The rapid annotation and identification by mass spectrometry techniques of flavonoids remains a challenge, due to their structural diversity and the limited availability of reference standards. This study applies a workflow to characterize two isoflavonoids, the orobol-C-glycosides analogs, using high-energy collisional dissociation (HCD)- and collision-induced dissociation (CID)-type fragmentation patterns, and also to evaluate the antioxidant effects of these compounds by ferric reducing antioxidant power (FRAP), 2,2′-azino-bis(3-ethylbenzothiazolin acid) 6-sulfonic acid (ABTS), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) methods. By the CID-type fragmentation, in positive mode and at all high-resolution mass spectrometry (HRMS) multiple stage, there were shown differences in the annotation of the compounds, mainly concerning some ratios of relative abundance. At CID-MS2 20 eV, the compounds could be efficiently characterized, because they present distinct base peaks [M + H]+ and [M + H–H2O]+ for the orobol-8-C- and orobol-6-C-glycoside, respectively. Similarly, by the HCD-type fragmentation, in HRMS2 stage, differences between orobol analogs in both mode of ionization were observed. However, the HR HCD-MS2 at 80 eV, in positive mode, generated more ions and each isomer presented different base peaks ions, [0,2X]+ for the orobol-8-C-glycoside and [0,3X]+ for the orobol-6-C-glycoside. By the DPPH, the 8-C-derivative showed a very close value compared with the standard rutin and, in the ABTS method, a higher radical-scavenging activity. In both methods, the EC50 of orobol-8-C-glycoside was almost twice better compared with orobol-6-C-glycoside. In FRAP, both C-glycosides showed a good capacity as Fe+3 reducing agents. We could realize that combined MS techniques, highlighting the positive mode of ionization, can be used to evaluate the isoflavones analogs being useful to differentiate between the isomeric flavones; therefore, these data are important to mass spectrometry dereplication studies become more efficient.

0

Long‐chain ferulic acid esters, such as eicosyl ferulate (1), show a complex and analytically valuable fragmentation behavior under negative‐ion electrospay collision‐induced dissociation ((‐)‐ESI‐CID) mass spectrometry, as studied by use of a high‐resolution (Orbitrap) mass spectrometer. In a strong contrast to the very simple fragmentation of the [M + H]+ ion, which is discussed briefly, the deprotonated molecule, [M ‐ H]‐, exhibits a rich secondary fragmentation chemistry. It first loses a methyl radical (MS2) and the ortho‐quinoid [M ‐ H ‐ Me]‐• radical anion thus formed then dissociates by loss of an extended series of neutral radicals, CnH2n+1• (n = 0‐16) from the long alkyl chain, in competition with the expulsion of CO and CO2 (MS3). The further fragmentation (MS4) of the [M ‐ H ‐ Me ‐ C3H7]‐ ion, discussed as an example, and the highly specific losses of alkyl radicals from the [M ‐ H ‐ Me ‐ CO]‐• and [M ‐ H ‐ Me ‐ CO2]‐• ions provide some mechanistic and structural insights.

Identification and structural determination of small molecules by mass spectrometry is an important step in chemistry and biochemistry. However, the chemically realistic annotation of a fragment ion spectrum can be a difficult challenge. We developed ChemFrag, for the detection of fragmentation pathways and the annotation of fragment ions with chemically reasonable structures. ChemFrag combines a quantum chemical with a rule‐based approach. For different doping substances as test instances, ChemFrag correctly annotates fragment ions. In most cases, the predicted fragments are chemically more realistic than those from purely combinatorial approaches, or approaches based on machine learning. The annotation generated by ChemFrag often coincides with spectra that have been manually annotated by experts. This is a major advance in peak annotation and allows a more precise automatic interpretation of mass spectra.

The molecular actions of mitogen-activated protein kinases (MAPKs) are ultimately accomplished by the substrate proteins where phosphorylation affects their molecular properties and function(s), but knowledge regarding plant MAPK substrates is currently still fragmentary. Here, we uncovered a previously uncharacterized protein family consisting of three proline/serine-rich proteins (PRPs) that are substrates of stress-related MAPKs. We demonstrated the importance of a MAPK docking domain necessary for protein–protein interaction with MAPKs and consequently also for phosphorylation. The main phosphorylated site was mapped to a residue conserved between all three proteins, which when mutated to a non-phosphorylatable form, differentially affected their protein stability. Together with their distinct gene expression patterns, this differential accumulation of the three proteins upon phosphorylation probably contributes to their distinct function(s). Transgenic over-expression of PRP, the founding member, led to plants with enhanced resistance to Pseudomonas syringae pv. tomato DC3000. Older plants of the over-expressing lines have curly leaves and were generally smaller in stature. This growth phenotype was lost in plants expressing the phosphosite variant, suggesting a phosphorylation-dependent effect. Thus, this novel family of PRPs may be involved in MAPK regulation of plant development and / or pathogen resistance responses. As datamining associates PRP expression profiles with hypoxia or oxidative stress and PRP-overexpressing plants have elevated levels of reactive oxygen species, PRP may connect MAPK and oxidative stress signaling.

Representative compounds with a 1,3‐dihydroxybenzene substructure belonging to different important polyphenol classes (stilbenes, flavones, isoflavones, flavonols, flavanones, flavanols, phloroglucinols, anthraquinones and bisanthraquinones) were investigated based on detailed high‐resolution tandem mass spectrometry measurements with an Orbitrap system under negative ion electrospray conditions. The mass spectral behaviour of these compound classes was compared among each other not only with respect to previously described losses of CO, CH2CO and C3O2 but also concerning the loss of CO2 and successive specific fragmentations. Furthermore, some unusual fragmentations such as the loss of a methyl radical during mass spectral decomposition are discussed. The obtained results demonstrate both similarities and differences in their mass spectral fragmentation under MSn conditions, allowing a characterization of the corresponding compound type.

Kelch repeat-containing proteins are involved in diverse cellular processes, but only a small subset of plant kelch proteins has been functionally characterized. Thiocyanate-forming protein (TFP) from field-penny cress, Thlaspi arvense (Brassicaceae), is a representative of specifier proteins, a group of kelch proteins involved in plant specialized metabolism. As components of the glucosinolate-myrosinase system of the Brassicaceae, specifier proteins determine the profile of bioactive products formed when plant tissue is disrupted and glucosinolates are hydrolyzed by myrosinases. Here, we describe the crystal structure of TaTFP at a resolution of 1.4 Å. TaTFP crystallized as homodimer. Each monomer forms a six-blade β-propeller with a wide “top” and a narrower “bottom” opening with distinct strand-connecting loops protruding far beyond the lower propeller surface. Molecular modeling and mutational analysis identified residues for glucosinolate aglucone and Fe2+ cofactor binding within these loops. As the first experimentally determined structure of a plant kelch protein, the crystal structure of TaTFP not only enables more detailed mechanistic studies on glucosinolate breakdown product formation, but also provides a new basis for research on the diverse roles and mechanisms of other kelch proteins in plants.

As components of the glucosinolate-myrosinase system, specifier proteins contribute to the diversity of chemical defenses that have evolved in plants of the Brassicales order as a protection against herbivores and pathogens. Glucosinolates are thioglucosides that are stored separately from their hydrolytic enzymes, myrosinases, in plant tissue. Upon tissue disruption, glucosinolates are hydrolyzed by myrosinases yielding instable aglucones that rearrange to form defensive isothiocyanates. In the presence of specifier proteins, other products, namely simple nitriles, epithionitriles and organic thiocyanates, can be formed instead of isothiocyanates depending on the glucosinolate side chain structure and the type of specifier protein. The biochemical role of specifier proteins is largely unresolved. We have used two thiocyanate-forming proteins and one epithiospecifier protein with different substrate/product specificities to develop molecular models that, in conjunction with mutational analyses, allow us to propose an active site and docking arrangements with glucosinolate aglucones that may explain some of the differences in specifier protein specificities. Furthermore, quantum-mechanical calculations support a reaction mechanism for benzylthiocyanate formation including a catalytic role of the TFP involved. These results may serve as a basis for further theoretical and experimental investigations of the mechanisms of glucosinolate breakdown that will also help to better understand the evolution of specifier proteins from ancestral proteins with functions outside glucosinolate metabolism.

Mass spectrometry (MS) is an important analytical technique for the detection and identification of small compounds. The main bottleneck in the interpretation of metabolite profiling or screening experiments is the identification of unknown compounds from tandem mass spectra.Spectral libraries for tandem MS, such as MassBank or NIST, contain reference spectra for many compounds, but their limited chemical coverage reduces the chance for a correct and reliable identification of unknown spectra outside the database domain.On the other hand, compound databases like PubChem or ChemSpider have a much larger coverage of the chemical space, but they cannot be queried with spectral information directly. Recently, computational mass spectrometry methods and in silico fragmentation prediction allow users to search such databases of chemical structures.We present a new strategy called MetFusion to combine identification results from several resources, in particular, from the in silico fragmenter MetFrag with the spectral library MassBank to improve compound identification. We evaluate the performance on a set of 1062 spectra and achieve an improved ranking of the correct compound from rank 28 using MetFrag alone, to rank 7 with MetFusion, even if the correct compound and similar compounds are absent from the spectral library. On the basis of the evaluation, we extrapolate the performance of MetFusion to the KEGG compound database.

A series of prevailing prenylated furanocoumarins from leaves of Dorstenia gigas and Dorstenia foetida (Moraceae) were investigated by liquid chromatography/electrospray tandem mass spectrometry. The mass spectral behavior of the furanocoumarins under positive ion electrospray conditions is discussed using both an ion trap and a triple quadrupole system. It is demonstrated that both methods represent valuable tools not only for the rapid classification of this type of compounds, but also with respect to their substitution pattern.