Proanthocyanidin structural details revealed by ultrahigh resolution FT-ICR MALDI-mass spectrometry, 1H-13C HSQC NMR, and thiolysis-HPLC-DAD

Authors: REEVES, SAVANAH - Miami University - Ohio; SOMOGYI, ARPAD - The Ohio State University; Zeller, Wayne; RAMELOT, THERESA - Rensselaer Polytechnic Institute; WRIGHTON, KELLY - Colorado State University; HAGERMAN, ANN - Miami University - Ohio

Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/29/2020
Publication Date: 11/10/2020
Citation: Reeves, S.G., Somogyi, A., Zeller, W.E., Ramelot, T.A., Wrighton, K.C., Hagerman, A.E. 2020. Proanthocyanidin structural details revealed by ultrahigh resolution FT-ICR MALDI-mass spectrometry, 1H-13C HSQC NMR, and thiolysis-HPLC-DAD. Journal of Agricultural and Food Chemistry. 68(47):14038-14048. https://doi.org/10.1021/acs.jafc.0c04877.
DOI: https://doi.org/10.1021/acs.jafc.0c04877

Interpretive Summary: Condensed tannins (proanthocyanidins) are a group of compounds that occur in some plant species. These compounds mediate food taste, plant defenses, soil chemistry and biology and have been shown to have positive impact on nitrogen feed efficiency and parasite mitigation in ruminants. Recent advances in laboratory and instrumental analysis now allows structural identification of the components in condensed tannin mixtures. However, it is imperative that these techniques are robust and of high integrity. We recently found that using one of these advanced methods (Fourier transform ion cyclotron resonance matrix assisted laser desorption ionization mass spectrometry, FT-ICR-MALDI-MS) mistakenly displayed evidence that the condensed tannin molecules isolated from Sorghum bicolor grain were connected to a galloyl group, an addendum often found in condensed tannins isolated from plant materials such as grapes or tea leaves, but previously unreported in sorghum grain. However, all of the additional chemical and spectroscopic tools that we employed supported the conclusion that Sorghum condensed tannin does not contain gallate ester groups. Proton NMR (nuclear magnetic resonance) spectroscopy revealed that the common chemical matrix compound used in the FT-ICR MALDI-MS analysis is tightly associated with the condensed tannin which leads to the appearance of the condensed tannin possessing gallate groups, and is actual an artifact of the analysis when conducted in this chemical matrix. In addition to highlighting the need to find better matrices to fully realize the potential of the ultrahigh resolution FT-ICR MALDI-MS method, our study re-emphasizes the importance of using more than one analytical technique to obtain structural information about complex natural products such as condensed tannins.

Technical Abstract: Proanthocyanidins (condensed tannins) are important in food chemistry, agriculture, and health, driving demand for improvements in structure determination. We used ultrahigh resolution Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) methods to determine the exact composition of individual species in heterogeneous mixtures of proanthocyanidin polymers from Sorghum bicolor grain and Neptunia lutea leaves. Fragmentation patterns obtained with FT-ICR ESI MS-MS (electrospray ionization) confirmed structural details from thiolysis-high-performance liquid chromatography (HPLC)- diode array detection (DAD) and 1H-13C heteronuclear single quantum coherence (HSQC) NMR. We found that A-type linkages were characteristic of shorter polymers in predominantly B-linked proanthocyanidin. We suggest that supramolecular complex formation between proanthocyanidins and matrix components such as 2,5-dihydroxybenzoic acid was responsible for anomalous 152 dalton peaks, incorrectly assigned as 3-O-galloylation, when using FT-ICR matrix-assisted laser desorption ionization (MALDI-MS). Our data illustrate the power of the ultrahigh resolution FT-ICR methods but include the caveat that MALDI-MS must be paired with complementary analytical tools to avoid artifacts.