obtained for laser excitation wavelengths corresponding to a high absorptivity of the matrix. Our findings thus point to completely different desorption/ionization processes with the two ion source geometries and laser excitation regimes. We hypothesize that for the AP case the material ablation comprises low-energy laser spallation and that rapid evaporative ionization of small droplets in the heated transfer capillary is responsible for charge production. From an analytical point of view, the production of multiply-charged peptide and protein ions with charge states that for the tested analytes (ranging by mass from 1 to 17kDa) almost fully resembled those generated by nano-electrospray ionization (ESI) could facilitate the coupling of laser-based ion sources with mass analyzers, such as orthogonal-extracting time-of-flight (QTOF) instruments or orbitraps which exhibit only limited m/z ranges.

Infrared MALDI mass spectrometry imaging of TLC-separated glycosphingolipids with emphasis on Shiga toxin receptors isolated from human colon epithelial cells

Thin-layer chromatography (TLC) combined with infrared MALDI mass spectrometry (IR-MALDI-MS) is a powerful analytical tool for detection and structural characterization of glyco- and phospholipids directly from the TLC plate. Here we coupled a pulsed IR-MALDI laser to a hybrid Synapt G2-S mass spectrometer (Waters) and obtained an effective focal spot size of ∼50×70μm2 in diameter. We used this new MALDI ion source configuration for TLC-IR-MALDI-MS imaging of neutral glycosphingolipids (GSLs), obtained from human colon epithelial HCT-8 cells, at 100μm step size. Our analytical focus was on the detection of globo-series GSLs globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), the main receptors for Shiga toxins (Stxs) produced by enterohemorrhagic Escherichia coli (EHEC). The direct TLC-IR-MALDI-MSI analysis allowed precise visualization of the chromatographic separation of the various lipoforms of Gb3Cer and Gb4Cer, with ceramide moieties mainly ranging from Cer (d18:1, C16:0) to Cer (d18:1, C24:0/C24:1) as well as those of their precursor GSLs glucosylceramide (GlcCer) and lactosylceramide (Lc2Cer). Reference TLC overlay immunostaining assays were conducted on parallel developed TLC lanes for confirmation of anomeric sugar configuration of proposed structures. Together, the adopted protocol provided a rapid and near-comprehensive overview of the GSL composition of the investigated cell line of high medical relevance. This possibility could also be highly useful in glycolipidomics studies of complex biological matrices.

Reply to "Comment on: MALDI ionization mechanisms investigated by comparison of isomers of dihydroxybenzoic acid".

Reply to "Comment on: MALDI ionization mechanisms investigated by comparison of isomers of dihydroxybenzoic acid".

Identification of Meliatoxins in Melia azedarach Extracts Using Mass Spectrometry for Quality Control.

Indiscriminate use of synthetic pesticides can be hazardous to both humans and the environment, but the use of natural products as a source of bio-based products, such as Melia azedarach extracts, is an interesting approach to overcome these hazards. Unfortunately, the limonoids found in M.azedarach with desired insecticidal properties (e.g. azadirachtin) may also be present with limonoids toxic to mammals. The goal of this report was to develop a fast and reliable MS-based experiment to characterize meliatoxins in crude extracts of M.azedarach, in order to provide unequivocal assessment of the safety for extracts for application in the field. MS and MS/MS experiments using MALDI ionization were evaluated as tools for the assignment of characteristic ions produced by each meliatoxin in crude extracts.The use of different experiments in combination, such as the analysis of fragment m/z 557 and [M + Na]+ (adducts ions m/z 681 and m/z 667), MALDI-MS can be used for detection of meliatoxins A1/B1 or A2/B2 in a crude extract and may be used to discriminate meliatoxins A from B, respectively. Subsequent MS/MS experiments can distinguish between the presence of group 1 and/or 2 in each class of meliatoxins classifying the proposed approach as a quick and efficient quality control method of meliatoxins in real M.azedarach samples.

Comment on: MALDI ionization mechanisms investigated by comparison of isomers of dihydroxybenzoic acid.

Ionization mechanism of matrix-assisted laser desorption/ionization was recently investigated by Kirmess et al. (J. Mass Spectrom. 2016, 51, 79). The authors compared the ion yields of dihydroxybenzoic acid isomers between experimental measurements and theoretical models and claimed that the predictions of chemical and physical dynamics model are in good agreement with experimental data, but the predictions of thermal model are not. Here, we show that wrong S1-S1 energy pooling rate constants and absorption cross sections were used in the aforementioned article. In addition, we suggest the authors to list the values of many parameters used in their calculations and describe how they obtained these values because these values are completely unknown.

Application of MALDI Mass Spectrometry in Natural Products Analysis.

This article presents the utility of mass spectrometry with a MALDI ionization source in natural products analysis. The advantages and drawbacks of this technique for natural products analyses will be presented and discussed. In addition, the structural determination of secondary metabolites using MALDI-MS/MS will be explored, which can guide MALDI experimental methods and stimulate new research in this area. Finally, several important approaches for MALDI data processing will be discussed.