A novel high molecular weight glutenin subunit encoded by the Glu-1B locus was identified in the French genotype Bagou, which we named 1B × 6.5. This subunit differed in SDS-PAGE from well-known 1B × 6 and 1B × 7 subunits, which are also encoded at this locus. Subunit 1B × 6.5 has a theoretical molecular weight of 88,322.83 Da, which is more mobile than 1B × 6 subunit, and isoelectric point (pI) of about 8.7, which is lower than that for 1B × 6 subunit. The specific primers were designed to amplify and sequence 2476 bp of the Glu-1B locus from genotype Bagou. A high level of similarity was found between the sequence encoding 1B × 6.5 and other x-type encoding alleles of this locus.

• Klíčová slova
• 1-D electrophoresis, 2-D electrophoresis, HMW glutenin, mass spectrometry, wheat,
• Publikační typ
• časopisecké články MeSH

Proteins play a major role in the three-dimensional organization of nuclear genome and its function. While histones arrange DNA into a nucleosome fiber, other proteins contribute to higher-order chromatin structures in interphase nuclei, and mitotic/meiotic chromosomes. Despite the key role of proteins in maintaining genome integrity and transferring hereditary information to daughter cells and progenies, the knowledge about their function remains fragmentary. This is particularly true for the proteins of condensed chromosomes and, in particular, chromosomes of plants. Here, we purified barley mitotic metaphase chromosomes by a flow cytometric sorting and characterized their proteins. Peptides from tryptic protein digests were fractionated either on a cation exchanger or reversed-phase microgradient system before liquid chromatography coupled to tandem mass spectrometry. Chromosomal proteins comprising almost 900 identifications were classified based on a combination of software prediction, available database localization information, sequence homology, and domain representation. A biological context evaluation indicated the presence of several groups of abundant proteins including histones, topoisomerase 2, POLYMERASE 2, condensin subunits, and many proteins with chromatin-related functions. Proteins involved in processes related to DNA replication, transcription, and repair as well as nucleolar proteins were found. We have experimentally validated the presence of FIBRILLARIN 1, one of the nucleolar proteins, on metaphase chromosomes, suggesting that plant chromosomes are coated with proteins during mitosis, similar to those of human and animals. These results improve significantly the knowledge of plant chromosomal proteins and provide a basis for their functional characterization and comparative phylogenetic analyses.

• Klíčová slova
• FIBRILLARIN 1, barley, chromatin, flow cytometric sorting, mass spectrometry, mitotic chromosome, perichromosomal layer, protein prediction,
• Publikační typ
• časopisecké články MeSH

The Dipteran family Tephritidae (true fruit flies) comprises more than 5000 species classified in 500 genera distributed worldwide. Tephritidae include devastating agricultural pests and highly invasive species whose spread is currently facilitated by globalization, international trade and human mobility. The ability to identify and exploit a wide range of host plants for oviposition, as well as effective and diversified reproductive strategies, are among the key features supporting tephritid biological success. Intraspecific communication involves the exchange of a complex set of sensory cues that are species- and sex-specific. Chemical signals, which are standing out in tephritid communication, comprise long-distance pheromones emitted by one or both sexes, cuticular hydrocarbons with limited volatility deposited on the surrounding substrate or on the insect body regulating medium- to short-distance communication, and host-marking compounds deposited on the fruit after oviposition. In this review, the current knowledge on tephritid chemical communication was analysed with a special emphasis on fruit fly pest species belonging to the Anastrepha, Bactrocera, Ceratitis, and Rhagoletis genera. The multidisciplinary approaches adopted for characterising tephritid semiochemicals, and the real-world applications and challenges for Integrated Pest Management (IPM) and biological control strategies are critically discussed. Future perspectives for targeted research on fruit fly chemical communication are highlighted.

• Klíčová slova
• cuticular hydrocarbons, host-marking pheromone, mating disruption, odours, olfaction, olfactory cues, pheromone, true fruit flies,
• Publikační typ
• časopisecké články MeSH
• přehledy 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

UNLABELLED: Determining disease-associated changes in protein glycosylation provides a better understanding of pathogenesis. This work focuses on human immunoglobulin A1 (IgA1), where aberrant O-glycosylation plays a key role in the pathogenesis of IgA nephropathy (IgAN). Normal IgA1 hinge region carries 3 to 6 O-glycans consisting of N-acetylgalactosamine (GalNAc) and galactose (Gal); both sugars may be sialylated. In IgAN patients, some O-glycans on a fraction of IgA1 molecules are Gal-deficient. Here we describe a sample preparation protocol with optimized cysteine alkylation of a Gal-deficient polymeric IgA1 myeloma protein prior to in-gel digestion and analysis of the digest by MALDI-TOF/TOF mass spectrometry (MS). Following a novel strategy, IgA1 hinge-region O-glycopeptides were fractionated by reversed-phase liquid chromatography using a microgradient device and identified by MALDI-TOF/TOF tandem MS (MS/MS). The acquired MS/MS spectra were interpreted manually and by means of our own software. This allowed assigning up to six O-glycosylation sites and demonstration, for the first time, of the distribution of isomeric O-glycoforms having the same molecular mass, but a different glycosylation pattern. The most abundant Gal-deficient O-glycoforms were GalNAc4Gal3 and GalNAc5Gal4 with one Gal-deficient site and GalNAc5Gal3 and GalNAc4Gal2 with two Gal-deficient sites. The most frequent Gal-deficient sites were at Ser230 and/or Thr236. BIOLOGICAL SIGNIFICANCE: In this work, we studied the O-glycosylation in the hinge region of human immunoglobulin A1 (IgA1). Aberrant glycosylation of the protein plays a key role in the pathogenesis of IgA nephropathy. Thus identification of the O-glycan composition of IgA1 is important for a deeper understanding of the disease mechanism, biomarker discovery and validation, and implementation and monitoring of disease-specific therapies. We developed a new procedure for elucidating the heterogeneity of IgA1 O-glycosylation. After running a polyacrylamide gel electrophoresis under denaturing conditions, the heavy chain of IgA1 was subjected to in-gel digestion by trypsin. O-glycopeptides were separated from the digest on capillary columns using a microgradient chromatographic device (replacing commonly used liquid chromatographs) and subjected to MALDI-TOF/TOF mass spectrometry (MS) and tandem mass spectrometry (MS/MS) involving post-source decay fragmentation. We show that the complete modification of cysteines by iodoacetamide prior to electrophoresis is critical for successful MS/MS analyses on the way to deciphering the microheterogeneity of O-glycosylation in IgA1. Similarly, the removal of the excess of the reagent is equally important. The acquired MS/MS allowed assigning up to six O-glycosylation sites and identification of isomeric O-glycoforms. We show that our simplified approach is efficient and has a high potential to provide a method for the rapid assessment of IgA1 heterogeneity that is a less expensive and yet corroborating alternative to LC-(high-resolution)-MS protocols. The novelty and biological significance reside in the demonstration, for the first time, of the distribution of the most abundant isoforms of HR O-glycopeptides of IgA1. As another new feature, we introduce a software solution for the interpretation of MS/MS data of O-glycopeptide isoforms, which provides the possibility of fast and easier data processing. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine.

• Klíčová slova
• ACN, CAM, CHCA, DTT, ECD, ETD, FEP, FT-ICR, Fourier transform ion cyclotron resonance, Glycopeptide, HR, Human immunoglobulin A1 (IgA1), IAM, IgA, IgA nephropathy, IgAN, LC, MALDI-TOF, MS, MS/MS, Mass spectrometry, Microgradient separation, O-glycosylation, PAM, PSD, RPLC, TFA, acetonitrile, carbamidomethylation, dithiothreitol, electron capture dissociation, electron transfer dissociation, fluorinated ethylene propylene, hinge region, immunoglobulin A, iodoacetamide, liquid chromatography, mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight, post-source decay, propionamidation, reversed-phase liquid chromatography, tandem mass spectrometry, trifluoroacetic acid, α-cyano-4-hydroxycinnamic acid,
• MeSH
• alkylace MeSH
• cystein krev   chemie   MeSH
• glykosylace MeSH
• IgA nefropatie krev   MeSH
• imunoglobulin A krev   chemie   MeSH
• lidé MeSH
• odběr biologického vzorku * MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• cystein MeSH
• imunoglobulin A MeSH