Biology subjects

Pabst, M.

Publications and source records attributed to Pabst, M..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Full-Length Single-Molecule Protein Fingerprinting

Proteins are the primary functional actors of the cell. Hence, their identification is pivotal to advance our understanding of cell biology and disease. Current protein analysis methods are of limited use for distinguishing proteoforms. In particular, mass spectrometric methods often provide only ambiguous information on post-translational modification sites, and sequences of co-existing modifications may not be resolved. Here we demonstrate FRET-based single-molecule protein fingerprinting to map the location of individual amino acids and a post-translational modification within single full-length protein molecules. Using an approach that relies on transient binding of fluorescently labeled DNA strands to probe the amino acids on a protein one by one we show that we can fingerprint intrinsically disordered proteins as well as folded globular proteins with sub-nanometer resolution. We anticipate that this technology will be used for proteoform identification in biological and translational research with ultimate sensitivity.

biophysics↗

bioRxiv2023

Aerobic denitrification as N2O source in microbial communities

Nitrous oxide (N2O) is a potent greenhouse gas of primarily microbial origin. Aerobic and anoxic emissions are commonly ascribed to nitrification and denitrification, respectively. Beyond this established dichotomy, we quantitatively prove that heterotrophic denitrification can significantly contribute to aerobic nitrogen turnover and N2O emissions in complex microbiomes exposed to frequent oxic/anoxic transitions. Planktonic, nitrification-inhibited denitrifying enrichments respired over a third of the influent organic substrate with nitrate at high oxygen concentrations. N2O accounted for up to one quarter of the aerobically respired nitrate. The constitutive detection of all denitrification enzymes in both anoxic and oxic periods highlight the selective advantage offered by metabolic preparedness in dynamic environments. We posit that aerobic denitrification and associated N2O formation is currently underestimated in dynamic microbial ecosystems.

microbiology↗

bioRxiv2022

Meta-omics profiling of full-scale groundwater rapid sand filters explains stratification of iron, ammonium and manganese removals

Rapid sand filters (RSF) are an established and widely applied technology for groundwater treatment. Yet, the underlying interwoven biological and physical-chemical reactions controlling the sequential removal of iron, ammonia and manganese remain poorly understood. To resolve the contribution and interactions between the individual reactions, we studied two full-scale drinking water treatment plant configurations, namely (i) one dual-media (anthracite and quartz sand) filter and (ii) two single-media (quartz sand) filters in series. In situ and ex situ activity tests were combined with mineral coating characterization and metagenome-guided metaproteomics along the depth of each filter. Both plants exhibited comparable performances and process compartmentalization, with most of ammonium and manganese removal occurring only after complete iron depletion. Within each compartment, the homogeneity of the media coating and genome-based microbial composition highlighted the effect of backwashing on filter media mixing. In stark contrast, intra-compartment contaminant removal was highly stratified following decreasing substrate availability along the filter height. This apparent and long-standing conflict was resolved by quantifying the expressed proteome at different filter heights, revealing a consistent stratification of proteins catalysing ammonia oxidation and protein-based relative abundances of nitrifying genera. This implies that microorganisms adapt their protein pool to the available nutrient load at a faster rate than the backwash mixing frequency. Ultimately, these results show the unique and complementary potential of metaproteomics to understand metabolic adaptations and interactions in highly dynamic ecosystems.

microbiology↗

bioRxiv2022

Comparative proteome analysis of different Saccharomyces cerevisiae strains during growth on sucrose and glucose

Both the identity and the amount of a carbon source present in laboratory or industrial cultivation media have major impacts on the growth and physiology of a microbial species. In the case of the yeast Saccharomyces cerevisiae, sucrose is arguably the most important sugar used in industrial biotechnology, whereas glucose is the most common carbon and energy source used in research, with many well-known and described regulatory effects, e.g. glucose repression. Here we compared the label-free proteomes of exponentially growing S. cerevisiae cells in a defined medium containing either sucrose or glucose as the sole carbon source. For this purpose, bioreactor cultivations were employed, and three different strains were investigated, namely: CEN.PK113-7D (a common laboratory strain), UFMG-CM-Y259 (a wild isolate), and JP1 (an industrial bioethanol strain). These strains present different physiologies during growth on sucrose; some of them reach higher specific growth rates on this carbon source, when compared to growth on glucose, whereas others display the opposite behavior. It was not possible to identify proteins that commonly presented either higher or lower levels during growth on sucrose, when compared to growth on glucose, considering the three strains investigated here, except for one protein, named Mnp1 - a mitochondrial ribosomal protein of the large subunit, which had higher levels on sucrose than on glucose, for all three strains. Interestingly, following a Gene Ontology overrepresentation and KEGG pathway enrichment analyses, an inverse pattern of enriched biological functions and pathways was observed for the strains CEN.PK113-7D and UFMG-CM-Y259, which is in line with the fact that whereas the CEN.PK113-7D strain grows faster on glucose than on sucrose, the opposite is observed for the UFMG-CM-Y259 strain.

systems biology↗

bioRxiv2022

Proteome dynamics during transition from exponential to stationary phase under aerobic and anaerobic conditions in yeast

The yeast Saccharomyces cerevisiae is a widely used eukaryotic model organism and a promising cell factory for industry. However, despite decades of research, the regulation of its metabolism is not yet fully understood, and its complexity represents a major challenge for engineering and optimising biosynthetic routes. Recent studies have demonstrated the potential of resource and proteomic allocation data in enhancing models for metabolic processes. However, comprehensive and accurate proteome dynamics data that can be used for such approaches are still very limited. Therefore, we performed a quantitative proteome dynamics study to comprehensively cover the transition from exponential to stationary phase for both aerobically and anaerobically grown yeast cells. The combination of highly controlled reactor experiments, biological replicates and standardised sample preparation procedures ensured reproducibility and accuracy. Additionally, we selected the CEN.PK lineage for our experiments because of its relevance for both fundamental and applied research. Together with the prototrophic, standard haploid strain CEN.PK113-7D, we also investigated an engineered strain with genetic minimisation of the glycolytic pathway, resulting in the quantitative assessment of over 1700 proteins across 54 proteomes. These proteins account for nearly 40% of the overall yeast proteome and approximately 99% of the total protein biomass. The anaerobic cultures showed remarkably less proteome-level changes compared to the aerobic cultures, during transition from the exponential to the stationary phase as a consequence of the lack of the diauxic shift in the absence of oxygen. These results support the notion that anaerobically growing cells lack time and resources to adapt to changes in the environment. This proteome dynamics study constitutes an important step towards better understanding of the impact of glucose exhaustion and oxygen on the complex proteome allocation process in yeast. Finally, the established proteome dynamics data provide a valuable resource for the development of resource allocation models as well as for metabolic engineering efforts.

systems biology↗

bioRxiv2022

Autoinsertion (LAiR): rapid functional reconstitution of integral membrane proteins into lipid bilayers

Functional investigation of purified integral membrane proteins (IMPs) is hampered by the need to insert these hydrophobic proteins from the detergent-solubilized state into liposomal membranes. Here we report reintegration of IMPs into a lipid environment within minutes, an order of magnitude faster than currently used standard techniques. The new approach yielded optimal results for IMPs solubilized in the detergent lauryl-maltose neopentyl glycol (LMNG) and is therefore termed LMNG Auto-insertion Reintegration (LAiR). LAiR displays superior performance to standard methods in terms of protein activity, long-term stability and proton tightness of proteoliposomes. LAiR reconstituted vectorial control of membrane-bound activity by the transmembrane ion motive force, a property particularly important in mitochondrial function, which was undetectable by standard reintegration methods. LAiR also preserved fragile IMP properties that are prone to disruption upon reintegration, including long-term multi-subunit integrity, inhibitor susceptibility, and higher-order oligomeric states. LAiR proved suitable for reintegration into liposomes as well as into surface-tethered membrane bilayers, and was compatible with IMPs and lipids from prokaryotic and eukaryotic sources. We anticipate a broad scope for LAiR as a powerful tool in fundamental research, pharmaceutical applications, and biotechnology.

biochemistry↗

bioRxiv2022

Enrichment and application of bacterial sialic acids containing polymers from the extracellular polymeric substances of Candidatus Accumulibacter

Pseudaminic and legionaminic acids are a subgroup of nonulosonic acids (NulOs) unique to bacterial species. There is a lack of advances in the study of these NulOs due to their complex synthesis and production. Recently, it was seen that "Candidatus Accumulibacter" can produce Pse or Leg analogues as part of its extracellular polymeric substances (EPS). In order to employ a "Ca. Accumulibacter" enrichment as production platform for bacterial sialic acids, it is necessary to determine which fractions of the EPS of "Ca. Accumulibacter" contain NulOs and how to enrich and/or isolate them. We extracted the EPS from granules enriched with "Ca. Accumulibcater" and used size-exclusion chromatography to separate them into different molecular weight fractions. This separation resulted in two high molecular weight (> 5,500 kDa) fractions dominated by polysaccharides, with a NulO content up to 4 times higher than the extracted EPS. This suggests that NulOs in "Ca. Accumulibacter" are likely located in high molecular weight polysaccharides. Additionally, it was seen that the extracted EPS and the NulO-rich fractions can bind and neutralize histones. This suggest that they can serve as source for sepsis treatment drugs, although further purification needs to be evaluated. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/508216v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@1f59ff7org.highwire.dtl.DTLVardef@d933fcorg.highwire.dtl.DTLVardef@1e3ff78org.highwire.dtl.DTLVardef@1994948_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LINulOs in "Ca. Accumulibacter" are likely located in high molecular weight polysaccharides. C_LIO_LISize exclusion chromatography allows to obtain high molecular weight polysaccharide-rich fractions enriched with NulOs. C_LIO_LIEPS and the NulOs-rich fractions can serve as source for sepsis treatment drugs. C_LI

microbiology↗

bioRxiv2022

Turnover of the extracellular polymeric matrix in an EBPR microbial community

Polyphosphate accumulating organisms (PAOs) are responsible for enhanced biological phosphate removal (EBPR) from wastewater, where they grow embedded in a matrix of extracellular polymeric substances (EPS). EPS comprise a mixture of biopolymers like polysaccharides or (glyco)proteins. Despite previous studies, little is known about the dynamics of EPS in mixed cultures, and their production by PAOs and potential consumption by flanking microbes. EPS are biodegradable and have been suggested to be a substrate for other organisms in the community. Studying EPS turnover can help elucidate their biosynthesis and biodegradation cycles. We analyzed the turnover of proteins and polysaccharides in the EPS of an enrichment culture of PAOs relative to the turnover of internal proteins. An anaerobic-aerobic sequencing batch reactor (SBR) simulating EBPR conditions was operated to enrich for PAOs. After achieving a stable culture, carbon source was switched to uniformly 13C-labelled acetate. Samples were collected at the end of each aerobic phase. EPS were extracted by alkaline treatment. 13C enrichment in proteins and sugars (after hydrolysis of polysaccharides) in the extracted EPS were measured by mass spectrometry. The average turnover rate of sugars and proteins (0.167 and 0.192 d-1 respectively) was higher than the expected value based on the solid removal rate (0.132 d-1), and no significant difference was observed between intracellular and secreted proteins. This indicates that EPS from the PAO enriched community is not selectively degraded by flanking populations under stable EBPR process conditions. Instead, we observed general decay of biomass, which corresponds to a value of 0.048 d-1. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/503576v1_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@925680org.highwire.dtl.DTLVardef@16d15ccorg.highwire.dtl.DTLVardef@b23a26org.highwire.dtl.DTLVardef@107d2b1_HPS_FORMAT_FIGEXP M_FIG C_FIG Key points- Proteins showed a higher turnover rate than carbohydrates. - Turnover of EPS was similar to the turnover of intracellular proteins. - EPS is not preferentially consumed by flanking populations.

microbiology↗

bioRxiv2022

Extracting and characterizing protein-free megabasepair DNA for in vitro experiments

Chromosome structure and function is studied in cells using imaging and chromosome-conformation-based methods as well as in vitro with a range of single-molecule techniques. Here we present a method to obtain genome-size (megabasepair length) deproteinated DNA for in vitro studies, which provides DNA substrates that are two orders of magnitude longer than typically studied in single-molecule experiments. We isolated chromosomes from bacterial cells and enzymatically digested the native proteins. Mass spectrometry indicated that 97-100% of DNA-binding proteins are removed from the sample. Upon protein removal, we observed an increase in the radius of gyration of the DNA polymers, while quantification of the fluorescence intensities showed that the length of the DNA objects remained megabasepair sized. In first proof-of-concept experiments using these deproteinated long DNA molecules, we observed DNA compaction upon adding the DNA-binding protein Fis or PEG crowding agents and showed that it is possible to track the motion of a fluorescently labelled DNA locus. These results indicate the practical feasibility of a genome-in-a-box approach to study chromosome organization from the bottom up.

biophysics↗

bioRxiv2022

Comparative metaproteomics demonstrates different views on the complex granular sludge microbiome

The tremendous progress in sequencing technologies has made 16S amplicon and whole metagenome sequencing routine in microbiome studies. Furthermore, advances in mass spectrometric techniques has expanded conventional proteomics into the field of microbial ecology. Commonly referred to as metaproteomics, this approach measures the gene products (i.e., proteins) to subsequently identify the actively-expressed metabolic pathways and the protein-biomass composition of complete microbial communities. However, more systematic studies on metaproteomic and genomic approaches are urgently needed, to determine the orthogonal character of these approaches. Here we describe a deep, comparative metaproteomic study on the complex aerobic granular sludge microbiome obtained from different wastewater treatment plants. Thereby, we demonstrate the different views that can be obtained on the central nutrient-removing organisms depending on the omic approach and reference sequence databases. Furthermore, we demonstrate a homogenized Genome Taxonomy Database (GTDB) that subsequently enables a more accurate interpretation of data from different omics approaches. Ultimately, our systematic study underscores the importance of metaproteomics in the characterization of complex microbiomes; and the necessity of accurate reference sequence databases to improve the comparison between approaches and accuracy in scientific reporting.

systems biology↗

bioRxiv2022

Comparison of two sampling, quenching and extraction methods for quantitative yeasts metabolomics

Knowing intracellular metabolite concentrations is important for fundamental metabolism research as well as for biotechnology. The first steps in a quantitative metabolomics workflow, i.e., sampling, quenching, and extraction, are key to obtaining unbiased quantitative data. A qualified quenching and extraction method not only needs to rapidly terminate the in vivo biochemical reaction activities to preserve the endogenous metabolite levels but also has to fully extract all metabolites from cells. Recently, two different filtration-based sampling, quenching, and extraction methods have been proposed and used for quantitative yeast metabolomics. One method integrates the quenching and extraction into one step using a methanol-acetonitrile-water solution after a filtration step, while the other --more conventional-- method quenches with cold methanol and extracts with boiling ethanol. In this study, we tested whether these two methods are equally well suited for quantitative metabolome analyses with yeast. Using isotope dilution mass spectrometry (IDMS) with GC-MS and LC-MS as analytical methods, in combination with precise quantification of cell volumes, we determined absolute concentrations of 63 intracellular metabolites covering amino acids, organic acids, phosphorylated sugars, and nucleotides in two S. cerevisiae strains with different physiology. By analyzing the data from samples generated with the two methods, we found that while both methods yielded essentially identical concentrations for amino and organic acids, the cold-solvent extraction yielded significantly lower concentrations for particularly phosphorylated sugars and nucleotides, presumably because of lower quenching or extraction efficiency of this method. Our results show that methanol-quenching combined with boiling-ethanol-extraction is the more accurate approach when aiming to quantify a broad range of different metabolites from yeast cells. The significant discrepancy observed between both common metabolite extraction methods demonstrates the importance of method optimization for quantitative studies in particular when working with microbes with rigid cell walls such as those found in yeast.

systems biology↗

bioRxiv2022

A systematic evaluation of yeast sample preparation protocols for spectral identifications, proteome coverage and post-isolation modifications

The importance of obtaining comprehensive and accurate information from cellular proteomics experiments asks for a systematic investigation of sample preparation protocols, particularly when working with unicellular organisms with strong cell walls, such as found in the model organism and cell factory S. cerevisiae. Sample preparation protocols may bias towards specific protein fractions or challenge the analysis of native protein modifications due to reagent-induced artefacts. Here, we performed a systematic comparison of sample preparation protocols using a matrix of different conditions commonly applied in whole cell lysate proteomics. The different protocols were evaluated for their overall fraction of identified spectra, proteome and amino acid sequence coverage, GO-term distribution and number of peptide modifications, by employing a combination of database and unrestricted modification search approaches. The best proteome and amino acid sequence coverage was achieved by using Urea combined with filter-aided or in-solution digestion protocols, where the overall outcomes were strongly influenced by the employed quenching procedure. Most importantly, the use of moderate incubation temperatures and times, circumvented excessive formation of modification artefacts. Extensive reagent-induced peptide modifications, however, were observed when using solvents such as acetone or additives such as formic acid. Moreover, several filter material-related modifications were observed when employing the filter-aided procedures. Ultimately, the best protocols enabled the identification of approximately 65-70% of all acquired fragmentation spectra, where additional de novo sequencing suggests that unidentified spectra were largely of too low spectral quality to provide confident spectrum matches. This study demonstrates the large impact of different sample preparation procedures on the proteomic analysis outcome, where the collected protocols and large sets of associated mass spectrometric raw data provide a resource to evaluate and design new protocols and guide the analysis of (native) peptide modifications in the model eukaryote yeast.

systems biology↗

bioRxiv2021

Looking for lipases and lipolytic organisms in low-temperature anaerobic reactors treating domestic wastewater

Poor lipid degradation limits low-temperature anaerobic treatment of domestic wastewater even when psychrophiles are used. We combined metagenomics and metaproteomics to find lipolytic bacteria and their potential, and actual, cold-adapted extracellular lipases in anaerobic membrane bioreactors treating domestic wastewater at 4{degrees}C and 15{degrees}C. Of the 40 recovered putative lipolytic metagenome-assembled genomes (MAGs), only three (Chlorobium, Desulfobacter, and Mycolicibacterium) were common and abundant (relative abundance [≥] 1%) in all reactors. Notably, some MAGs that represented aerobic autotrophs contained lipases. Therefore, we hypothesised that the lipases we found are not always associated with exogenous lipid degradation and can have other roles such as polyhydroxyalkanoates (PHA) accumulation/degradation and interference with the outer membranes of other bacteria. Metaproteomics did not provide sufficient proteome coverage for relatively lower abundant proteins such as lipases though the expression of fadL genes, long-chain fatty acid transporters, was confirmed for four genera (Dechloromonas, Azoarcus, Aeromonas and Sulfurimonas), none of which were recovered as putative lipolytic MAGs. Metaproteomics also confirmed the presence of 15 relatively abundant ([≥]1%) genera in all reactors, of which at least 6 can potentially accumulate lipid/polyhydroxyalkanoates. For most putative lipolytic MAGs, there was no statistically significant correlation between the read abundance and reactor conditions such as temperature, phase (biofilm and bulk liquid), and feed type (treated by ultraviolet light or not). Results obtained by metagenomics and metaproteomics did not confirm each other and further work is required to identify the true lipid degraders in these systems.

molecular biology↗

bioRxiv2021

Functional single cell proteomic profiling of cells with abnormal DNA damage response dynamics

Tumor heterogeneity is an important source of cancer therapy resistance. Single cell proteomics has the potential to decipher protein content leading to heterogeneous cellular phenotypes. Single-Cell ProtEomics by Mass Spectrometry (SCoPE-MS) is a recently developed, promising, unbiased proteomic profiling techniques, which allows profiling several tens of single cells for >1000 proteins per cell. However, a method to link single cell proteomes with cellular behaviors is needed to advance this type of profiling technique. Here, we developed a microscopy-based functional single cell proteomic profiling technology, called FUNpro, to link the proteome of individual cells with phenotypes of interest, even if the phenotypes are dynamic or the cells of interest are sparse. FUNpro enables one i) to screen thousands of cells with subcellular resolution and monitor (intra)cellular dynamics using a custom-built microscope, ii) to real-time analyze (intra)cellular dynamics of individual cells using an integrated cell tracking algorithm, iii) to promptly isolate the cells displaying phenotypes of interest, and iv) to single cell proteomically profile the isolated cells. We applied FUNpro to proteomically profile a newly identified small subpopulation of U2OS osteosarcoma cells displaying an abnormal, prolonged DNA damage response (DDR) after ionizing radiation (IR). With this, we identified PDS5A and PGAM5 proteins contributing to the abnormal DDR dynamics and helping the cells survive after IR.

bioengineering↗

bioRxiv2021

Modular, synthetic chromosomes as new tools for large scale engineering of metabolism

The construction of powerful cell factories requires intensive genetic engineering for the addition of new functionalities and the remodeling of native pathways and processes. The present study demonstrates the feasibility of extensive genome reprogramming using modular, specialized de novo-assembled neochromosomes in yeast. The in vivo assembly of linear and circular neochromosomes, carrying 20 native and 21 heterologous genes, enabled the first de novo production in a microbial cell factory of anthocyanins, plant compounds with a broad range pharmacological properties. Turned into exclusive expression platforms for heterologous and essential metabolic routes, the neochromosomes mimic native chromosomes regarding mitotic and genetic stability, copy number, harmlessness for the host and editability by CRISPR/Cas9. This study paves the way for future microbial cell factories with modular genomes in which core metabolic networks, localized on satellite, specialized neochromosomes can be swapped for alternative configurations and serve as landing pads for the addition of functionalities.

bioengineering↗

bioRxiv2021

A yeast with muscle does not run faster: full humanization of the glycolytic pathway in Saccharomyces cerevisiae

While transplantation of single genes in yeast plays a key role in elucidating gene functionality in metazoans, technical challenges hamper the humanization of full pathways and processes. Empowered by advances in synthetic biology, this study demonstrates the feasibility and implementation of full humanization of glycolysis in yeast. Single gene and full pathway transplantation revealed the remarkable conservation of both glycolytic and moonlighting functions and, combined with evolutionary strategies, brought to light novel, context-dependent responses. Remarkably, human hexokinase 1 and 2, but not 4, required mutations in their catalytic or allosteric sites for functionality in yeast, while hexokinase 3 was unable to complement its yeast ortholog. Comparison with human tissues cultures showed the preservation of turnover numbers of human glycolytic enzymes in yeast and human cell cultures. This demonstration of transplantation of an entire, essential pathway paves the way to the establishment of species, tissue and disease-specific metazoan models. One Sentence SummaryThis work demonstrates the successful humanization of an entire pathway in Saccharomyces cerevisiae and establishes an attractive strategy to study (human) glycolysis architecture and regulation. HighlightsO_LIThe successful humanization of the entire glycolytic pathway in yeast offers new microbial models for both fundamental and applied studies. C_LIO_LIBoth glycolytic and moonlighting functions and turnover numbers of glycolytic enzymes are highly conserved between yeast and human. C_LIO_LIFunctionality of human hexokinases 1 and 2 in yeast requires mutations in the catalytic or allosteric binding sites. C_LIO_LICombination of single gene and full transplantation with laboratory evolution reveals context-dependent activity and evolution of glycolytic enzymes. C_LI

systems biology↗

bioRxiv2021

Adaptation of anammox bacteria to low temperature via gradual acclimation and cold shocks: distinctions in protein expression, membrane composition and activities

Anammox bacteria enable an efficient removal of nitrogen from sewage in processes involving partial nitritation and anammox (PN/A) or nitrification, partial denitrification, and anammox (N-PdN/A). In mild climates, anammox bacteria must be adapted to [≤]15 {degrees}C, typically by gradual temperature decrease; however, this takes months or years. To reduce the time necessary for the adaptation, an unconventional method of cold shocks is promising, involving hours-long exposure of anammox biomass to extremely low temperatures. We compared the efficacies of gradual temperature decrease and cold shocks to increase the metabolic activity of anammox (fed batch reactor, planktonic "Ca. Kuenenia"). We assessed the cold shock mechanism on the level of protein expression (quantitative shot-gun proteomics, LC-HRMS/MS) and structure of membrane lipids (UPLC-HRMS/MS). The shocked culture was more active (0.66{+/-}0.06 vs 0.48{+/-}0.06 kg-N/kg-VSS/d) and maintained the relative content of N-respiration proteins at levels consistent levels with the initial state, whereas the content of these proteins decreased in gradually acclimated culture. Cold shocks also induced a more efficient up-regulation of cold shock proteins (e.g. CspB, TypA, ppiD). Ladderane lipids characteristic for anammox evolved to a similar end-point in both cultures which confirms their role in anammox bacteria adaptation to cold and indicates a three-pronged adaptation mechanism involving ladderane lipids (ladderane alkyl length, introduction of shorter non-ladderane alkyls, polar headgroup). Overall, we show the outstanding potential of cold shocks for low-temperature adaptation of anammox bacteria and provide yet unreported detailed mechanisms of anammox adaptation to low temperatures. HighlightsO_LIAnammox bacteria were adapted to low T by gradual acclimation and cold shocks C_LIO_LIThe shocked culture was more active (0.66{+/-}0.06 vs 0.48{+/-}0.06 kg-N/kg-VSS/d) C_LIO_LIN-respiration proteins content decreased in gradually acclimated bacteria C_LIO_LISeveral cold shock proteins were upregulated more efficiently by cold shocks C_LIO_LIAt {downarrow}T, anammox adjusted ladderane membrane lipid composition in three aspects C_LI Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

bioengineering↗

bioRxiv2021

Time grid-based isomer specific N-glycan analysis and detection of bisecting Lewis X in human brain

The importance of protein glycosylation in the biomedical field demands for methods capable of resolving and identifying isomeric structures of N-glycans. However, the unambiguous identification of isomeric structures from complex mixtures is currently not reasonably realized even by the most sophisticated approaches. Here we present a novel approach which uses stable isotope labelled reference N-glycans to establish a retention time grid (glyco-TiGr) on porous graphitized carbon. This furthermore enables retention as the primary criterion for the structural assignment of isomeric N-glycans. Moreover, we biosynthesized forty natural isomers of the fundamental N-glycan type consisting of five hexoses, four N-acetylhexosamines and one fucose residue. Nearly all of these isomers occupied unique positions on the retention time grid. Reference glycan assisted retention time determination with deci-minute accuracy narrowed the assignment space to very few, often only one possible glycan isomer. Application of the glyco-TiGr approach revealed yet undescribed isomers of Lewis x determinants in multimeric human IgA and hybrid type N-glycans in human brain with galactose and even fucose linked to the bisecting N-acetylglucosamine. Thus, the brain N-glycome displayed a degree of sophistication commensurate with this organs role.

biochemistry↗