Advances in structure elucidation of small molecules using mass spectrometry

The structural elucidation of small molecules using mass spectrometry plays an important role in modern life sciences and bioanalytical approaches. This review covers different soft and hard ionization techniques and figures of merit for modern mass spectrometers, such as mass resolving power, mass accuracy, isotopic abundance accuracy, accurate mass multiple-stage MS(n) capability, as well as hybrid mass spectrometric and orthogonal chromatographic approaches. The latter part discusses mass spectral data handling strategies, which includes background and noise subtraction, adduct formation and detection, charge state determination, accurate mass measurements, elemental composition determinations, and complex data-dependent setups with ion maps and ion trees. The importance of mass spectral library search algorithms for tandem mass spectra and multiple-stage MS(n) mass spectra as well as mass spectral tree libraries that combine multiple-stage mass spectra are outlined. The successive chapter discusses mass spectral fragmentation pathways, biotransformation reactions and drug metabolism studies, the mass spectral simulation and generation of in silico mass spectra, expert systems for mass spectral interpretation, and the use of computational chemistry to explain gas-phase phenomena. A single chapter discusses data handling for hyphenated approaches including mass spectral deconvolution for clean mass spectra, cheminformatics approaches and structure retention relationships, and retention index predictions for gas and liquid chromatography. The last section reviews the current state of electronic data sharing of mass spectra and discusses the importance of software development for the advancement of structure elucidation of small molecules

In-depth analysis of crocetin ester glycosides from dried/processed stigmas of Crocus sativus L. by HPLC-ESI-MS n (n = 2, 3)

Introduction: Saffron stigmas from Crocus sativus L. (Iridaceae) are used as a drug in folk medicine, as a food additive and as a dying agent for at least 3500 years. Despite this long-term use the chemical composition of saffron seems still to be not fully known. Objective: An analytical strategy for detailed investigations of aqueous saffron extract is developed based on reverse-phase high-performance liquid chromatography electrospray ionisation (HPLC-ESI) multistage mass spectrometry (MS n ) for crocins. Methods: Commercially available stigmas are analysed by reverse-phase HPLC in combination with ESI/three-dimensional (3D)-ion trap mass spectrometry (MS) and MS n (n = 2 and 3). Sodium chloride is added to the analyte solution ready for injection to promote abundant [M + Na] + adduct ions of crocins, being ideal precursor ions for low-energy collision-induced dissociation (CID)-MS 2/3 . Results: This strategy allows the detailed structural elucidation of known as well as previously unknown crocin derivatives (molecular mass of the aglycon, oligosaccharide chain length and linkage determination). The two isomeric trisaccharide substituents neapolitanose and gentiotriose are distinguished based on linkage-specific cross-ring cleavage for the first time. Furthermore, crocins containing up to six hexose units are also observed. Five novel crocin ester glycosides shifted by a mass difference of −40 Da indicate the presence of the here newly described C 17 -aglycon, termed norcrocetin (crocetin = C 20 ). Conclusions: These findings indicate the action of at least two different carotenoid cleavage dioxygenases (CCD2 and tentatively CCD4) during biosynthesis of this new bis-apocarotenoid aglycon (norcrocetin) and the existence of even higher glycosylated crocin derivatives at trace level. © 2019 John Wiley & Sons, Ltd

In-chain neutral hydrocarbon loss from crocin apocarotenoid ester glycosides and the crocetin aglycon (Crocus sativus L.) by ESI-MSn (n = 2, 3)

The stigmas of Crocus sativus L. have been used as spice and colorant agent (i.e. saffron) for more than 4000 years. For an updated structural investigation of the aglycon present in the glycosylated crocetin apocarotenoids (i.e. crocins), seven representative derivatives ranging from one up to five glucosyl-residues with a maximum number of three monosaccharides per glycosylation site (glucose, gentiobiose, gentiotriose and neapolitanose) were isolated and purified by high-performance liquid chromatography. The compounds selected for further mass spectrometric investigation include glucosyl-, bis-glucosyl-, gentiobiosyl-, gentiobiosyl-glucosyl-, bis-gentiobiosyl-, gentiobiosyl-gentiotriosyl- and gentiobiosyl-neapolitanosyl-crocetin. Electrospray ionization in combination with low-energy collision-induced dissociation/tandem mass spectrometry of sodiated crocin precursor ions utilizing either a 3D-ion trap (MSn, n = 2, 3) or a QqTOF instrument, with the latter providing accurate mass determination with an accuracy of ±1-3 ppm or better at a resolution of 10 000 (full width at half maximum), was used. Major fragmentation pathways included loss of either one or two carbohydrate substituents leading to the sodiated aglycon without interglycosidic bond cleavage during in MS2-experiments. All sodiated precursor ions and major product ions were accompanied by a loss of 92 Da, which was elucidated as C7H8-loss from the aglycon by skeletal rearrangement via an eight-membered transition state as previously described for intact C40-carotenoids. Copyright © 2013 John Wiley & Sons, Ltd. Copyright © 2013 John Wiley & Sons, Ltd

Processed stigmas of Crocus sativus L. imaged by MALDI-based MS

The processed, i.e. dried under certain conditions, stigmas of Crocus sativus L. are one of the most expensive plant parts used commercially. For the color, aroma and biological activity a very complex mixture of glycolipids termed crocins are responsible. Therefore studying structural composition and distribution in the commercial plant material is of great interest. We showed successfully the application of a MALDI-based mass spectrometric imaging (MSI) approach for stigmas towards different crocin species. MSI opens up the investigation of processed plant materials in various fields allowing studying the processing in detail as well as adulteration attempts (which are quite frequent due to the price of the material). Furthermore, we could demonstrate that a similar number of crocins present in stigmas could be detected by MALDI MSI compared to the classical approach of analyzing the solvent-extract of stigmas by MALDI-MS. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinhei

Comparison of different tandem mass spectrometric techniques (ESI-IT, ESI- and IP-MALDI-QRTOF and vMALDI-TOF/RTOF) for the analysis of crocins and picrocrocin from the stigmas of Crocus sativus L.

RATIONALE The expensive spice saffron originating from the stigmas of Crocus sativus L. and also applied in traditional Chinese medicine (TCM) constitutes a complex mixture of glycoconjugates varying not only in the aglycon structure, but also in glycosylation pattern. Therefore, various tandem mass spectrometric techniques were evaluated for their usefulness in structural elucidation. METHODS Three selected constituents of the stigmas of Crocus sativus L., trans- and cis-crocin-4 as well as picrocrocin, were isolated and purified by HPLC and finally analyzed by ESI-MS (ion trap, QqRTOF), IP-MALDI-MS (QqRTOF) and vMALDI-MS (TOF/RTOF) in combination with tandem mass spectrometry in collision energy regimes ranging from a few eV (LE) to 20 keV (HE) collisions for the first time. These data aid in structurally elucidating minor, unknown glycoconjugates originating from this plant-derived spice. RESULTS LE-CID of isomeric crocins on either an ion trap with ESI or a QqRTOF-instrument with ESI or IP-MALDI as desorption/ionization technique only yielded a limited number of structurally diagnostic sodiated product ions related to the carbohydrate moiety as well as to the intact aglycon in contrast to true HE-CID. The low MW constituent picrocrocin did not yield useful LE-CID spectra, but showed a high number of structurally diagnostic product ions by HE-CID utilizing a vMALDI TOF/RTOF-instrument. CONCLUSIONS The highest number of structurally diagnostic product ions allowing also determination of the carbohydrate linkage of the gentiobiose-moiety of isomeric crocins ( 0,4A 2, 3,5A 2 product ions indicating a 1→6 carbohydrate linkage) was only achievable by HE-CID. Fragmentation of the aglycon was not observed by any collision energy regime applied. Copyright © 2012 John Wiley & Sons, Ltd

Primary structure of cyanelle peptidoglycan of Cyanophora paradoxa: a prokaryotic cell wall as part of an organelle envelope.

The peptidoglycan layer surrounding the photosynthetic organelles (cyanelles) of the protist Cyanophora paradoxa is thought to be a relic of their cyanobacterial ancestors. The separation of muropeptides by gel filtration and reverse-phase high-performance liquid chromatography revealed four different muropeptide monomers. A number of muropeptides were identical in retention behavior to muropeptides of Escherichia coli, while others had remarkably long retention times with respect to their sizes, as indicated by gel filtration. Molecular mass determination by plasma desorption and matrix-assisted laser desorption ionization mass spectrometry showed that these unusual muropeptides had molecular masses greater by 112 Da or a multiple thereof than those of ones common to both species. Fast atom bombardment-tandem mass spectrometry of these reduced muropeptide monomers allowed the localization of the modification to D-glutamic acid. High-resolution fast atom bombardment-mass spectrometry and amino acid analysis revealed N-acetylputrescine to be the substituent (E. Pittenauer, E. R. Schmid, G. Allmaier, B. Pfanzagl, W. Löffelhardt, C. Quintela, M. A. de Pedro, and W. Stanek, Biol. Mass Spectrom. 22:524-536, 1993). In addition to the 4 monomers already known, 8 dimers, 11 trimers, and 6 tetramers were characterized. An average glycan chain length of 51 disaccharide units was determined by the transfer of [U-14C]galactose to the terminal N-acetylglucosamine residues of cyanelle peptidoglycan. The muropeptide pattern is discussed with respect to peptidoglycan biosynthesis and processing

Dynamics of Pd Dopant Atoms inside Au Nanoclusters during Catalytic CO Oxidation

Doping gold nanoclusters with palladium has been reported to increase their catalytic activity and stability. PdAu24 nanoclusters, with the Pd dopant atom located at the center of the Au cluster core, were supported on titania and applied in catalytic CO oxidation, showing significantly higher activity than supported monometallic Au25 nanoclusters. After pretreatment, operando DRIFTS spectroscopy detected CO adsorbed on Pd during CO oxidation, indicating migration of the Pd dopant atom from the Au cluster core to the cluster surface. Increasing the number of Pd dopant atoms in the Au structure led to incorporation of Pd mostly in the S-(M-S)n protecting staples, as evidenced by in situ XAFS. A combination of oxidative and reductive thermal pretreatment resulted in the formation of isolated Pd surface sites within the Au surface. The combined analysis of in situ XAFS, operando DRIFTS, and ex situ XPS thus revealed the structural evolution of bimetallic PdAu nanoclusters, yielding a Pd single-site catalyst of 2.7 nm average particle size with improved CO oxidation activity.N.B. thanks the TUW Innovative Project GIP165CDGC. G.R. acknowledges financial support by the Austrian Science Fund (FWF) via grants SFB FOXSI (F4502), DK+ Solids4Fun (W1243), and Single Atom Catalysis (I 4434-N). The authors thank Stephan Pollitt for his advice in synthesizing Pd-doped Au nanoclusters. ALBA synchrotron is acknowledged for beamtime at CLAESS beamline (Proposal ID: 2019023443) and Dr. Vlad Martin-Diaconesu for assistance during measurements