Enumeration of carboxyl groups carried on individual components of humic systems using deuteromethylation and Fourier transform mass spectrometry
Here, we report a novel approach to enumeration of carboxylic groups carried by individual molecules of humic substances using selective chemical modification and isotopic labeling (deuteromethylation) and high-resolution electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FTICR MS). Esterification was conducted with a use of thionyl chloride–deuteromethanol reagent under mild conditions to avoid transesterification. The deuteromethylated products were subjected to solid phase extraction using PPL Bond Elute cartridges prior to FTICR MS analysis. An amount of carboxyl groups in the individual molecular component was estimated from the length of identified deuteromethylation series. The method allowed for discerning between compounds with close elemental compositions possessing different protolytic properties. We found that different carboxylic moieties occupy distinct regions in molecular space of humic substances (HS) projected onto Van Krevelen diagram. These locations do not depend on the source of the humic material and can be assigned to carboxyl-rich alicyclic molecules (5 to 6 COOH), hydrolyzable tannins (3-4 COOH), lignins (1 to 2 COOH), condensed tannins and lignans (0 to 1 COOH), and carbohydrates (0 COOH). At the same time, the alignment pattern of these carboxylated species along the structural evolution lines in Van Krevelen diagrams was characteristic to the specific transformation processes undergone by the humic materials in the different environments. The obtained data enable mapping of molecular ensemble of HS with regards to their specific acidic compartments and might be used for directed fractionation of HS.
KeywordsHumic substances FTICR MS Isotopic labeling Modification Carboxylic group 1H NMR spectroscopy
We would like to acknowledge the Interdisciplinary Research Centre for Magnetic Tomography and Spectroscopy of the Lomonosov MSU and personally Dr. Borisova N.E. for the assistance in 1H NMR analysis.
This work was partially supported by the Russian Foundation for Basic Research (grants 16-04-01753-a and 16-33-00914-mol-a). 1H NMR spectroscopy studies were supported by the Russian Science Foundation (grant 16-14-00167), and the high-resolution mass spectrometry study was funded by the Russian Science Foundation grant no. 14-24-00114.
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Conflict of interest
The authors declare that they have no competing interests.
- 6.Perminova IV, Grechishcheva NY, Kovalevskii DV, Kudryavtsev AV, Petrosyan VS, Matorin DN. Quantification and prediction of the detoxifying properties of humic substances related to their chemical binding to polycyclic aromatic hydrocarbons. Environ Sci Technol. 2001;35:3841–8. doi: 10.1021/es001699b.CrossRefGoogle Scholar
- 10.Perdue EM, Benner R. Marine organic matter. In: N. Senesi, B. Xing and PMH (ed) Biophys. Process. Involv. Nat. Nonliving Org. Matter Environ. Syst. Wiley. 2009; 407–449.Google Scholar
- 12.Klučáková M, Kaláb M, Pekař M, Lapčík L. Study of structure and properties of humic and fulvic acids. II. Study of adsorption of Cu2+ ions to humic acids extracted from lignite. J Polym Mater. 2002;19:287–94.Google Scholar
- 19.Noyes TI, Leenheer JA. Proton nuclear-magnetic-resonance studies of fulvic acid from the Suwannee River. Humic Subst. Suwannee River, Georg. Interact. Prop. Propos. Struct. Open-File Rep. 87-557. 1989; 231-250, 8 fig, 5 tab, 23 ref.Google Scholar
- 27.Kovalevskii DV, Permin AB, Perminova IV, Konnov DV, Petrosyan VS. Quantitative 1H NMR spectroscopic determination of exchangeable and backbone protons of humic substances. Moscow Univ Chem Bull. 1999;40:375–80.Google Scholar
- 28.Hertkorn N, Kettrup A. Molecular level structural analysis of natural organic matter and of humic substances by multinuclear and higher dimensional NMR spectroscopy. In: Use Humic Subst. to Remediat. Polluted Environ. From Theory to Pract. Springer-Verlag, Berlin/Heidelberg. 2005; 391–435.Google Scholar
- 33.Perminova IV, Dubinenkov IV, Kononikhin AS, Konstantinov AI, Zherebker AY, Andzhushev MA, et al. Molecular mapping of sorbent selectivities with respect to isolation of Arctic dissolved organic matter as measured by Fourier transform mass spectrometry. Environ Sci Technol. 2014;48:7461–8. doi: 10.1021/es5015423.CrossRefGoogle Scholar
- 41.Kostyukevich Y, Kononikhin A, Popov I, Spasskiy A, Nikolaev E. In ESI-source H/D exchange under atmospheric pressure for peptides and proteins of different molecular weights from 1 to 66 kDa: the role of the temperature of the desolvating capillary on H/D exchange. J Mass Spectrom. 2015;50:49–55. doi: 10.1002/jms.3535.CrossRefGoogle Scholar
- 42.Zherebker AY, Airapetyan D, Konstantinov AI, Kostyukevich YI, Kononikhin AS, Popov IA, et al. Synthesis of model humic substances: a mechanistic study using controllable H/D exchange and Fourier transform ion cyclotron resonance mass spectrometry. Analyst. 2015;140:4708–19. doi: 10.1039/c5an00602c.CrossRefGoogle Scholar
- 43.Zherebker A, Kostyukevich Y, Kononikhin A, Roznyatovsky VA, Popov I, Grishin YK, et al. High desolvation temperature facilitates the ESI-source H/D exchange at non-labile sites of hydroxybenzoic acids and aromatic amino acids. Analyst. 2016;141:2426–34. doi: 10.1039/c5an02676h.CrossRefGoogle Scholar
- 44.Kostyukevich Y, Kononikhin A, Popov I, Kharybin O, Perminova I, Konstantinov A, et al. Enumeration of labile hydrogens in natural organic matter by use of hydrogen/deuterium exchange Fourier transform ion cyclotron resonance mass spectrometry. Anal Chem. 2013;85:11007–13. doi: 10.1021/ac402609x.CrossRefGoogle Scholar
- 45.Kostyukevich Y, Kononikhin A, Zherebker A, Popov I, Perminova I, Nikolaev E. Enumeration of non-labile oxygen atoms in dissolved organic matter by use of 16O/18O exchange and Fourier transform ion-cyclotron resonance mass spectrometry. Anal Bioanal Chem. 2014;406:6655–64. doi: 10.1007/s00216-014-8097-9.CrossRefGoogle Scholar
- 46.Swift RS. Organic matter characterization. In: Sparks DL, Page AL, Helmke PA, Loeppert RH, Soltanpour PN, Tabatabai MA, Johnson CT, MES, editors. Methods soil Anal. Part 3. Chem. methods, SSSA Book Series 5. Madison: SSSA; 1996. p. 1036.Google Scholar
- 50.Kunenkov EV, Kononikhin AS, Perminova IV, Hertkorn N, Gaspar A, Schmitt-Kopplin P, et al. Total mass difference statistics algorithm: a new approach to identification of high-mass building blocks in electrospray ionization Fourier transform ion cyclotron mass spectrometry data of natural organic matter. Anal Chem. 2009;81:10106–15. doi: 10.1021/ac901476u.CrossRefGoogle Scholar
- 53.Sleighter RL, Hatcher PG. Fourier transform mass spectrometry for the molecular level characterization of natural organic matter: instrument capabilities, applications, and limitations. INTECH Open Access Publisher. 2011.Google Scholar
- 59.Stubbins A, Spencer RGM, Chen H, Hatcher PG, Mopper K, Hernes PJ, et al. Illuminated darkness: molecular signatures of Congo River dissolved organic matter and its photochemical alteration as revealed by ultrahigh precision mass spectrometry. Limnol Oceanogr. 2010;55:1467–77. doi: 10.4319/lo.2010.55.4.1467.CrossRefGoogle Scholar
- 61.Kovács K, Gáspár A, Sajgó C, Schmitt-Kopplin P, Tombácz E. Comparison of humic substances isolated from thermal water and surface water by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. Eur J Mass Spectrom (Chichester, Eng). 2010;16:625–30. doi: 10.1255/ejms.1087.CrossRefGoogle Scholar
- 64.D’Andrilli J, Foreman CM, Marshall AG, McKnight DM. Characterization of IHSS Pony Lake fulvic acid dissolved organic matter by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry and fluorescence spectroscopy. Org Geochem. 2013;65:19–28. doi: 10.1016/j.orggeochem.2013.09.013.CrossRefGoogle Scholar