The endoplasmic reticulum (ER) is an extensive network of intracellular membranes. Its major functions include proteosynthesis, protein folding, post-transcriptional modification and sorting of proteins within the cell, and lipid anabolism. Moreover, several studies have suggested that it may be involved in regulating intracellular auxin homeostasis in plants by modulating its metabolism. Therefore, to study auxin metabolome in the ER, it is necessary to obtain a highly enriched (ideally, pure) ER fraction. Isolation of the ER is challenging because its biochemical properties are very similar to those of other cellular endomembranes. Most published protocols for ER isolation use density gradient ultracentrifugation, despite its suboptimal resolving power. Here we present an optimised protocol for ER isolation from Arabidopsis thaliana seedlings for the subsequent mass spectrometric determination of ER-specific auxin metabolite profiles. Auxin metabolite analysis revealed highly elevated levels of active auxin form (IAA) within the ER compared to whole plants. Moreover, samples prepared using our optimised isolation ER protocol are amenable to analysis using various "omics" technologies including analyses of both macromolecular and low molecular weight compounds from the same sample.

• Klíčová slova
• auxin, density gradient centrifugation, endoplasmic reticulum, mass spectrometry, subcellular fractionation,
• MeSH
• Arabidopsis cytologie   metabolismus   MeSH
• endoplazmatické retikulum metabolismus   MeSH
• kyseliny indoloctové metabolismus   MeSH
• metabolom MeSH
• metabolomika metody   MeSH
• proteiny huseníčku analýza   metabolismus   MeSH
• proteomika metody   MeSH
• rostlinné buňky MeSH
• semenáček cytologie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• indoleacetic acid MeSH Prohlížeč
• kyseliny indoloctové MeSH
• proteiny huseníčku MeSH

Photosystem II (PSII) complexes are organized into large supercomplexes with variable amounts of light-harvesting proteins (Lhcb). A typical PSII supercomplex in plants is formed by four trimers of Lhcb proteins (LHCII trimers), which are bound to the PSII core dimer via monomeric antenna proteins. However, the architecture of PSII supercomplexes in Norway spruce[Picea abies (L.) Karst.] is different, most likely due to a lack of two Lhcb proteins, Lhcb6 and Lhcb3. Interestingly, the spruce PSII supercomplex shares similar structural features with its counterpart in the green alga Chlamydomonas reinhardtii [Kouřil et al. (2016) New Phytol. 210, 808-814]. Here we present a single-particle electron microscopy study of isolated PSII supercomplexes from Norway spruce that revealed binding of a variable amount of LHCII trimers to the PSII core dimer at positions that have never been observed in any other plant species so far. The largest spruce PSII supercomplex, which was found to bind eight LHCII trimers, is even larger than the current largest known PSII supercomplex from C. reinhardtii. We have also shown that the spruce PSII supercomplexes can form various types of PSII megacomplexes, which were also identified in intact grana membranes. Some of these large PSII supercomplexes and megacomplexes were identified also in Pinus sylvestris, another representative of the Pinaceae family. The structural variability and complexity of LHCII organization in Pinaceae seems to be related to the absence of Lhcb6 and Lhcb3 in this family, and may be beneficial for the optimization of light-harvesting under varying environmental conditions.

• Klíčová slova
• Picea abies, Pinus sylvestris, clear native polyacrylamide electrophoresis, grana membrane, megacomplex, photosystem II, single-particle electron microscopy, supercomplex,
• MeSH
• fotosystém II (proteinový komplex) chemie   metabolismus   MeSH
• smrk metabolismus   MeSH
• světlosběrné proteinové komplexy chemie   metabolismus   MeSH
• terciární struktura proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fotosystém II (proteinový komplex) MeSH
• světlosběrné proteinové komplexy 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

α-Galactosidases are assigned to the class of hydrolases and the subclass of glycoside hydrolases (GHs). They belong to six GH families and include the only characterized α-galactosidases from yeasts (GH 27, Saccharomyces cerevisiae). The present study focuses on an investigation of the lactose-inducible α-galactosidase produced by Papiliotrema flavescens. The enzyme was present on the surface of cells and in the cytosol. Its temperature optimum was about 60 °C and the pH optimum was 4.8; the pH stability ranged from 3.2 to 6.6. This α-galactosidase also exhibited transglycosylation activity. The cytosol α-galactosidase with a molecular weight about 110 kDa, was purified using a combination of liquid chromatography techniques. Three intramolecular peptides were determined by the partial structural analysis of the sequences of the protein isolated, using MALDI-TOF/TOF mass spectrometry. The data obtained recognized the first yeast α-galactosidase, which belongs to the GH 36 family. The bioinformatics analysis and homology modeling of a 210 amino acids long C-terminal sequence (derived from cDNA) confirmed the correctness of these findings. The study was also supplemented by the screening of capsular cryptococcal yeasts, which produce the surface lactose-inducible α- and β-galactosidases. The production of the lactose-inducible α-galactosidases was not found to be a general feature within the yeast strains examined and, therefore, the existing hypothesis on the general function of this enzyme in cryptococcal capsule rearrangement cannot be confirmed.

• Klíčová slova
• Cryptococcus flavescens, GH 36 family, Lactose-inducible, Long-chain, Papiliotrema flavescens, α-Galactosidase,
• MeSH
• alfa-galaktosidasa chemie   genetika   izolace a purifikace   metabolismus   MeSH
• Basidiomycota klasifikace   enzymologie   genetika   růst a vývoj   MeSH
• Cryptococcus MeSH
• cytosol enzymologie   MeSH
• DNA fungální genetika   MeSH
• fungální proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• geny hub genetika   MeSH
• glykosidhydrolasy metabolismus   MeSH
• komplementární DNA MeSH
• koncentrace vodíkových iontů MeSH
• konformace proteinů MeSH
• laktosa metabolismus   MeSH
• molekulární modely MeSH
• molekulová hmotnost MeSH
• sekvence aminokyselin MeSH
• sekvenční analýza proteinů MeSH
• sekvenční seřazení MeSH
• stabilita enzymů MeSH
• substrátová specifita MeSH
• teplota MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• alfa-galaktosidasa MeSH
• DNA fungální MeSH
• fungální proteiny MeSH
• glykosidhydrolasy MeSH
• komplementární DNA MeSH
• laktosa MeSH

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