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Publikation

Paudel, G.; Bilova, T.; Schmidt, R.; Greifenhagen, U.; Berger, R.; Tarakhovskaya, E.; Stöckhardt, S.; Balcke, G. U.; Humbeck, K.; Brandt, W.; Sinz, A.; Vogt, T.; Birkemeyer, C.; Wessjohann, L.; Frolov, A.; Osmotic stress is accompanied by protein glycation in Arabidopsis thaliana J. Exp. Bot. 67, 6283-6295, (2016) DOI: 10.1093/jxb/erw395

Among the environmental alterations accompanying oncoming climate changes, drought is the most important factor influencing crop plant productivity. In plants, water deficit ultimately results in the development of oxidative stress and accumulation of osmolytes (e.g. amino acids and carbohydrates) in all tissues. Up-regulation of sugar biosynthesis in parallel to the increasing overproduction of reactive oxygen species (ROS) might enhance protein glycation, i.e. interaction of carbonyl compounds, reducing sugars and α-dicarbonyls with lysyl and arginyl side-chains yielding early (Amadori and Heyns compounds) and advanced glycation end-products (AGEs). Although the constitutive plant protein glycation patterns were characterized recently, the effects of environmental stress on AGE formation are unknown so far. To fill this gap, we present here a comprehensive in-depth study of the changes in Arabidopsis thaliana advanced glycated proteome related to osmotic stress. A 3 d application of osmotic stress revealed 31 stress-specifically and 12 differentially AGE-modified proteins, representing altogether 56 advanced glycation sites. Based on proteomic and metabolomic results, in combination with biochemical, enzymatic and gene expression analysis, we propose monosaccharide autoxidation as the main stress-related glycation mechanism, and glyoxal as the major glycation agent in plants subjected to drought.
Publikation

Greifenhagen, U.; Frolov, A.; Blüher, M.; Hoffmann, R.; Site-specific analysis of advanced glycation end products in plasma proteins of type 2 diabetes mellitus patients Anal. Bioanal. Chem. 408, 5557-5566, (2016) DOI: 10.1007/s00216-016-9651-4

Advanced glycation end products (AGEs) are posttranslational modifications formed non-enzymatically from the reaction of carbohydrates and their degradation products with proteins. Accumulation of AGEs is associated with the progression of severe diabetic complications, for example, and elevated tissue levels of AGEs might even predict these pathologies. As AGE formation is often site-specific, mapping of these modification sites may reveal more sensitive and specific markers than the global tissue level. Here, 42 AGE modifications were identified in a bottom-up proteomic approach by tandem mass spectrometry, which corresponded to 36 sites in 22 high to medium abundant proteins in individual plasma samples obtained from type 2 diabetes mellitus (T2DM) patients with long disease duration (>10 years). Major modifications were glarg (11 modification sites) and carboxymethylation (5) of arginine and formylation (8), acetylation (7), and carboxymethylation (7) of lysine residues. Relative quantification of these sites in plasma samples obtained from normoglycemic individuals (n = 47) and patients with T2DM being newly diagnosed (n = 47) or of medium (2–5 years, n = 20) and long disease duration (>10 years, n = 20) did not reveal any significant differences.
Publikation

Gladilovich, V.; Greifenhagen, U.; Sukhodolov, N.; Selyutin, A.; Singer, D.; Thieme, D.; Majovsky, P.; Shirkin, A.; Hoehenwarter, W.; Bonitenko, E.; Podolskaya, E.; Frolov, A.; Immobilized metal affinity chromatography on collapsed Langmuir-Blodgett iron(III) stearate films and iron(III) oxide nanoparticles for bottom-up phosphoproteomics J. Chromatogr. A 1443, 181-190, (2016) DOI: 10.1016/j.chroma.2016.03.044

Phosphorylation is the enzymatic reaction of site-specific phosphate transfer from energy-rich donors to the side chains of serine, threonine, tyrosine, and histidine residues in proteins. In living cells, reversible phosphorylation underlies a universal mechanism of intracellular signal transduction. In this context, analysis of the phosphoproteome is a prerequisite to better understand the cellular regulatory networks. Conventionally, due to the low contents of signaling proteins, selective enrichment of proteolytic phosphopeptides by immobilized metal affinity chromatography (IMAC) is performed prior to their LC–MS or -MS/MS analysis. Unfortunately, this technique still suffers from low selectivity and compromised analyte recoveries. To overcome these limitations, we propose IMAC systems comprising stationary phases based on collapsed Langmuir-Blodgett films of iron(III) stearate (FF) or iron(III) oxide nanoparticles (FO) and mobile phases relying on ammonia, piperidine and heptadecafluorooctanesulfonic acid (PFOS). Experiments with model phosphopeptides and phosphoprotein tryptic digests showed superior binding capacity, selectivity and recovery for both systems in comparison to the existing commercial analogs. As evidenced by LC–MS/MS analysis of the HeLa phosphoproteome, these features of the phases resulted in increased phosphoproteome coverage in comparison to the analogous commercially available phases, indicating that our IMAC protocol is a promising chromatographic tool for in-depth phosphoproteomic research.
Publikation

Bilova, T.; Lukasheva, E.; Brauch, D.; Greifenhagen, U.; Paudel, G.; Tarakhovskaya, E.; Frolova, N.; Mittasch, J.; Balcke, G. U.; Tissier, A.; Osmolovskaya, N.; Vogt, T.; Wessjohann, L. A.; Birkemeyer, C.; Milkowski, C.; Frolov, A.; A Snapshot of the Plant Glycated Proteome: STRUCTURAL, FUNCTIONAL, AND MECHANISTIC ASPECTS J. Biol. Chem. 291, 7621-7636, (2016) DOI: 10.1074/jbc.M115.678581

Glycation is the reaction of carbonyl compounds (reducing sugars and α-dicarbonyls) with amino acids, lipids, and proteins, yielding early and advanced glycation end products (AGEs). The AGEs can be formed via degradation of early glycation intermediates (glycoxidation) and by interaction with the products of monosaccharide autoxidation (autoxidative glycosylation). Although formation of these potentially deleterious compounds is well characterized in animal systems and thermally treated foods, only a little information about advanced glycation in plants is available. Thus, the knowledge of the plant AGE patterns and the underlying pathways of their formation are completely missing. To fill this gap, we describe the AGE-modified proteome of Brassica napus and characterize individual sites of advanced glycation by the methods of liquid chromatography-based bottom-up proteomics. The modification patterns were complex but reproducible: 789 AGE-modified peptides in 772 proteins were detected in two independent experiments. In contrast, only 168 polypeptides contained early glycated lysines, which did not resemble the sites of advanced glycation. Similar observations were made with Arabidopsis thaliana. The absence of the early glycated precursors of the AGE-modified protein residues indicated autoxidative glycosylation, but not glycoxidation, as the major pathway of AGE formation. To prove this assumption and to identify the potential modifying agents, we estimated the reactivity and glycative potential of plant-derived sugars using a model peptide approach and liquid chromatography-mass spectrometry-based techniques. Evaluation of these data sets together with the assessed tissue carbohydrate contents revealed dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, ribulose, erythrose, and sucrose as potential precursors of plant AGEs.
Bücher und Buchkapitel

Bilova, T.; Greifenhagen, U.; Paudel, G.; Lukasheva, E.; Brauch, D.; Osmolovskaya, N.; Tarakhovskaya, E.; Balcke, G. U.; Tissier, A.; Vogt, T.; Milkowski, C.; Birkemeyer, C.; Wessjohann, L.; Frolov, A.; Glycation of Plant Proteins under Environmental Stress — Methodological Approaches, Potential Mechanisms and Biological Role (Shanker, A. K. & Shanker, C., eds.). 295-316, (2016) DOI: 10.5772/61860

Environmental stress is one of the major factors reducing crop productivity. Due to the oncoming climate changes, the effects of drought and high light on plants play an increasing role in modern agriculture. These changes are accompanied with a progressing contamination of soils with heavy metals. Independent of their nature, environmental alterations result in development of oxidative stress, i.e. increase of reactive oxygen species (ROS) contents, and metabolic adjustment, i.e. accumulation of soluble primary metabolites (amino acids and sugars). However, a simultaneous increase of ROS and sugar concentrations ultimately results in protein glycation, i.e. non-enzymatic interaction of reducing sugars or their degradation products (α-dicarbonyls) with proteins. The eventually resulting advanced glycation end-products (AGEs) are known to be toxic and pro-inflammatory in mammals. Recently, their presence was unambiguously demonstrated in vivo in stressed Arabidopsis thaliana plants. Currently, information on protein targets, modification sites therein, mediators and mechanisms of plant glycation are being intensively studied. In this chapter, we comprehensively review the methodological approaches for plant glycation research and discuss potential mechanisms of AGE formation under stress conditions. On the basis of these patterns and additional in vitro experiments, the pathways and mechanisms of plant glycation can be proposed.

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