Community evaluation of glycoproteomics informatics solutions reveals high-performance search strategies for serum glycopeptide analysis.

Glycoproteomics is a powerful yet analytically challenging research tool. Software packages aiding the interpretation of complex glycopeptide tandem mass spectra have appeared, but their relative performance remains untested. Conducted through the HUPO Human Glycoproteomics Initiative, this community study, comprising both developers and users of glycoproteomics software, evaluates solutions for system-wide glycopeptide analysis. The same mass spectrometrybased glycoproteomics datasets from human serum were shared with participants and the relative team performance for N- and O-glycopeptide data analysis was comprehensively established by orthogonal performance tests. Although the results were variable, several high-performance glycoproteomics informatics strategies were identified. Deep analysis of the data revealed key performance-associated search parameters and led to recommendations for improved 'high-coverage' and 'high-accuracy' glycoproteomics search solutions. This study concludes that diverse software packages for comprehensive glycopeptide data analysis exist, points to several high-performance search strategies and specifies key variables that will guide future software developments and assist informatics decision-making in glycoproteomics.

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.

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.

Capillary electrophoresis/mass spectrometry relevant to pharmaceutical and biotechnological applications.

Advanced analytical techniques play a crucial role in the pharmaceutical and biotechnological field. In this context, capillary electrophoresis/mass spectrometry (CE/MS) has attracted attention due to efficient and selective separation in combination with powerful detection allowing identification and detailed characterization. Method developments and applications of CE/MS have been focused on questions not easily accessible by liquid chromatography/mass spectrometry (LC/MS) as the analysis of intact proteins, carbohydrates, and various small molecules, including peptides. Here, recent approaches and applications of CE/MS relevant to (bio)pharmaceuticals are reviewed and discussed to show actual developments and future prospects. Based on other reviews on related subjects covering large parts of previous works, the paper is focused on general ideas and contributions of the last 2 years; for the analysis of glycans, the period is extended back to 2006.

Toward a screening method for the analysis of small intact proteins by CE-ESI-TOF MS.

Capillary electrophoresis-mass spectrometry (CE-MS) more and more gains in importance as an analytical technique for the identification and characterization of intact proteins in the biopharmaceutical area. Thus, a CE-ESI-MS method was optimized and validated systematically with respect to the improved screening and characterization of intact proteins. The optimization was accomplished by variation of different CE-MS parameters, such as capillary coating, background electrolyte, sheath liquid, and nebulizer gas pressure, while monitoring both the resolution and signal intensities. Achievable separation is discussed quantitatively in the context of the coating and the resulting EOF, the protein mobilities, and the suction effect of the sprayer. The observed precisions of the optimized method regarding the migration times (mean RSD = 1.4%) and peak areas (mean RSD = 12.3%) and an extensive principal component analysis revealed that the presented method is reliable and useful for the quantitation of intact proteins and protein isoforms. The applicability of this method to various proteins showing different characteristics (pI value, molecular mass, hydrophobicity, etc.) is discussed. The presented method will contribute to the improved characterization of a large variety of intact proteins in the biomedical and pharmaceutical area.

Metaproteome analysis to determine the metabolically active part of a thermophilic microbial community producing biogas from agricultural biomass.

Complex consortia of microorganisms are responsible for biogas production. A lot of information about the taxonomic structure and enzymatic potential of such communities has been collected by a variety of gene-based approaches, yet little is known about which of all the assumable metabolic pathways are active throughout the process of biogas formation. To tackle this problem, we established a protocol for the metaproteomic analysis of samples taken from biogas reactors fed with agricultural biomass. In contrast to previous studies where an anaerobic digester was fed with synthetic wastewater, the complex matrix in this study required the extraction of proteins with liquid phenol and the application of paper bridge loading for 2-dimensional gel electrophoresis. Proteins were subjected to nanoHPLC (high-performance liquid chromatography) coupled to tandem mass spectrometry for characterization. Several housekeeping proteins as well as methanogenesis-related enzymes were identified by a MASCOT search and de novo sequencing, which proved the feasibility of our approach. The establishment of such an approach is the basis for further metaproteomic studies of biogas-producing communities. In particular, the apparent status of metabolic activities within the communities can be monitored. The knowledge collected from such experiments could lead to further improvements of biogas production.

Multivariate statistics for the differentiation of erythropoietin preparations based on intact glycoforms determined by CE-MS.

Owing to the increasing number of erythropoietin biosimilars being approved, the comparison of different erythropoietin preparations in the pharmaceutical area is gaining in importance. Erythropoietin has a distinct natural heterogeneity arising from its glycosylation, which shows strong composition variations. This heterogeneity increases the complexity of the analysis of erythropoietin considerably, but may also be used to distinguish different preparations. Here, a method is presented for the differentiation of various erythropoietin preparations by capillary electrophoresis-mass spectrometry and the subsequent application of multivariate statistics. Relative peak areas of selected intact erythropoietin isoforms were used as variables in principal component analysis and hierarchical agglomerative clustering. Both of these strategies were suited for the clear differentiation of all erythropoietin preparations, including marketed products and preproduction preparations, which differ in the manufacturer, the production cell line, and the batch number. By this means, even closely related preparations were distinguished on the basis of the combined information on the antennarity, the sialoform, and the acetylation of the observed isoforms.