Metallomics Integratedaccepted Biometal Manuscript Science
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View Article Online View Journal Metallomics IntegratedAccepted biometal Manuscript science This article can be cited before page numbers have been issued, to do this please use: A. Raab, M. RONZAN and J. Feldmann, Metallomics, 2017, DOI: 10.1039/C7MT00098G. Volume 8 Number 1 January 2016 Pages 1–136 This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Metallomics Accepted Manuscripts are published online shortly after www.rsc.org/metallomics acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the author guidelines. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s ISSN 1756-5901 standard Terms & Conditions and the ethical guidelines, outlined in our author and reviewer resource centre, still apply. In no PAPER David P. Giedroc et al. The S2 Cu(I) site in CupA from Streptococcus pneumoniae is required for cellular copper resistance Indexed in event shall the Royal Society of Chemistry be held responsible Medline! for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. rsc.li/metallomics Page 1 of 22 Metallomics View Article Online DOI: 10.1039/C7MT00098G 1 2 3 Comprehensive non-target analysis identifies 54 sulphur containing compounds in garlic. 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Manuscript 26 27 28 29 30 31 32 33 34 35 Accepted 36 37 38Published on 07 September 2017. Downloaded by University of Aberdeen 12/09/2017 09:14:29. 39 40 41 42 43 44 45 46 Metallomics 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Metallomics Page 2 of 22 View Article Online DOI: 10.1039/C7MT00098G 1 2 3 4 1 Sulphur fertilization influences the sulphur species composition 5 6 2 in Allium sativum: sulphomics using HPLC-ICPMS/MS-ESI- 7 8 3 MS/MS 9 4 Andrea Raab, Marilena Ronzan, Joerg Feldmann 10 11 5 TESLA (Trace Element Speciation Laboratory), University of Aberdeen, Chemistry, 12 6 Meston Walk, Aberdeen, AB243UE, Scotland, UK 13 14 7 15 16 17 8 Abstract 18 19 9 Garlic ( A. sativum ) contains a large number of small sulphur (S)-containing 20 10 metabolites, which are important for its taste and smell and vary with A. sativum 21 11 variety and growth conditions. 22 23 12 This study was designed to investigate the influence of different sulphur-fertilization 24 13 regimes on the low molecular weight S-species by attempting the first sulphur mass 25 Manuscript 26 14 balance in A. sativum roots and bulbs using HPLC-ICPMS/MS-ESI-MS/MS. 27 28 15 Species unspecific quantification of acid soluble S-containing metabolites was 29 16 achieved using HPLC-ICP-MS/MS. For identification of the compounds high 30 31 17 resolution ESI-MS (Orbitrap LTQ and q-TOF) was used. 32 33 18 The plants contained up to 54 separated sulphur-containing compounds, which 34 19 constitute about 80 % of the total sulphur present in A. sativum . Roots and bulbs of Accepted 35 20 A.sativum contained the same compounds, but not necessarily the same amounts 36 21 and proportions. The S-containing metabolites in the roots reacted more sensitive to 37 38Published on 07 September 2017. Downloaded by University of Aberdeen 12/09/2017 09:14:29. 22 manipulations of sulphur fertilization than those compounds in the bulbs. In addition 39 23 to known compounds (eg. γ-glutamyl-S-1-propenylcysteine) we were able to identify 40 24 and partially quantify 31 compounds. Three as yet undescribed S-containing 41 42 25 compounds were also identified and quantified for the first time. Putative structures 43 26 were assigned to the oxidised forms of S-1-propenylmercaptoglutathione, S-2- 44 27 propenylmercaptoglutathione, S-allyl/propenyl-containing PC-2 and 2-amino-3-[(2- 45 28 carboxypropyl)sulfanyl]propanoic acid. 46 Metallomics 47 29 48 The parallel use of ICP-MS/MS as sulphur-specific detector and ESI-MS as 49 30 molecular detector simplifies the identification and quantification of sulphur 50 31 containing metabolites without species specific standards. This non-target analysis 51 32 approach enables a mass balance approach and identifies the occurrence of so far 52 53 33 unidentified organosulphur compounds. The experiments showed that the sulphur- 54 34 fertilization regime does not influence sulphur-speciation, but the concentration of 55 35 some S-containing compounds in roots is depending on the sulphur fertilization. 56 57 36 58 59 60 Page 3 of 22 Metallomics View Article Online DOI: 10.1039/C7MT00098G 1 2 3 37 Significance to Metallomics 4 5 38 Sulphur is not a metal, but the similarity to Se which is featured in the journal should 6 39 make an S-based study eligible. The multitude of S-containing metabolites in allium 7 8 40 is difficult to quantify using traditional methods. We developed a species 9 41 independent quantification method coupled with simultaneous identification using 10 42 HPLC-ICPMS/ESI-MS to give a holistic (sulphomic) view on the acid soluble low 11 43 molecular weight S-metabolites. This is in the spirit of non-metal non target 12 1 13 44 speciation analysis as laid out recently. 14 15 45 16 17 46 Introduction 18 47 19 20 48 A wide variety of Allium sativum (garlic) is cultivated worldwide for use as spice in 21 2 22 49 the kitchen and for their medical properties. Like all alliums garlic contains a variety 23 50 of volatile and non-volatile sulphur (S) containing metabolites, which are mainly 24 51 responsible for its typical smell and taste. Several sulphur containing phytochemicals 25 Manuscript 52 26 present in A. sativum show at least in vitro medical properties, namely alliin and 3 27 53 allicin, supporting the use of garlic in traditional medicine. Suggestions of health 28 54 benefits resulting from consumption of garlic range from reducing the risk of coronary 29 55 heart disease to anti-cancer properties.4,5 Allicin, the major volatile S-species 30 31 56 produced by crushing garlic and first identified in 1944 by Cavallito, was shown to 32 57 have significant bacteriostatic activity in vitro .2 Clinical trials have, however, so far 33 58 failed to show conclusive evidence for significant health benefits.6 34 Accepted 35 59 Research in sulphur-containing compounds (S-containing compounds) of garlic 36 37 60 focuses predominantly on alliin, allicin and some of their major derivatives. The 38Published on 07 September 2017. Downloaded by University of Aberdeen 12/09/2017 09:14:29. 61 presence of some di- and tripeptides of the γ-glutamyl cysteinyl family containing an 39 62 S-allyl or S-propenyl moiety is known. The best known are γ-glutamyl-S-allyl-L- 40 63 cysteine (GSAC), γ-glutamyl-S-1-propenyl-L-cysteine (GSPC) and γ-L-glutamyl-S- 41 5,7,8 42 64 methyl-L-cysteine (GSMC). Whether they are precursors and / or sulphur storage 43 65 peptides, especially during favourable growing conditions, for the eventual formation 44 66 of alliin and allicin, is one example of what is not known in the biosynthesis of S- 45 67 46 containing molecules. Metallomics 47 48 68 The main reasons for this lack of knowledge are the use of unspecific analytical 49 69 techniques for the determination of non-volatile S-containing compounds; mainly 50 70 HPLC-UV with quantification & identification of compounds by synthetic standards is 51 71 used. Rarely ESI-MS is used for identification of S-containing compounds in garlic. 52 8,7 53 72 Quantification is always done using synthesised standard compounds. When ESI- 54 73 MS is used for identification of unknown S-containing compounds high accuracy not 55 74 only with regard to m/z , but also the isotopic pattern is required. The latter is 56 75 57 important, since the sulphur isotopic pattern is not very different from the isotopic 58 76 pattern contributed by carbon, oxygen, nitrogen and hydrogen present in the 59 60 Metallomics Page 4 of 22 View Article Online DOI: 10.1039/C7MT00098G 1 2 3 77 compound. 32 S constitutes about 95 % of the present sulphur with 33 S contributing 4 78 about 0.8 %, 34 S 4.2% and 36 S 0.02%. Sulphur has also a small mass-deficiency, a 5 -1 6 79 compound of similar composition (m: 305 g mol ) containing one sulphur atom is -1 7 80 about 0.0905 g mol lighter than a compound containing only C,N,O and H. Both 8 81 mass defect and isotope pattern shift can be applied to identify unknown compounds 9 82 when using high-resolution accurate MS instruments.9 But a non-target analysis with 10 11 83 mass balance approach is not possible using molecular mass spectrometry. 12 13 84 The aim of this feasibility study was to test firstly whether the parallel use of ICP- 14 85 MS/MS and ESI-MS is advantageous for quantification and identification of S- 15 86 containing compounds in roots and bulbs of A. sativum without the use of species 16 17 87 specific standards.