Post-translational modifications (PTMs) of biomacromolecules can be useful for understanding the processes by which a relatively small number of individual genes in a particular genome can generate enormous biological complexity in different organisms. The proteomes of barley and the brewing process were investigated by different techniques. However, their diverse and complex PTMs remain understudied. As standard analytical approaches have limitations, innovative analytical approaches need to be developed and applied in PTM studies. To make further progress in this field, it is necessary to specify the sites of modification, as well as to characterize individual isoforms with increased selectivity and sensitivity. This review summarizes advances in the PTM analysis of barley proteins, particularly those involving mass spectrometric detection. Our focus is on monitoring phosphorylation, glycation, and glycosylation, which critically influence functional behavior in metabolism and regulation in organisms.

• Klíčová slova
• barley, mass spectrometry, post-translational modification, protein,
• MeSH
• fosforylace MeSH
• glykosylace MeSH
• ječmen (rod) * genetika   MeSH
• posttranslační úpravy proteinů MeSH
• proteom chemie   MeSH
• proteomika metody   MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• proteom MeSH

Proteins are directly involved in plant phenotypic response to ever changing environmental conditions. The ability to produce multiple mature functional proteins, i.e., proteoforms, from a single gene sequence represents an efficient tool ensuring the diversification of protein biological functions underlying the diversity of plant phenotypic responses to environmental stresses. Basically, two major kinds of proteoforms can be distinguished: protein isoforms, i.e., alterations at protein sequence level arising from posttranscriptional modifications of a single pre-mRNA by alternative splicing or editing, and protein posttranslational modifications (PTMs), i.e., enzymatically catalyzed or spontaneous modifications of certain amino acid residues resulting in altered biological functions (or loss of biological functions, such as in non-functional proteins that raised as a product of spontaneous protein modification by reactive molecular species, RMS). Modulation of protein final sequences resulting in different protein isoforms as well as modulation of chemical properties of key amino acid residues by different PTMs (such as phosphorylation, N- and O-glycosylation, methylation, acylation, S-glutathionylation, ubiquitinylation, sumoylation, and modifications by RMS), thus, represents an efficient means to ensure the flexible modulation of protein biological functions in response to ever changing environmental conditions. The aim of this review is to provide a basic overview of the structural and functional diversity of proteoforms derived from a single gene in the context of plant evolutional adaptations underlying plant responses to the variability of environmental stresses, i.e., adverse cues mobilizing plant adaptive mechanisms to diminish their harmful effects.

• Klíčová slova
• biological functions, crops, environmental stresses, protein isoforms, protein posttranslational modifications (PTMs), protein-protein interactions,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Angiosperm mature pollen represents a quiescent stage with a desiccated cytoplasm surrounded by a tough cell wall, which is resistant to the suboptimal environmental conditions and carries the genetic information in an intact stage to the female gametophyte. Post pollination, pollen grains are rehydrated, activated, and a rapid pollen tube growth starts, which is accompanied by a notable metabolic activity, synthesis of novel proteins, and a mutual communication with female reproductive tissues. Several angiosperm species (Arabidopsis thaliana, tobacco, maize, and kiwifruit) were subjected to phosphoproteomic studies of their male gametophyte developmental stages, mostly mature pollen grains. The aim of this review is to compare the available phosphoproteomic studies and to highlight the common phosphoproteins and regulatory trends in the studied species. Moreover, the pollen phosphoproteome was compared with root hair phosphoproteome to pinpoint the common proteins taking part in their tip growth, which share the same cellular mechanisms.

• Klíčová slova
• kinase motif, male gametophyte, phosphoproteomics, pollen tube, root hair, signal transduction,
• MeSH
• fosfoproteiny metabolismus   MeSH
• opylení * MeSH
• proteom metabolismus   MeSH
• proteomika * MeSH
• pylová láčka metabolismus   MeSH
• rostlinné proteiny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• fosfoproteiny MeSH
• proteom MeSH
• rostlinné proteiny MeSH

Mass spectrometry (MS) is a powerful and sensitive method often used for the identification of phosphoproteins. However, in phosphoproteomics, there is an identified need to compensate for the low abundance, insufficient ionization, and suppression effects of non-phosphorylated peptides. These may hamper the subsequent liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis, resulting in incomplete phosphoproteome characterization, even when using high-resolution instruments. To overcome these drawbacks, we present here an effective microgradient chromatographic technique that yields specific fractions of enriched phosphopeptides compatible with LC-MS/MS analysis. The purpose of our study was to increase the number of identified phosphopeptides, and thus, the coverage of the sample phosphoproteome using the reproducible and straightforward fractionation method. This protocol includes a phosphopeptide enrichment step followed by the optimized microgradient fractionation of enriched phosphopeptides and final LC-MS/MS analysis of the obtained fractions. The simple fractionation system consists of a gas-tight microsyringe delivering the optimized gradient mobile phase to reversed-phase microcolumn. Our data indicate that combining the phosphopeptide enrichment with the microgradient separation is a promising technique for in-depth phosphoproteomic analysis due to moderate input material requirements and more than 3-fold enhanced protein identification.

• Klíčová slova
• acetonitrile, enrichment, fractionation, gradient, mass spectrometry, phosphopeptides, titanium dioxide,
• MeSH
• acetonitrily chemie   MeSH
• chemická frakcionace metody   MeSH
• chromatografie kapalinová metody   MeSH
• fosfopeptidy chemie   MeSH
• fosfoproteiny metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• proteom MeSH
• proteomika MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• titan chemie   MeSH
• tlak MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• acetonitrile MeSH Prohlížeč
• acetonitrily MeSH
• fosfopeptidy MeSH
• fosfoproteiny MeSH
• proteom MeSH
• titan MeSH
• titanium dioxide MeSH Prohlížeč

This work reports highly selective phosphopeptide enrichment using amorphous TiO2 nanotubes (TiO2NTs) and the same material decorated with superparamagnetic Fe3O4 nanoparticles (TiO2NTs@Fe3O4NPs). TiO2NTs and TiO2NTs@Fe3O4NPs materials were applied for phosphopeptide enrichment both from a simple peptide mixture (tryptic digest of bovine serum albumin and α-casein) and from a complex peptide mixture (tryptic digest of Jurkat T cell lysate). The obtained enrichment efficiency and selectivity for phosphopeptides of TiO2NTs and TiO2NTs@Fe3O4NPs were increased to 28.7 and 25.3%, respectively, as compared to those of the well-established TiO2 microspheres. The enrichment protocol was extended for a second elution step facilitating the identification of additional phosphopeptides. It further turned out that both types of amorphous TiO2 nanotubes provide qualitatively new physicochemical features that are clearly advantageous for highly selective phosphopeptide enrichment. This has been confirmed experimentally resulting in substantial reduction of non-phosphorylated peptides in the enriched samples. In addition, TiO2NTs@Fe3O4NPs combine high selectivity and ease of handling due to the superparamagnetic character of the material. The presented materials and performances are further promising for applications toward a whole range of other types of biomolecules to be treated in a similar fashion.

• Publikační typ
• časopisecké články MeSH

Overview of pollen development. Male gametophyte development of angiosperms is a complex process that requires coordinated activity of different cell types and tissues of both gametophytic and sporophytic origin and the appropriate specific gene expression. Pollen ontogeny is also an excellent model for the dissection of cellular networks that control cell growth, polarity, cellular differentiation and cell signaling. This article describes two sequential phases of angiosperm pollen ontogenesis-developmental phase leading to the formation of mature pollen grains, and a functional or progamic phase, beginning with the impact of the grains on the stigma surface and ending at double fertilization. Here we present an overview of important cellular processes in pollen development and explosive pollen tube growth stressing the importance of reserves accumulation and mobilization and also the mutual activation of pollen tube and pistil tissues, pollen tube guidance and the communication between male and female gametophytes. We further describe the recent advances in regulatory mechanisms involved such as posttranscriptional regulation (including mass transcript storage) and posttranslational modifications to modulate protein function, intracellular metabolic signaling, ionic gradients such as Ca(2+) and H(+) ions, cell wall synthesis, protein secretion and intercellular signaling within the reproductive tissues.

• Klíčová slova
• Flowering plants, Male gametophyte, Pollen development, Pollen tube growth,
• MeSH
• Magnoliopsida růst a vývoj   metabolismus   MeSH
• pyl růst a vývoj   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Tobacco mature pollen has extremely desiccated cytoplasm, and is metabolically quiescent. Upon re-hydration it becomes metabolically active and that results in later emergence of rapidly growing pollen tube. These changes in cytoplasm hydration and metabolic activity are accompanied by protein phosphorylation. In this study, we subjected mature pollen, 5-min-activated pollen, and 30-min-activated pollen to TCA/acetone protein extraction, trypsin digestion and phosphopeptide enrichment by titanium dioxide. The enriched fraction was subjected to nLC-MS/MS. We identified 471 phosphopeptides that carried 432 phosphorylation sites, position of which was exactly matched by mass spectrometry. These 471 phosphopeptides were assigned to 301 phosphoproteins, because some proteins carried more phosphorylation sites. Of the 13 functional groups, the majority of proteins were put into these categories: transcription, protein synthesis, protein destination and storage, and signal transduction. Many proteins were of unknown function, reflecting the fact that male gametophyte contains many specific proteins that have not been fully functionally annotated. The quantitative data highlighted the dynamics of protein phosphorylation during pollen activation; the identified phosphopeptides were divided into seven groups based on the regulatory trends. The major group comprised mature pollen-specific phosphopeptides that were dephosphorylated during pollen activation. Several phosphopeptides representing the same phosphoprotein had different regulation, which pinpointed the complexity of protein phosphorylation and its clear functional context. Collectively, we showed the first phosphoproteomics data on activated pollen where the position of phosphorylation sites was clearly demonstrated and regulatory kinetics was resolved.

• MeSH
• fosfoproteiny chemie   metabolismus   MeSH
• kinetika MeSH
• proteomika metody   MeSH
• pyl metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• rostlinné proteiny chemie   metabolismus   MeSH
• tabák genetika   metabolismus   MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfoproteiny MeSH
• rostlinné proteiny MeSH