GNPS MSn study of polyphenol standards

Abstract: High-mass resolution multi-stage mass spectrometry (MS(n)) fragmentation was tested for differentiation and identification of metabolites, using a series of 121 polyphenolic molecules. The MS(n) fragmentation approach is based on the systematic breakdown of compounds, forming a so-called spectral tree. A chip-based nanoelectrospray ionization source was used combined with an ion-trap, providing reproducible fragmentation, and accurate mass read-out in an Orbitrap Fourier transform (FT) MS enabling rapid assignment of elemental formulas to the molecular ions and all fragment ions derived thereof. The used protocol resulted in reproducible MS(n) fragmentation trees up to MS(5). Obtained results were stable over a 5 month time period, a concentration change of 100-fold, and small changes in normalized collision energy, which is key to metabolite annotation and helpful in structure and substructure elucidation. Differences in the hydroxylation and methoxylation patterns of polyphenolic core structures were found to be reflected by the differential fragmentation of the entire molecule, while variation in a glycosylation site displayed reproducible differences in the relative intensities of fragments originating from the same aglycone fragment ion. Accurate MS(n)-based spectral tree data are therefore a powerful tool to distinguish metabolites with similar elemental formula, thereby assisting compound identification in complex biological samples such as crude plant extracts.
Sample and short method description - Chemicals: Phenolic standards were obtained from Apin (Oxon, U.K.), Extrasynthese (Genay, France), Sigma (St. Louis), Fluka (Dorset, U.K.), and Acros (Geel, Belgium). The purity of all compounds was more than 98%. Quercetin-glucosides were produced in-house through bacterial expression of the tomato UDP-glucose flavonoid glycosyl transferase enzyme. The purified protein was incubated with quercetin aglycone and UDP-glucose as substrates for 24 h, allowing the production of a series of mono-, di-, and triglucosides. These quercetin-glucosides were separated by preparative LC and their structures determined using Q-TOF accurate mass MS and 2D-NMR. HPLC grade solvents were obtained from Biosolve (Valkenswaard, The Netherlands) and Merck-Schuchardt (Hohenbrunn, Germany). Ultrapure water was made in purification units present in-house.
Preparation of Flavonoid Standard Solutions: From each polyphenolic molecule, a stock solution of 2.5 ?g/mL in 75% MeOH in H2O acidified with 0.1% formic acid was prepared and stored at ?20 °C. Working solutions were prepared by filling 96 wells plates (Abgene) with 40 ?L of each stock solution, after which the plates were sealed with thermo foil.
Mass Spectrometry and Data Handling: A chip-based nanoelectrospray ionization source (Triversa NanoMate, Advion BioSciences) was used for automated direct sample infusion into a LTQ-Orbitrap hybrid mass spectrometer (Thermo Fisher Scientific) used in negative and positive ionization mode. For each unique m/z value, a separate Xcalibur method was prepared. The LTQ was programmed to use a window of 10 D to isolate the mass of interest in MS1. The data-dependent fragmentation was set as follows: MS2 fragmentation of most intense ion in MS1; MS3 fragmentation of the 5 most intense fragment ions in MS2; MS4 fragmentation of the 5 most intense fragment ions in each MS3; MS5 fragmentation of the 3 most intense fragment ions in each MS4. The spectrum list with accurate m/z values was used to export the MSn spectral tree fragmentation data from Xcalibur into Excel where it was processed.