ABSTRACT: Ethyl Acetate fraction of Streptomyces sp. CBMAI 2042 was investigated for identifying cyclodepsipeptides using electrospray ionisation tandem mass spectrometry (ESI-MS/MS). Without prior isolation, the structural determination was achieved on the basis of mass fragmentation pattern and comparison with the previously established data. The ESI-MS of the fraction in the positive ion mode gave clusters of singly and doubly charged molecular ion peaks. The ESI-MS spectrum showed peaks for the presence of the cyclodepsipeptides Valinomycin, Montanastatin and at least 5 structural analogues never reported before.
Project description:Ethyl Acetate fraction of Streptomyces sp. CBMAI 2042 was investigated for identifying cyclodepsipeptides using electrospray ionisation tandem mass spectrometry (ESI-MS/MS). Without prior isolation, the structural determination was achieved on the basis of mass fragmentation pattern and comparison with the previously established data. The ESI-MS of the fraction in the positive ion mode gave clusters of singly and doubly charged molecular ion peaks. The ESI-MS spectrum showed peaks for the presence of the cyclodepsipeptides Valinomycin, Montanastatin and at least 5 structural analogues never reported before.
Project description:RATIONALE:Although mass spectrometry (MS) is routinely used to determine deamination in peptide mixtures, the effects of the choice of ionisation source have not yet been investigated. In particular, matrix-assisted laser desorption/ionisation (MALDI) has become a popular tool with which to measure levels of glutamine deamidation in ancient proteins. Here we use model synthetic peptides to rigorously compare MALDI and electrospray ionisation (ESI). METHODS:We used two synthetic peptides, with glutamine (Q) in one substituted for glutamic acid (E) in the other, to investigate the suitability of MALDI and ESI sources for the assessment of deamidation in peptides using MS. We also compared measurements of the same Q- and E-containing peptide mixtures using two different mass analysers (time-of-flight (TOF) and Fourier transform ion cyclotron resonance (FT-ICR)). RESULTS:When standard mixtures of the Q- and E-containing peptides were analysed using MALDI, under-representation of the E-containing peptide was observed. This observation was consistent between analyses carried out using either TOF or FT-ICR-MS. When the same mixtures were analysed using ESI FT-ICR-MS, no ionisation bias was observed. CONCLUSIONS:MALDI may not be a suitable ionisation method for the determination of deamidation in peptide mixtures. However, ESI was successfully used to determine the ratio in known mixtures of Q- and E-containing peptides. These preliminary observations warrant further investigation into ionisation bias when measuring deamidation in other peptide sequences.
Project description:In natural-product drug discovery, finding new compounds is the main task, and thus fast dereplication of known compounds is essential. This is usually performed by manual liquid chromatography-ultraviolet (LC-UV) or visible light-mass spectroscopy (Vis-MS) interpretation of detected peaks, often assisted by automated identification of previously identified compounds. We used a 15 min high-performance liquid chromatography-diode array detection (UHPLC-DAD)-high-resolution MS method (electrospray ionization (ESI)(+) or ESI(-)), followed by 10-60 s of automated data analysis for up to 3000 relevant elemental compositions. By overlaying automatically generated extracted-ion chromatograms from detected compounds on the base peak chromatogram, all major potentially novel peaks could be visualized. Peaks corresponding to compounds available as reference standards, previously identified compounds, and major contaminants from solvents, media, filters etc. were labeled to differentiate these from compounds only identified by elemental composition. This enabled fast manual evaluation of both known peaks and potential novel-compound peaks, by manual verification of: the adduct pattern, UV-Vis, retention time compared with log D, co-identified biosynthetic related compounds, and elution order. System performance, including adduct patterns, in-source fragmentation, and ion-cooler bias, was investigated on reference standards, and the overall method was used on extracts of Aspergillus carbonarius and Penicillium melanoconidium, revealing new nitrogen-containing biomarkers for both species.
Project description:A mechanistic investigation of the acid-catalysed redox-neutral oxoarylation reaction of ynamides using electrospray ionisation mass-spectrometry (ESI-MS) and quantum chemical calculations (DFT and MP2) is presented. This study reveals the diversity of pathways and products available from an otherwise deceptively simple-looking, classical transformation: fragmentation, an unusual meta-arylation and competing ?-carbonyl cation pathways are some of the alternatives unveiled by ESI-MS and mechanistic experiments. Detailed calculations explain the observed trends and rationalise the results.
Project description:Metabolomics experiments using Mass Spectrometry (MS) technology measure the mass to charge ratio (m/z) and intensity of ionised molecules in crude extracts of complex biological samples to generate high dimensional metabolite 'fingerprint' or metabolite 'profile' data. High resolution MS instruments perform routinely with a mass accuracy of < 5 ppm (parts per million) thus providing potentially a direct method for signal putative annotation using databases containing metabolite mass information. Most database interfaces support only simple queries with the default assumption that molecules either gain or lose a single proton when ionised. In reality the annotation process is confounded by the fact that many ionisation products will be not only molecular isotopes but also salt/solvent adducts and neutral loss fragments of original metabolites. This report describes an annotation strategy that will allow searching based on all potential ionisation products predicted to form during electrospray ionisation (ESI).Metabolite 'structures' harvested from publicly accessible databases were converted into a common format to generate a comprehensive archive in MZedDB. 'Rules' were derived from chemical information that allowed MZedDB to generate a list of adducts and neutral loss fragments putatively able to form for each structure and calculate, on the fly, the exact molecular weight of every potential ionisation product to provide targets for annotation searches based on accurate mass. We demonstrate that data matrices representing populations of ionisation products generated from different biological matrices contain a large proportion (sometimes > 50%) of molecular isotopes, salt adducts and neutral loss fragments. Correlation analysis of ESI-MS data features confirmed the predicted relationships of m/z signals. An integrated isotope enumerator in MZedDB allowed verification of exact isotopic pattern distributions to corroborate experimental data.We conclude that although ultra-high accurate mass instruments provide major insight into the chemical diversity of biological extracts, the facile annotation of a large proportion of signals is not possible by simple, automated query of current databases using computed molecular formulae. Parameterising MZedDB to take into account predicted ionisation behaviour and the biological source of any sample improves greatly both the frequency and accuracy of potential annotation 'hits' in ESI-MS data.
Project description:Synthetic nucleotide and nucleic acid analogues are useful research tools and modern therapeutics. Hence, methods for the rapid and unambiguous identification of mononucleotides derived from organic syntheses or biological materials are of broad interest. Here, we analysed over 150 mononucleotides (mostly nucleoside 5'-mono-, 5'-di-, and 5'-triphosphates) and their structurally related nucleobase-, phosphate-, and ribose-modified analogues by electrospray tandem mass spectrometry (ESI/MS/MS), identifying characteristic fragmentation ions that may be helpful in structure determination. While positive-ion mode yielded fragments derived mainly from nucleobases, negative-ion mode provided insight into the structures of phosphoryl and phosphoribosyl moieties, enabling the determination of structural features such as the number of phosphate groups and the presence of ribose or phosphate substitutions. Based on these data, we proposed fragmentation pathways that were confirmed by experiments with [18O]-isotopologues. We demonstrated the utility of ESI(-)/MS/MS in the analysis of structurally related compounds by analysing isomeric and isobaric nucleotides and applying ESI(-)/MS/MS to rapid identification of nucleotide synthesis products. We formulated general rules regarding nucleotide structure-fragmentation pattern relationships and indicating characteristic fragmentation ions for the interpretation of ESI(-)/MS/MS spectra of nucleotides and their analogues. The ESI(-)/MS/MS spectra of all nucleotides are available in an on-line database, msTide, at www.msTide-db.com.
Project description:Herein, we present an approach for the rapid, straightforward and economical preparation of a tailored reactor device using three-dimensional (3D) printing, which can be directly linked to a high-resolution electrospray ionisation mass spectrometer (ESI-MS) for real-time, in-line observations. To highlight the potential of the setup, supramolecular coordination chemistry was carried out in the device, with the product of the reactions being recorded continuously and in parallel by ESI-MS. Utilising in-house-programmed computer control, the reactant flow rates and order were carefully controlled and varied, with the changes in the pump inlets being mirrored by the recorded ESI-MS spectra.
Project description:A high resolution ion mobility time-of-flight mass spectrometer with electrospray ionization source (ESI-IM-MS) was evaluated as an analytical method for rapid analysis of complex biological samples such as human blood metabolome was investigated. The hybrid instrument (IM-MS) provided an average ion mobility resolving power of ~90 and a mass resolution of ~1500 (at m/z 100). A few µL of whole blood was extracted with methanol, centrifuged and infused into the IM-MS via an electrospray ionization source. Upon IM-MS profiling of the human blood metabolome approximately 1,100 metabolite ions were detected and 300 isomeric metabolites separated in short analyses time (30 minutes). Estimated concentration of the metabolites ranged from the low micromolar to the low nanomolar level. Various classes of metabolites (amino acids, organic acids, fatty acids, carbohydrates, purines and pyrimidines etc) were found to form characteristic mobility-mass correlation curves (MMCC) that aided in metabolite identification. Peaks corresponding to various sterol derivatives, estrogen derivatives, phosphocholines, prostaglandins, and cholesterol derivatives detected in the blood extract were found to occupy characteristic two dimensional IM-MS space. Low abundance metabolite peaks that can be lost in MS random noise were resolved from noise peaks by differentiation in mobility space. In addition, the peak capacity of MS increased six fold by coupling IMS prior to MS analysis.
Project description:As an essential medicine and tea source in many countries, Plumula Nelumbinis potentially exerts its major biological activities through its alkaloids. However, the activities of Plumula Nelumbinis are not fully understood due to the lack of studies on its chemical components.To establish an ultra-performance liquid chromatography combined with diode-array detector (UPLC/DAD) method, coupled to an electrospray ionisation with quadrupole time-of-flight mass spectrometry (ESI/QTOF/MS) method, for the separation and identification of Plumula Nelumbinis alkaloids.The eluant from an UPLC separation of an ethanol extract of Plumula Nelumbinis was directly infused into an ESI/QTOF/MS system. Both positive and negative ion modes of ESI with low and high collision energy (CE) were used to obtain sufficient MS information.Twenty-one alkaloids were tentatively identified based on their chromatographic characteristics, UV spectra, exact mass, MS fragments and literature reports. They consist of six bis-1-benzyltetrahydroisoquinoline, eleven benzyltetrahydroisoquinoline (including two glycoalkaloids and two quaternary ammoniums), two aporphine, one proaporphine and one indole alkaloids. Eleven were identified in Plumula Nelumbinis for the first time and seven were first reported in Nelumbo nucifera Gaertn. Five compounds, namely norcoclaurine-4'-O-glucoside, norcoclaurine-6-O-glucoside, isolotusine, 6-demethyl-4-demethylN-methylcoclaurine and N-norisoliensinine, were characterised and proposed as new compounds.The established UPLC/DAD?-?ESI/QTOF/MS method is efficient for systematic identification of the alkaloids in Plumula Nelumbinis extract.
Project description:Intrinsically disordered proteins do not adopt well-defined native structures and therefore present an intriguing challenge in terms of structural elucidation as they are relatively inaccessible to traditional approaches such as NMR and X-ray crystallography. Many members of this important group of proteins have a distinct biological function and frequently undergo a conformational change on binding to their physiological targets which can in turn modulate their function. Furthermore, many intrinsically unstructured proteins are associated with a wide range of major diseases including cancer and amyloid-related disorders. Here, electrospray ionisation-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) has been used to probe the conformational characteristics of two intrinsically disordered proteins: apo-cytochrome c and apo-osteocalcin. Both proteins are structured in their holo-states when bound to their respective substrates, but disordered in their apo-states. Here, the conformational properties of the holo- and the apo-protein forms for both species have been analysed and their mass spectral data and ion mobility spectrometry-derived collision cross-sectional areas, indicative of their physical size, compared to study the relationship between substrate binding and tertiary structure. In both cases, the intrinsically unstructured apo-states populated multiple conformations with larger cross-sectional areas than their holo-analogues, suggesting that intrinsic disorder in proteins does not preclude the formation of preferred conformations. Additionally, analysis of truncated analogues of osteocalcin has located the region of the protein responsible for the conformational changes detected upon metal cation binding. Together, the data illustrate the scope and utility of ESI-IMS-MS for studying the characteristics and properties of intrinsically disordered proteins whose analysis by other techniques is limited.