Protein glycation is usually referred to as an array of non-enzymatic post-translational modifications formed by reducing sugars and carbonyl products of their degradation. The resulting advanced glycation end products (AGEs) represent a heterogeneous group of covalent adducts, known for their pro-inflammatory effects in mammals, and impacting on pathogenesis of metabolic diseases and ageing. In plants, AGEs are the markers of tissue ageing and response to environmental stressors, the most prominent of which is drought. Although water deficit enhances protein glycation in leaves, its effect on seed glycation profiles is still unknown. Moreover, the effect of drought on biological activities of seed protein in mammalian systems is still unstudied with respect to glycation. Therefore, here we address the effects of a short-term drought on the patterns of seed protein-bound AGEs and accompanying alterations in pro-inflammatory properties of seed protein in the context of seed metabolome dynamics. A short-term drought, simulated as polyethylene glycol-induced osmotic stress and applied at the stage of seed filling, resulted in the dramatic suppression of primary seed metabolism, although the secondary metabolome was minimally affected. This was accompanied with significant suppression of NF-kB activation in human SH-SY5Y neuroblastoma cells after a treatment with protein hydrolyzates, isolated from the mature seeds of drought-treated plants. This effect could not be attributed to formation of known AGEs. Most likely, the prospective anti-inflammatory effect of short-term drought is related to antioxidant effect of unknown secondary metabolite protein adducts, or down-regulation of unknown plant-specific AGEs due to suppression of energy metabolism during seed filling.

Metabolomics, which is typically referred to as the post-genomic methodology addressing low-molecular-weight metabolites, became a powerful tool in post-genomic research over the last two decades. Indeed, the state-of-the-art metabolomics relies on several well-established complementary platforms—nuclear magnetic resonance (NMR) spectroscopy, liquid and gas chromatography coupled on-line with mass spectrometry (LC- and GC-MS, respectively), and capillary electrophoresis–mass spectrometry (CE-MS). Among them, GC-MS represents one of the oldest and most well-established techniques currently employed in the metabolomics of volatile compounds and non-volatiles—polar low-molecular-weight metabolites, which can be efficiently converted in volatile form by comprehensive derivatization of polar functional groups. Currently, GC-MS is established as the principal analytical method for characterizing primary plant metabolism, although other methods also contribute significantly to determining the complete metabolite profile. Therefore, here, we address the role of GC-MS in plant metabolomics and its potential for the profiling of low-molecular-weight metabolites. Further, we comprehensively review the methods of sample preparation with special emphasis on extraction and derivatization approaches, which are currently employed to improve the method performance and its metabolome coverage.

Metabolomics, which is typically referred to as the post-genomic methodology addressing low molecular weight metabolites, became a powerful tool in post-genomic research over the last two decades. Indeed, the state of the art metabolomics relies on several well-established complemen-tary platforms – nuclear magnetic resonance (NMR) spectroscopy, liquid and gas chromatog-raphy coupled on-line to mass spectrometry (LC- and GC-MS, respectively), capillary electropho-resis-mass spectrometry (CE-MS). Among them, GC-MS represents one of the oldest and most well-established techniques currently employed in metabolomics of volatile compounds and non-volatiles – polar low-molecular weight metabolites, which can be efficiently converted in volatile form by comprehensive derivatization of polar functional groups. Currently, GC-MS is recog-nized as the core analytical technique in characterization of plant primary metabolome, although the other methods contribute to the whole metabolite profile essentially. Therefore, here we ad-dress the role of GC-MS in plant metabolomics and its potential for profiling of low molecular weight metabolites. Further, we comprehensively review the methods of sample preparation with a special emphasis on the extraction and derivatization approaches, which are currently employed to improve the method performance and its metabolome coverage.

Because of ongoing climate change, drought is becoming the major factor limiting productivity of all plants, including legumes. As these protein-rich crops form symbiotic associations with rhizobial bacteria — root nodules — they readily lose their productivity under drought conditions. Understanding the underlying molecular mechanisms might give access to new strategies to preserve the productivity of legume crops under dehydration. As was shown recently, development of drought response is accompanied by alterations in the patterns of protein glycation and formation of advanced glycation end products (AGEs) that might be a part of unknown regulatory mechanisms. Therefore, here we address the effects of moderate drought on protein dynamics and AGE patterns in pea (Pisum sativum) root nodules. For this, plants inoculated with rhizobial culture were subjected to osmotic stress for one week, harvested, the total protein fraction was isolated from root nodules by phenol extraction, analyzed by bottom-up LC-MS-based proteomics, and AGE patterns were characterized. Surprisingly, despite the clear drought-related changes in phenotype and stomatal conductivity, only minimal accompanying expressional changes (14 rhizobial and 14 pea proteins, mostly involved in central metabolism and nitrogen fixation) could be observed. However, 71 pea and 97 rhizobial proteins (mostly transcription factors, ABC transporters and effector enzymes) were glycated, with carboxymethylation being the major modification type. Thereby, the numbers of glycated sites in nodule proteins dramatically decreased upon stress application. It might indicate an impact of glycation in regulation of transport, protein degradation, central, lipid and nitrogen metabolism. The data are available at Proteome Xchange (accession: PXD024042). advanced glycation end products (AGEs), drought stress, glycation, pea (Pisum sativum), polyethylene glycol (PEG), proteomics, root nodules

Decades ago, the introduction of GC-MS marked a significant advancement in primary plant metabolite studies. Here, in our review, we will delve into critical aspects of the workflow, spanning the selection of an analytical platform, sample preparation, analytical acquisition, and data processing and interpretation. The exceptional separation capabilities of GC, characterized by remarkable chromatographic resolution, render it ideal for analysis of the complex plant metabolome, including the separation of isomeric compounds. The diversity of analytical platforms allows the investigation of plant metabolomes using targeted and non-targeted approaches. GC-MS, equipped with efficient extraction methods and reliable derivatization protocols for semi- and non-volatile compounds, enables qualitative and quantitative analysis of these molecules. The stability of derivatives forms the foundation for the robustness and reproducibility of GC-MS methods, and their mass spectra provide characteristic fragments for confident identification and sensitive quantification of individual metabolites. There has been key progress in the advancement of GC-MS approaches to studying plant metabolism. However, the presence of artifacts during GC-MS analysis, particularly during derivatization, is a challenge that requires careful validations, which frequently necessitate additional investigations. The feasible solutions that were achieved to overcome the limitations in GC-MS-based studies are a particular focus of the present discussion.

Magnetic resonance (MR) phenomena have proven themselves in high-precision spectroscopy with NMR and in in situ tomography with MRI. Fabrication of the novel devices and experiments largely relies on multi-step procedures that are time-consuming and equipment-intensive. Digital light processing (DLP), in part, offers a single-step fabrication, significantly dropping the costs, and allows rapid prototyping of the devices within a few micron resolution. However, the selection of DLP resins must be carefully considered. Many DLP resins undergo chemical degradation in liquid state MR, resulting in spectral artifacts; with only a few outstanding cases holding up in aggressive solvents. In this study, we investigate the effect of solvents on the most utilized and market-available resins and compare the reliability of DLP-processed devices for MR. Specifically, we selected and assessed the best resins and 3D-printed them following the designs of standard NMR tubes, with circular and hexagonal cross-sections. Finally, we also tested them in an example application, namely Signal Amplification by Reversible Exchange (SABRE) hyperpolarization with parahydrogen. This not only demonstrated that the tubes can be pressurized up to ∼5 bar, but also their suitability for NMR experiments, including a radiofrequency amplification by stimulated emission of radiation (RASER) experiment.