High-resolution longitudinal N- and O-glycoprofiling of human monocyte-to-macrophage transition.

Protein glycosylation impacts the development and function of innate immune cells. The glycophenotypes and the glycan remodelling associated with the maturation of macrophages from monocytic precursor populations remain incompletely described. Herein, label-free porous graphitised carbon-liquid chromatography-tandem mass spectrometry (PGC-LC-MS/MS) was employed to profile with high resolution the N- and O-glycome associated with human monocyte-to-macrophage transition. Primary blood-derived CD14+ monocytes were differentiated ex vivo in the absence of strong anti- and proinflammatory stimuli using a conventional 7-day granulocyte-macrophage colony-stimulating factor differentiation protocol with longitudinal sampling. Morphology and protein expression monitored by light microscopy and proteomics validated the maturation process. Glycomics demonstrated that monocytes and macrophages display similar N-glycome profiles, comprising predominantly paucimannosidic (Man1-3GlcNAc2Fuc0-1, 22.1-30.8%), oligomannosidic (Man5-9GlcNAc2, 29.8-35.7%) and α2,3/6-sialylated complex-type N-glycans with variable core fucosylation (27.6-39.1%). Glycopeptide analysis validated conjugation of these glycans to human proteins, while quantitative proteomics monitored the glycoenzyme expression levels during macrophage differentiation. Significant interperson glycome variations were observed suggesting a considerable physiology-dependent or heritable heterogeneity of CD14+ monocytes. Only few N-glycome changes correlated with the monocyte-to-macrophage transition across donors including decreased core fucosylation and reduced expression of mannose-terminating (paucimannosidic-/oligomannosidic-type) N-glycans in macrophages, while lectin flow cytometry indicated that more dramatic cell surface glycan remodelling occurs during maturation. The less heterogeneous core 1-rich O-glycome showed a minor decrease in core 2-type O-glycosylation but otherwise remained unchanged with macrophage maturation. This high-resolution glycome map underpinning normal monocyte-to-macrophage transition, the most detailed to date, aids our understanding of the molecular makeup pertaining to two vital innate immune cell types and forms an important reference for future glycoimmunological studies.

The Fine Art of Destruction: A Guide to In-Depth Glycoproteomic Analyses-Exploiting the Diagnostic Potential of Fragment Ions.

The unambiguous mass spectrometric identification and characterization of glycopeptides is crucial to elucidate the micro- and macroheterogeneity of glycoproteins. Here, combining lower and stepped collisional energy fragmentation for the in-depth and site-specific analysis of N- and O-glycopeptides is proposed. Using a set of four representative and biopharmaceutically relevant glycoproteins (IgG, fibrinogen, lactotransferrin, and ribonuclease B), the benefits and limitations of the developed workflow are highlighted and a state-of-the-art blueprint for conducting high-quality in-depth N- and O-glycoproteomic analyses is provided. Further, a modified and improved version of cotton hydrophilic interaction liquid chromatography-based solid phase extraction for glycopeptide enrichment is described. For the unambiguous identification of N-glycopeptides, the use of a conserved yet, rarely employed-fragmentation signature [Mpeptide +H+0,2 X GlcNAc]+ is proposed. It is shown for the first time that this fragmentation signature can consistently be found across all N-glycopeptides, but not on O-glycopeptides. Moreover, the use of the relative abundance of oxonium ions to retrieve glycan structure information, for example, differentiation of hybrid- and high-mannose-type N-glycans or differentiation between antenna GlcNAc and bisecting GlcNAc, is systematically and comprehensively evaluated. The findings may increase confidence and comprehensiveness in manual and software-assisted glycoproteomics.

glyXtoolMS: An Open-Source Pipeline for Semiautomated Analysis of Glycopeptide Mass Spectrometry Data.

For glycoproteomic analyses several web tools and standalone software packages have been developed over the recent years. These tools support or replace the time-consuming, cumbersome, and error-prone manual spectra analysis and glycopeptide identification. However, existing software tools are usually tailored to one fragmentation technique and only present the final analysis results. This makes manual inspection and correction of intermediate results difficult or even impossible. We solved this problem by dividing the analysis tasks into modular tools with defined functions, which are executed within a software pipeline with a graphical editor. This gives users a maximum of flexibility and control over the progress of analyses. Here, we present the open-source Python software suite glyXtoolMS, developed for the semiautomated analysis of N- and O-glycopeptide fragmentation data. glyXtoolMS is built around the pipeline engine of OpenMS (TOPPAS) and provides a glycopeptide analysis toolbox for the analysis, interpretation, and visualization of glycopeptide spectra. The toolbox encompasses (a) filtering of fragment spectra using a scoring scheme for oxonium ions, (b) in silico digest of protein sequences to collect glycopeptide candidates, (c) precursor matching to possible glycan compositions and peptide sequences, and finally, (d) an annotation tool for glycopeptide fragment ions. The resulting analysis file can be visualized by the glyXtool MS Evaluator, enabling further manual analysis, including inspection, verification, and various other options. Using higher-energy collisional dissociation data from human immunoglobulin γ (IgG) and human fibrinogen tryptic digests, we show that glyXtoolMS enables a fast, flexible, and transparent analysis of N- and O-glycopeptide samples, providing the user a versatile tool even for explorative data analysis. glyXtoolMS is freely available online on https://github.com/glyXera/glyXtoolMS licensed under the GPL-3.0 open-source license. The test data are available via ProteomeXchange with identifier PXD009716.

Improvement of electrospray stability in negative ion mode for nano-PGC-LC-MS glycoanalysis via post-column make-up flow.

Analysis of glycans via a porous graphitized carbon liquid chromatography (PGC-LC) coupled with electrospray ionization (tandem) mass spectrometry (ESI-MS(/MS)) is a powerful analytical method in the field of glycomics. Isobaric glycan structures can be identified reliably with the help of PGC-LC separation and subsequent identification by ESI-MS(/MS) in negative ion mode. In an effort to adapt PGC-LC-ESI-MS(/MS) to the nano-scale operation, spray instability along the nano-PGC-LC gradient was repeatedly observed on an LTQ Orbitrap Elite mass spectrometer equipped with a standard nano-electrospray ionization source. A stable electrospray was achieved with the implementation of a post-column make-up flow (PCMF). Thereby, acetonitrile was used to supplement the eluate from the nano-PGC-LC column. The improved spray stability enhanced detection and resolution of glycans during the analysis. This was in particular the case for smaller O-glycans which elute early in the high aqueous content regime of the nano-PGC-LC elution gradient. This study introduces PCMF as an easy-to-use instrumental adaptation to significantly improve spray stability in negative ion mode nano-PGC-LC-ESI-MS(/MS)-based analysis of glycans.

p97/VCP promotes Cullin-RING-ubiquitin-ligase/proteasome-dependent degradation of IκBα and the preceding liberation of RelA from ubiquitinated IκBα.

Cullin-RING-ubiquitin-ligase (CRL)-dependent ubiquitination of the nuclear factor kappa B (NF-κB) inhibitor IκBα and its subsequent degradation by the proteasome usually precede NF-κB/RelA nuclear activity. Through removal of the CRL-activating modification of their cullin subunit with the ubiquitin (Ub)-like modifier NEDD8, the COP9 signalosome (CSN) opposes CRL Ub-ligase activity. While RelA phosphorylation was observed to mediate NF-κB activation independent of Ub-proteasome-pathway (UPP)-dependent turnover of IκBα in some studies, a strict requirement of the p97/VCP ATPase for both, IκBα degradation and NF-κB activation, was reported in others. In this study, we thus aimed to reconcile the mechanism for tumour necrosis factor (TNF)-induced NF-κB activation. We found that inducible phosphorylation of RelA is accomplished in an IKK-complex-dependent manner within the NF-κB/RelA-IκBα-complex contemporaneous with the phosphorylation of IκBα, and that RelA phosphorylation is not sufficient to dissociate NF-κB/RelA from IκBα. Subsequent to CRL-dependent IκBα ubiquitination functional p97/VCP is essentially required for efficient liberation of (phosphorylated) RelA from IκBα, preceding p97/VCP-promoted timely and efficient degradation of IκBα as well as simultaneous NF-κB/RelA nuclear translocation. Collectively, our data add new facets to the knowledge about maintenance of IκBα and RelA expression, likely depending on p97/VCP-supported scheduled basal NF-κB activity, and the mechanism of TNF-induced NF-κB activation.

State-of-the-Art Glycomics Technologies in Glycobiotechnology.

Glycosylation affects the properties of biologics; thus regulatory bodies classified it as critical quality attribute and force biopharma industry to capture and control it throughout all phases, from R&D till end of product lifetime. The shift from originators to biosimilars further increases importance and extent of glycoanalysis, which thus increases the need for technology platforms enabling reliable high-throughput and in-depth glycan analysis. In this chapter, we will first summarize on established glycoanalytical methods based on liquid chromatography focusing on hydrophilic interaction chromatography, capillary electrophoresis focusing on multiplexed capillary gel electrophoresis, and mass spectrometry focusing on matrix-assisted laser desorption; we will then highlight two emerging technologies based on porous graphitized carbon liquid chromatography and on ion-mobility mass spectrometry as both are highly promising tools to deliver an additional level of information for in-depth glycan analysis; additionally we elaborate on the advantages and challenges of different glycoanalytical technologies and their complementarity; finally, we briefly review applications thereof to biopharmaceutical products. This chapter provides an overview of current state-of-the-art analytical approaches for glycan characterization of biopharmaceuticals that can be employed to capture glycoprotein heterogeneity in a biopharmaceutical context.

Site-specific N-glycosylation analysis of animal cell culture-derived Zika virus proteins.

Here, we present for the first time, a site-specific N-glycosylation analysis of proteins from a Brazilian Zika virus (ZIKV) strain. The virus was propagated with high yield in an embryo-derived stem cell line (EB66, Valneva SE), and concentrated by g-force step-gradient centrifugation. Subsequently, the sample was proteolytically digested with different enzymes, measured via a LC-MS/MS-based workflow, and analyzed in a semi-automated way using the in-house developed glyXtoolMS software. The viral non-structural protein 1 (NS1) was glycosylated exclusively with high-mannose structures on both potential N-glycosylation sites. In case of the viral envelope (E) protein, no specific N-glycans could be identified with this method. Nevertheless, N-glycosylation could be proved by enzymatic de-N-glycosylation with PNGase F, resulting in a strong MS-signal of the former glycopeptide with deamidated asparagine at the potential N-glycosylation site N444. This confirmed that this site of the ZIKV E protein is highly N-glycosylated but with very high micro-heterogeneity. Our study clearly demonstrates the progress made towards site-specific N-glycosylation analysis of viral proteins, i.e. for Brazilian ZIKV. It allows to better characterize viral isolates, and to monitor glycosylation of major antigens. The method established can be applied for detailed studies regarding the impact of protein glycosylation on antigenicity and human pathogenicity of many viruses including influenza virus, HIV and corona virus.

Comprehensive N-glycosylation analysis of the influenza A virus proteins HA and NA from adherent and suspension MDCK cells.

Glycosylation is considered as a critical quality attribute for the production of recombinant biopharmaceuticals such as hormones, blood clotting factors, or monoclonal antibodies. In contrast, glycan patterns of immunogenic viral proteins, which differ significantly between the various expression systems, are hardly analyzed yet. The influenza A virus (IAV) proteins hemagglutinin (HA) and neuraminidase (NA) have multiple N-glycosylation sites, and alteration of N-glycan micro- and macroheterogeneity can have strong effects on virulence and immunogenicity. Here, we present a versatile and powerful glycoanalytical workflow that enables a comprehensive N-glycosylation analysis of IAV glycoproteins. We challenged our workflow with IAV (A/PR/8/34 H1N1) propagated in two closely related Madin-Darby canine kidney (MDCK) cell lines, namely an adherent MDCK cell line and its corresponding suspension cell line. As expected, N-glycan patterns of HA and NA from virus particles produced in both MDCK cell lines were similar. Detailed analysis of the HA N-glycan microheterogeneity showed an increasing variability and a higher complexity for N-glycosylation sites located closer to the head region of the molecule. In contrast, NA was found to be exclusively N-glycosylated at site N73. Almost all N-glycan structures were fucosylated. Furthermore, HA and NA N-glycan structures were exclusively hybrid- and complex-type structures, to some extent terminated with alpha-linked galactose(s) but also with blood group H type 2 and blood group A epitopes. In contrast to the similarity of the overall glycan pattern, differences in the relative abundance of individual structures were identified. This concerned, in particular, oligomannose-type, alpha-linked galactose, and multiantennary complex-type N-glycans.

Efficient influenza A virus production in high cell density using the novel porcine suspension cell line PBG.PK2.1.

Seasonal and pandemic influenza respiratory infections are still a major public health issue. Vaccination is the most efficient way to prevent influenza infection. One option to produce influenza vaccines is cell-culture based virus propagation. Different host cell lines, such as MDCK, Vero, AGE1.CR or PER.C6 cells have been shown to be a good substrate for influenza virus production. With respect to the ease of scale-up, suspension cells should be preferred over adherent cells. Ideally, they should replicate different influenza virus strains with high cell-specific yields. Evaluation of new cell lines and further development of processes is of considerable interest, as this increases the number of options regarding the design of manufacturing processes, flexibility of vaccine production and efficiency. Here, PBG.PK2.1, a new mammalian cell line that was developed by ProBioGen AG (Germany) for virus production is presented. The cells derived from immortal porcine kidney cells were previously adapted to growth in suspension in a chemically-defined medium. Influenza virus production was improved after virus adaptation to PBG.PK2.1 cells and optimization of infection conditions, namely multiplicity of infection and trypsin concentration. Hemagglutinin titers up to 3.24 log10(HA units/100 µL) were obtained in fed-batch mode in bioreactors (700 mL working volume). Evaluation of virus propagation in high cell density culture using a hollow-fiber based system (ATF2) demonstrated promising performance: Cell concentrations of up to 50 × 106 cells/mL with viabilities exceeding 95%, and a maximum HA titer of 3.93 log10(HA units/100 µL). Analysis of glycosylation of the viral HA antigen expressed showed clear differences compared to HA produced in MDCK or Vero cell lines. With an average cell-specific productivity of 5000 virions/cell, we believe that PBG.PK2.1 cells are a very promising candidate to be considered for next-generation influenza virus vaccine production.

Improvement of the glycoproteomic toolbox with the discovery of a unique C-terminal cleavage specificity of flavastacin for N-glycosylated asparagine.

To determine all potential N-glycosylation sites of a glycoprotein, one central aspect of every bottom-up N-glycoproteomic strategy is to generate suitable N-glycopeptides that can be detected and analyzed by mass spectrometry. Specific proteases, such as trypsin, bear the potential of generating N-glycopeptides that either carry more than one N-glycosylation site or are too long to be readily analyzed by mass spectrometry- both due to the lack of tryptic cleavage sites near the N-glycosylation site. Here, we present a newly identified cleavage specificity of flavastacin, a protease from Flavobacterium menigosepticum, which - up to now - was only reported to cleave peptide bonds N-terminal to aspartic acid residues. In contrast to literature, we could not confirm this N-terminal specificity of flavastacin for aspartic acid. However, for the first time, we show a unique cleavage specificity of flavastacin towards the C-terminus of N-glycosylated asparagine residues. Implemented in an N-glycoproteomic workflow the use of flavastacin can thus not only render data analysis much easier, it can also significantly increase the confidence of MS-based N-glycoproteomic analyses. We demonstrate this newly discovered specificity of flavastacin by in-depth LC-MS(/MS) analysis of complex-type glycosylated human lactotransferrin and bovine serum albumin peptides and N-glycopeptides that were generated by trypsin and flavastacin digestion. Following to this work, further elucidation of the efficiency, specificity and mode of action of flavastacin is needed, but we believe that our discovery has great potential to facilitate and improve the characterization of N-glycoproteomes.