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Publikation

Leonova, T.; Ihling, C.; Saoud, M.; Frolova, N.; Rennert, R.; Wessjohann, L. A.; Frolov, A.; Does filter-aided sample preparation provide sufficient method linearity for quantitative plant shotgun proteomics? Front. Plant Sci. 13, 874761, (2022) DOI: 10.3389/fpls.2022.874761

Due to its outstanding throughput and analytical resolution, gel-free LC-based shotgun proteomics represents the gold standard of proteome analysis. Thereby, the efficiency of sample preparation dramatically affects the correctness and reliability of protein quantification. Thus, the steps of protein isolation, solubilization, and proteolysis represent the principal bottleneck of shotgun proteomics. The desired performance of the sample preparation protocols can be achieved by the application of detergents. However, these compounds ultimately compromise reverse-phase chromatographic separation and disrupt electrospray ionization. Filter-aided sample preparation (FASP) represents an elegant approach to overcome these limitations. Although this method is comprehensively validated for cell proteomics, its applicability to plants and compatibility with plant-specific protein isolation protocols remain to be confirmed. Thereby, the most important gap is the absence of the data on the linearity of underlying protein quantification methods for plant matrices. To fill this gap, we address here the potential of FASP in combination with two protein isolation protocols for quantitative analysis of pea (Pisum sativum) seed and Arabidopsis thaliana leaf proteomes by the shotgun approach. For this aim, in comprehensive spiking experiments with bovine serum albumin (BSA), we evaluated the linear dynamic range (LDR) of protein quantification in the presence of plant matrices. Furthermore, we addressed the interference of two different plant matrices in quantitative experiments, accomplished with two alternative sample preparation workflows in comparison to conventional FASP-based digestion of cell lysates, considered here as a reference. The spiking experiments revealed high sensitivities (LODs of up to 4 fmol) for spiked BSA and LDRs of at least 0.6 × 102. Thereby, phenol extraction yielded slightly better recoveries, whereas the detergent-based method showed better linearity. Thus, our results indicate the very good applicability of FASP to quantitative plant proteomics with only limited impact of the protein isolation technique on the method’s overall performance.
Publikation

Balarynová, J.; Klčová, B.; Sekaninová, J.; Kobrlová, L.; Cechová, M. Z.; Krejčí, P.; Leonova, T.; Gorbach, D.; Ihling, C.; Smržová, L.; Trněný, O.; Frolov, A.; Bednář, P.; Smýkal, P.; The loss of polyphenol oxidase function is associated with hilum pigmentation and has been selected during pea domestication New Phytol. 235, 1807–1821, (2022) DOI: 10.1111/nph.18256

Seed coats serve as protective tissue to the enclosed embryo. As well as mechanical there are also chemical defence functions. During domestication, the property of the seed coat was altered including the removal of the seed dormancy. We used a range of genetic, transcriptomic, proteomic and metabolomic approaches to determine the function of the pea seed polyphenol oxidase (PPO) gene. Sequencing analysis revealed one nucleotide insertion or deletion in the PPO gene, with the functional PPO allele found in all wild pea samples, while most cultivated peas have one of the three nonfunctional ppo alleles. PPO functionality cosegregates with hilum pigmentation. PPO gene and protein expression, as well as enzymatic activity, was downregulated in the seed coats of cultivated peas. The functionality of the PPO gene relates to the oxidation and polymerisation of gallocatechin in the seed coat. Additionally, imaging mass spectrometry supports the hypothesis that hilum pigmentation is conditioned by the presence of both phenolic precursors and sufficient PPO activity. Taken together these results indicate that the nonfunctional polyphenol oxidase gene has been selected during pea domestication, possibly due to better seed palatability or seed coat visual appearance.
Publikation

Frolova, N.; Soboleva, A.; Nguyen, V. D.; Kim, A.; Ihling, C.; Eisenschmidt-Bönn, D.; Mamontova, T.; Herfurth, U. M.; Wessjohann, L. A.; Sinz, A.; Birkenmeyer, C.; Frolov, A.; Probing glycation potential of dietary sugars in human blood by anintegrated in vitro approach Food Chem. 347, 128951, (2021) DOI: 10.1016/j.foodchem.2020.128951

Glycation is referred to as the interaction of protein amino and guanidino groups with reducing sugars and carbonyl products of their degradation. Resulting advanced glycation end-products (AGEs) contribute to pathogenesis of diabetes mellitus and neurodegenerative disorders. Upon their intestinal absorption, dietary sugars and á-dicarbonyl compounds interact with blood proteins yielding AGEs. Although the differences in glycation potential of monosaccharides are well characterized, the underlying mechanisms are poorly understood. To address this question, D-glucose, D-fructose and L-ascorbic acid were incubated with human serum albumin (HSA). The sugars and á-dicarbonyl intermediates of their degradation were analyzed in parallel to protein glycation patterns (exemplified with hydroimidazolone modifications of arginine residues and products of their hydrolysis) by bottom-up proteomics and computational chemistry. Glycation of HSA with sugars revealed 9 glyoxal- and 14 methylglyoxal-derived modification sites. Their dynamics was sugar-specific and depended on concentrations of á-dicarbonyls, their formation kinetics, and presence of stabilizing residues in close proximity to the glycation sites.
Publikation

Leonova, T.; Popova, V.; Tsarev, A.; Henning, C.; Antonova, K.; Rogovskaya, N.; Vikhnina, M.; Baldensperger, T.; Soboleva, A.; Dinastia, E.; Dorn, M.; Shiroglasova, O.; Grishina, T.; Balcke, G. U.; Ihling, C.; Smolikova, G.; Medvedev, S.; Zhukov, V. A.; Babakov, V.; Tikhonovich, I. A.; Glomb, M. A.; Bilova, T.; Frolov, A.; Does Protein Glycation Impact on the Drought-Related Changes in Metabolism and Nutritional Properties of Mature Pea (Pisum sativum L.) Seeds? Int. J. Mol. Sci. 21, 567, (2020) DOI: 10.3390/ijms21020567

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.
Publikation

Soboleva, A.; Mavropulo-Stolyarenko, G.; Karonova, T.; Thieme, D.; Hoehenwarter, W.; Ihling, C.; Stefanov, V.; Grishina, T.; Frolov, A.; Multiple Glycation Sites in Blood Plasma Proteins as an Integrated Biomarker of Type 2 Diabetes Mellitus Int. J. Mol. Sci. 20, 2329, (2019) DOI: 10.3390/ijms20092329

Type 2 diabetes mellitus (T2DM) is one of the most widely spread metabolic diseases. Because of its asymptomatic onset and slow development, early diagnosis and adequate glycaemic control are the prerequisites for successful T2DM therapy. In this context, individual amino acid residues might be sensitive indicators of alterations in blood glycation levels. Moreover, due to a large variation in the half-life times of plasma proteins, a generalized biomarker, based on multiple glycation sites, might provide comprehensive control of the glycemic status across any desired time span. Therefore, here, we address the patterns of glycation sites in highly-abundant blood plasma proteins of T2DM patients and corresponding age- and gender-matched controls by comprehensive liquid chromatography-mass spectrometry (LC-MS). The analysis revealed 42 lysyl residues, significantly upregulated under hyperglycemic conditions. Thereby, for 32 glycation sites, biomarker behavior was demonstrated here for the first time. The differentially glycated lysines represented nine plasma proteins with half-lives from 2 to 21 days, giving access to an integrated biomarker based on multiple protein-specific Amadori peptides. The validation of this biomarker relied on linear discriminant analysis (LDA) with random sub-sampling of the training set and leave-one-out cross-validation (LOOCV), which resulted in an accuracy, specificity, and sensitivity of 92%, 100%, and 85%, respectively.
Publikation

Mamontova, T.; Afonin, A. M.; Ihling, C.; Soboleva, A.; Lukasheva, E.; Sulima, A. S.; Shtark, O. Y.; Akhtemova, G. A.; Povydysh, M. N.; Sinz, A.; Frolov, A.; Zhukov, V. A.; Tikhonovich, I. A.; Profiling of Seed Proteome in Pea (Pisum sativum L.) Lines Characterized with High and Low Responsivity to Combined Inoculation with Nodule Bacteria and Arbuscular Mycorrhizal Fungi Molecules 24, 1603, (2019) DOI: 10.3390/molecules24081603

Legume crops represent the major source of food protein and contribute to human nutrition and animal feeding. An essential improvement of their productivity can be achieved by symbiosis with beneficial soil microorganisms—rhizobia (Rh) and arbuscular mycorrhizal (AM) fungi. The efficiency of these interactions depends on plant genotype. Recently, we have shown that, after simultaneous inoculation with Rh and AM, the productivity gain of pea (Pisum sativum L) line K-8274, characterized by high efficiency of interaction with soil microorganisms (EIBSM), was higher in comparison to a low-EIBSM line K-3358. However, the molecular mechanisms behind this effect are still uncharacterized. Therefore, here, we address the alterations in pea seed proteome, underlying the symbiosis-related productivity gain, and identify 111 differentially expressed proteins in the two lines. The high-EIBSM line K-8274 responded to inoculation by prolongation of seed maturation, manifested by up-regulation of proteins involved in cellular respiration, protein biosynthesis, and down-regulation of late-embryogenesis abundant (LEA) proteins. In contrast, the low-EIBSM line K-3358 demonstrated lower levels of the proteins, related to cell metabolism. Thus, we propose that the EIBSM trait is linked to prolongation of seed filling that needs to be taken into account in pulse crop breeding programs. The raw data have been deposited to the ProteomeXchange with identifier PXD013479.
Publikation

Matamoros, M. A.; Kim, A.; Peñuelas, M.; Ihling, C.; Griesser, E.; Hoffmann, R.; Fedorova, M.; Frolov, A.; Becana, M.; Protein Carbonylation and Glycation in Legume Nodules Plant Physiol. 177, 1510-1528, (2018) DOI: 10.1104/pp.18.00533

Nitrogen fixation is an agronomically and environmentally important process catalyzed by bacterial nitrogenase within legume root nodules. These unique symbiotic organs have high metabolic rates and produce large amounts of reactive oxygen species that may modify proteins irreversibly. Here, we examined two types of oxidative posttranslational modifications of nodule proteins: carbonylation, which occurs by direct oxidation of certain amino acids or by interaction with reactive aldehydes arising from cell membrane lipid peroxides; and glycation, which results from the reaction of Lys and Arg residues with reducing sugars or their auto-oxidation products. We used a strategy based on the enrichment of carbonylated peptides by affinity chromatography followed by liquid chromatography-tandem mass spectrometry to identify 369 oxidized proteins in bean (Phaseolus vulgaris) nodules. Of these, 238 corresponded to plant proteins and 131 to bacterial proteins. Lipid peroxidation products induced most carbonylation sites. This study also revealed that carbonylation has major effects on two key nodule proteins. Metal-catalyzed oxidation caused inactivation of malate dehydrogenase and aggregation of leghemoglobin. In addition, numerous glycated proteins were identified in vivo, including three key nodule proteins: sucrose synthase, glutamine synthetase, and glutamate synthase. Label-free quantification identified 10 plant proteins and 18 bacterial proteins as age-specifically glycated. Overall, our results suggest that the selective carbonylation or glycation of crucial proteins involved in nitrogen metabolism, transcriptional regulation, and signaling may constitute a mechanism to control cell metabolism and nodule senescence.
Publikation

Frolov, A.; Didio, A.; Ihling, C.; Chantzeva, V.; Grishina, T.; Hoehenwarter, W.; Sinz, A.; Smolikova, G.; Bilova, T.; Medvedev, S.; The effect of simulated microgravity on the Brassica napus seedling proteome Funct. Plant Biol. 45, 440-452, (2018) DOI: 10.1071/FP16378

The magnitude and the direction of the gravitational field represent an important environmental factor affecting plant development. In this context, the absence or frequent alterations of the gravity field (i.e. microgravity conditions) might compromise extraterrestrial agriculture and hence space inhabitation by humans. To overcome the deleterious effects of microgravity, a complete understanding of the underlying changes on the macromolecular level is necessary. However, although microgravity-related changes in gene expression are well characterised on the transcriptome level, proteomic data are limited. Moreover, information about the microgravity-induced changes in the seedling proteome during seed germination and the first steps of seedling development is completely missing. One of the valuable tools to assess gravity-related issues is 3D clinorotation (i.e. rotation in two axes). Therefore, here we address the effects of microgravity, simulated by a two-axial clinostat, on the proteome of 24- and 48-h-old seedlings of oilseed rape (Brassica napus L.). The liquid chromatography-MS-based proteomic analysis and database search revealed 95 up- and 38 downregulated proteins in the tryptic digests obtained from the seedlings subjected to simulated microgravity, with 42 and 52 annotations detected as being unique for 24- and 48-h treatment times, respectively. The polypeptides involved in protein metabolism, transport and signalling were annotated as the functional groups most strongly affected by 3-D clinorotation.
Publikation

Frolov, A.; Mamontova, T.; Ihling, C.; Lukasheva, E.; Bankin, M.; Chantseva, V.; Vikhnina, M.; Soboleva, A.; Shumilina, J.; Mavropolo-Stolyarenko, G.; Grishina, T.; Osmolovskaya, N.; Zhukov, V.; Hoehenwarter, W.; Sinz, A.; Tikhononovich, I.; Wessjohann, L.; Bilova, T.; Smolikova, G.; Medvedev, S.; Mining seed proteome: from protein dynamics to modification profiles Biol. Commun. 63, 43-58, (2018) DOI: 10.21638/spbu03.2018.106

In the modern world, crop plants represent a major source of daily consumed foods. Among them, cereals and legumes — i.e. the crops accumulating oils, carbohydrates and proteins in their seeds — dominate in European agriculture, tremendously impacting global protein consumption and biodiesel production. Therefore, the seeds of crop plants attract the special attention of biologists, biochemists, nutritional physiologists and food chemists. Seed development and germination, as well as age- and stress-related changes in their viability and nutritional properties, can be addressed by a variety of physiological and biochemical methods. In this context, the methods of functional genomics can be applied to address characteristic changes in seed metabolism, which can give access to stress-resistant genotypes. Among these methods, proteomics is one of the most effective tools, allowing mining metabolism changes on the protein level. Here we discuss the main methodological approaches of seed proteomics in the context of physiological changes related to environmental stress and ageing. We provide a comprehensive comparison of gel- and chromatographybased approaches with a special emphasis on advantages and disadvantages of both strategies in characterization of the seed proteome.

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