Analytical and Bioanalytical Chemistry / Determination of true ratios of different N-glycan structures in electrospray ionization mass spectrometry

Nicht verf\ufcgbarAn ideal method for the analysis of N-glycans would both identify the isomeric structure and deliver a true picture of the relative, if not absolute, amounts of the various structures in one sample. Porous graphitic carbon chromatography coupled with electrospray ionization mass spectrometry (ESI-MS) detection has emerged as a method with a particularly high potential of resolving isomeric oligosaccharides, but little attention has so far been paid to quantitation of the results obtained. In this work, we isolated a range of structures from Man5 to complex type N-glycans with zero to four sialic acids and blended them into an equimolar \u201cglyco tune mix\u201d. When subjected to liquid chromatography\u2013ESI-MS in positive and negative modes, the glyco tune mix clearly demonstrated the futility of quantitation of N-glycans of different overall composition, different number of sialic acids, and strongly differing size without compensation for their very different molar responses. Relative quantitation of human plasma N-glycans was performed with correction factors deduced from this external glyco tune mix. Addition of just one isotope-coded internal standard with enzymatically added 13C-galactose led to absolute quantification in the same experiment

The borderlands of waking:quantifying the transition from reflective thought to hallucination in sleep onset

We lose waking consciousness spontaneously and regularly over the circadian cycle. It seems that every time we fall asleep, reflective thinking gradually gives way to our interactions with an imaginary, hallucinatory world that brings multimodal experiences in the absence of adequate external stimuli. The present study investigates this transition, proposing a new measure of hallucinatory states. Reflective thinking and motor imagery were quantified in 150 mentation reports provided by 16 participants after forced awakenings from different physiology-monitored time intervals after sleep onset. Cognitive agency analysis and motor agency analysis – which are objective (grammatical–semantic) tools derived from linguistic theories – show (i) a decrease in reflective thinking which sleepers would need to acknowledge the hallucinatory quality of their state, and (ii) an increase in motor imagery, indicating interactions with a hallucinatory world. By mapping these spontaneous changes in human consciousness onto physiology, we can in the long run explore the conditions of its decline, and possibilities for treatment

„Cztery kamienie to wdzięczność ojczyzny” – pomniki poświęcone poległym żołnierzom versus kultura pamięci w Górnej Austrii od czasów pierwszej wojny światowej do współczesności

Kriegerdenkmäler spiegeln auf besondere Weise sowohl Brüche als auch Kontinuitäten wider, welche die politischen Umwälzungen des Ersten Weltkrieges, des Dritten Reichs sowie der Nachkriegszeit in der österreichischen Erinnerungskultur hinterlassen haben. Dieser Beitrag widmet sich einer Reihe von Denkmälern einer spezifischen Region in Oberösterreich. Die Analyse der Inschriften führt den ambivalenten und oberflächlich neutralen Charakter von Kriegerdenkmälern vor Augen: Deren sprachliche Botschaften lassen zwischen den Extrempolen ‚Helden‘ und ‚Opfer‘ bis heute kaum Platz für gesellschaftlich schonungslose und gleichzeitig historisch reflektierte Perspektiven auf die Gewaltexzesse des 20. Jahrhunderts.War memorials reflect in a special way both the breaks and continuities which the political upheavals of the First World War, the Third Reich and the post-war period left in the Austrian culture of remembrance. In this article, the main focus is on a sample of war memorials in a specific region of Upper Austria. An analysis of the inscriptions shows the ambivalence and superficial neutrality characteristic of war memorials: Their messages based on the extreme poles of ‘heroes’ and ‘victims’, leave little space for socially direct and historically reflective perspectives on the violent excesses of the 20th century.Pomniki poległych odzwierciedlają w szczególny sposób zarówno trwałość jak i niestabilność spuścizny, jaką pozostawiły w austriackiej kulturze pamięci polityczne zawirowania okresu pierwszej wojny światowej, Trzeciej Rzeszy a także okresu po drugiej wojnie światowej. Artykuł poświęcony jest pomnikom specyficznego regionu: Górnej Austrii. Analiza inskrypcji nagrobnych unaocznia zarówno powierzchowność pamięci zbiorowej jak i ambiwalentny charakter pomników poległych żołnierzy. Ich językowe przesłania – nadające żołnierzom ekstremalne funkcje, oscylujące pomiędzy rolą ‘bohaterów’ i ‘ofiar’ – do dnia dzisiejszego nie dopuszczają społecznie koniecznej i jednocześnie historycznie pogłębionej refleksji o zbrodniach XX wieku

Transcranial direct current stimulation of the motor cortex in waking resting state induces motor imagery

This study investigates if anodal and cathodal transcranial direct current stimulation (tDCS) of areas above the motor cortex (C3) influences spontaneous motor imagery experienced in the waking resting state. A randomized triple-blinded design was used, combining neurophysiological techniques with tools of quantitative mentation report analysis from cognitive linguistics. The results indicate that while spontaneous motor imagery rarely occurs under sham stimulation, general and athletic motor imagery (classified as athletic disciplines), is induced by anodal tDCS. This insight may have implications beyond basic consciousness research. Motor imagery and corresponding motor cortical activation have been shown to benefit later motor performance. Electrophysiological manipulations of motor imagery could in the long run be used for rehabilitative tDCS protocols benefitting temporarily immobile clinical patients who cannot perform specific motor imagery tasks – such as dementia patients, infants with developmental and motor disorders, and coma patients.<br/

Effects of N-glycans on the structure of human IgA2

The transition of IgA antibodies into clinical development is crucial because they have the potential to create a new class of therapeutics with superior pathogen neutralization, cancer cell killing, and immunomodulation capacity compared to IgG. However, the biological role of IgA glycans in these processes needs to be better understood. This study provides a detailed biochemical, biophysical, and structural characterization of recombinant monomeric human IgA2, which varies in the amount/locations of attached glycans. Monomeric IgA2 antibodies were produced by removing the N-linked glycans in the CH1 and CH2 domains. The impact of glycans on oligomer formation, thermal stability, and receptor binding was evaluated. In addition, we performed a structural analysis of recombinant IgA2 in solution using Small Angle X-Ray Scattering (SAXS) to examine the effect of glycans on protein structure and flexibility. Our results indicate that the absence of glycans in the Fc tail region leads to higher-order aggregates. SAXS, combined with atomistic modeling, showed that the lack of glycans in the CH2 domain results in increased flexibility between the Fab and Fc domains and a different distribution of open and closed conformations in solution. When binding with the Fcα-receptor, the dissociation constant remains unaltered in the absence of glycans in the CH1 or CH2 domain, compared to the fully glycosylated protein. These results provide insights into N-glycans' function on IgA2, which could have important implications for developing more effective IgA-based therapeutics in the future

Engineering vacuolar sorting pathways for efficient secretion of recombinant proteins

Recombinant protein production is an expanding branch of biotechnology with increasing economic importance. Currently, 20% of biopharmaceutical proteins and approximately half of the industrial enzymes are produced in yeasts. Many proteins are efficiently secreted by yeast systems, reaching product titers in the g L-1 range. The expression of more complex proteins, however, may overwhelm the folding and secretion capacity of the host cells. This triggers the unfolded protein response (UPR), which aims at restoring endoplasmic reticulum (ER) homeostasis. The UPR, in turn, is thought to activate ER-associated protein degradation (ERAD). Alternatively, trafficking of correctly folded proteins can be hampered on their way to the cell exterior leading e.g. to missorting and subsequent degradation in the vacuole. The methylotrophic yeast Pichia pastoris (Komagataella spp.) is a popular microbial host for the production of recombinant proteins. Vacuolar protein sorting has not been investigated in detail so far in P. pastoris, although there were a few indications that vacuolar mistargeting of recombinant products might occur also in this yeast. Thus we engineered the vacuolar sorting pathways in P. pastoris and investigated their impact on extracellular product titers as well as intracellular localization of the recombinant secretory product. Thereby, differences between vps (vacuolar protein sorting) mutant strains disrupted in genes involved either in the CORVET or the HOPS tethering complexes became obvious. Moreover, we were able to show that engineering of the vacuolar sorting pathways has a positive impact on heterologous protein secretion, however, in some cases simultaneous inactivation of specific vacuolar proteases was necessary. Taken together, these studies allowed us to gain deeper insight into the pathways leading to intracellular degradation of recombinant secretory proteins. Based on these findings, approaches how to efficiently adapt the host cell’s secretion capacity will be presented, which confirm that impairment of vacuolar protein sorting is an effective means of enhancing secretion of heterologous proteins

Engineering, expression in transgenic plants and characterisation of e559, a rabies virus-neutralising monoclonal antibody.

Rabies post-exposure prophylaxis (PEP) currently comprises administration of rabies vaccine together with rabies immunoglobulin (RIG) of either equine or human origin. In the developing world, RIG preparations are expensive, often in short supply, and of variable efficacy. Therefore, we are seeking to develop a monoclonal antibody cocktail to replace RIG. Here, we describe the cloning, engineering and production in plants of a candidate monoclonal antibody (E559) for inclusion in such a cocktail. The murine constant domains of E559 were replaced with human IgG1κ constant domains and the resulting chimeric mouse-human genes were cloned into plant expression vectors for stable nuclear transformation of Nicotiana tabacum. The plant-expressed, chimeric antibody was purified and biochemically characterized, was demonstrated to neutralize rabies virus in a fluorescent antibody virus neutralization assay, and conferred protection in a hamster challenge model

Systems-level organization of yeast methylotrophic lifestyle

BACKGROUND: Some yeasts have evolved a methylotrophic lifestyle enabling them to utilize the single carbon compound methanol as a carbon and energy source. Among them, Pichia pastoris (syn. Komagataella sp.) is frequently used for the production of heterologous proteins and also serves as a model organism for organelle research. Our current knowledge of methylotrophic lifestyle mainly derives from sophisticated biochemical studies which identified many key methanol utilization enzymes such as alcohol oxidase and dihydroxyacetone synthase and their localization to the peroxisomes. C1 assimilation is supposed to involve the pentose phosphate pathway, but details of these reactions are not known to date. RESULTS: In this work we analyzed the regulation patterns of 5,354 genes, 575 proteins, 141 metabolites, and fluxes through 39 reactions of P. pastoris comparing growth on glucose and on a methanol/glycerol mixed medium, respectively. Contrary to previous assumptions, we found that the entire methanol assimilation pathway is localized to peroxisomes rather than employing part of the cytosolic pentose phosphate pathway for xylulose-5-phosphate regeneration. For this purpose, P. pastoris (and presumably also other methylotrophic yeasts) have evolved a duplicated methanol inducible enzyme set targeted to peroxisomes. This compartmentalized cyclic C1 assimilation process termed xylose-monophosphate cycle resembles the principle of the Calvin cycle and uses sedoheptulose-1,7-bisphosphate as intermediate. The strong induction of alcohol oxidase, dihydroxyacetone synthase, formaldehyde and formate dehydrogenase, and catalase leads to high demand of their cofactors riboflavin, thiamine, nicotinamide, and heme, respectively, which is reflected in strong up-regulation of the respective synthesis pathways on methanol. Methanol-grown cells have a higher protein but lower free amino acid content, which can be attributed to the high drain towards methanol metabolic enzymes and their cofactors. In context with up-regulation of many amino acid biosynthesis genes or proteins, this visualizes an increased flux towards amino acid and protein synthesis which is reflected also in increased levels of transcripts and/or proteins related to ribosome biogenesis and translation. CONCLUSIONS: Taken together, our work illustrates how concerted interpretation of multiple levels of systems biology data can contribute to elucidation of yet unknown cellular pathways and revolutionize our understanding of cellular biology. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12915-015-0186-5) contains supplementary material, which is available to authorized users

Absolute and relative quantitation of amylase/trypsin-inhibitors by LC-MS/MS from wheat lines obtained by CRISPR-Cas9 and RNAi

Quantitation of wheat proteins is still a challenge, especially regarding amylase/trypsin-inhibitors (ATIs). A selection of ATIs was silenced in the common wheat cultivar Bobwhite and durum wheat cultivar Svevo by RNAi and gene editing, respectively, in order to reduce the amounts of ATIs. The controls and silenced lines were analyzed after digestion to peptides by LC-MS/MS with different approaches to evaluate changes in composition of ATIs. First, a targeted method with stable isotope dilution assay (SIDA) using labeled peptides as internal standards was applied. Additionally, four different approaches for relative quantitation were conducted, in detail, iTRAQ labeled and label free quantitation (LFQ) combined with data dependent acquisition (DDA) and data independent acquisition (DIA). Quantitation was performed manually (Skyline and MASCOT) and with different proteomics software tools (PLGS, MaxQuant, and PEAKS X Pro). To characterize the wheat proteins on protein level, complementary techniques as high-performance liquid chromatography (HPLC) and gel electrophoresis were performed. The targeted approach with SIDA was able to quantitate all ATIs, even at low levels, but an optimized extraction is necessary. The labeled iTRAQ approach revealed an indistinct performance. LFQ with low resolution equipment (IonTrap) showed similar results for major ATIs, but low abundance ATIs as CM1, were not detectable. DDA measurements with an Orbitrap system and evaluation using MaxQuant showed that the relative quantitation was dependent on the wheat species. The combination of manual curation of the MaxQuant search with Skyline revealed a very good performance. The DIA approach with analytical flow found similar results compared to absolute quantitation except for some minor ATIs, which were not detected. Comparison of applied methods revealed that peptide selection is a crucial step for protein quantitation. Wheat proteomics faces challenges due to the high genetic complexity, the close relationship to other cereals and the incomplete, redundant protein database requiring sensitive, precise and accurate LC-MS/MS methods