A patient-based medaka alg2 mutant as a model for hypo-N-glycosylation.

Defects in the evolutionarily conserved protein-glycosylation machinery during embryonic development are often fatal. Consequently, congenital disorders of glycosylation (CDG) in human are rare. We modelled a putative hypomorphic mutation described in an alpha-1,3/1,6-mannosyltransferase (ALG2) index patient (ALG2-CDG) to address the developmental consequences in the teleost medaka (Oryzias latipes). We observed specific, multisystemic, late-onset phenotypes, closely resembling the patient's syndrome, prominently in the facial skeleton and in neuronal tissue. Molecularly, we detected reduced levels of N-glycans in medaka and in the patient's fibroblasts. This hypo-N-glycosylation prominently affected protein abundance. Proteins of the basic glycosylation and glycoprotein-processing machinery were over-represented in a compensatory response, highlighting the regulatory topology of the network. Proteins of the retinal phototransduction machinery, conversely, were massively under-represented in the alg2 model. These deficiencies relate to a specific failure to maintain rod photoreceptors, resulting in retinitis pigmentosa characterized by the progressive loss of these photoreceptors. Our work has explored only the tip of the iceberg of N-glycosylation-sensitive proteins, the function of which specifically impacts on cells, tissues and organs. Taking advantage of the well-described human mutation has allowed the complex interplay of N-glycosylated proteins and their contribution to development and disease to be addressed.

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.

Glycosyltransferase POMGNT1 deficiency strengthens N-cadherin-mediated cell-cell adhesion.

Defects in protein O-mannosylation lead to severe congenital muscular dystrophies collectively known as α-dystroglycanopathy. A hallmark of these diseases is the loss of the O-mannose-bound matriglycan on α-dystroglycan, which reduces cell adhesion to the extracellular matrix. Mutations in protein O-mannose ß1,2-N-acetylglucosaminyltransferase 1 (POMGNT1), which is crucial for the elongation of O-mannosyl glycans, have mainly been associated with muscle-eye-brain (MEB) disease. In addition to defects in cell-extracellular matrix adhesion, aberrant cell-cell adhesion has occasionally been observed in response to defects in POMGNT1. However, specific molecular consequences of POMGNT1 deficiency on cell-cell adhesion are largely unknown. We used POMGNT1 knockout HEK293T cells and fibroblasts from an MEB patient to gain deeper insight into the molecular changes in POMGNT1 deficiency. Biochemical and molecular biological techniques combined with proteomics, glycoproteomics, and glycomics revealed that a lack of POMGNT1 activity strengthens cell-cell adhesion. We demonstrate that the altered intrinsic adhesion properties are due to an increased abundance of N-cadherin (N-Cdh). In addition, site-specific changes in the N-glycan structures in the extracellular domain of N-Cdh were detected, which positively impact on homotypic interactions. Moreover, in POMGNT1-deficient cells, ERK1/2 and p38 signaling pathways are activated and transcriptional changes that are comparable with the epithelial-mesenchymal transition (EMT) are triggered, defining a possible molecular mechanism underlying the observed phenotype. Our study indicates that changes in cadherin-mediated cell-cell adhesion and other EMT-related processes may contribute to the complex clinical symptoms of MEB or α-dystroglycanopathy in general and suggests that the impact of changes in O-mannosylation on N-glycosylation has been underestimated.

A Bacterial Mannose Binding Lectin as a Tool for the Enrichment of C- and O-Mannosylated Peptides.

Mass spectrometry (MS) easily detects C-mannosylated peptides from purified proteins but not from complex biological samples. Enrichment of specific glycopeptides by lectin affinity prior to MS analysis has been widely applied to support glycopeptide identification but was until now not available for C-mannosylated peptides. Here, we used the α-mannose-specific Burkholderia cenocepacia lectin A (BC2L-A) and show that, in addition to its previously demonstrated high-mannose N-glycan binding capability, this lectin is able to retain C- and O-mannosylated peptides. Besides testing binding abilities to standard peptides, we applied BC2L-A affinity to enrich C-mannosylated peptides from complex samples of tryptic digests of HEK293 and MCF10A whole cell extracts, which led to the identification of novel C-mannosylation sites. In conclusion, BC2L-A enabled specific enrichment of C- and O-mannosylated peptides and might have superior properties over other mannose binding lectins for this purpose.

[Physician Assistants - Eine effektive und sinnvolle Erweiterung des herzchirurgischen Behandlungsteams]. / Positionspapier der Deutschen Gesellschaft für Thorax-, Herz- und Gefäßchirurgie zum Einsatz von Physician Assistants in der Herzchirurgie.

High-quality care of cardiac surgical patients requires the employment and recruiting of qualified medical professionals with minimal fluctuation of staff members. This aspect becomes increasingly difficult due to the current shortage of skilled professionals as well as the present framework conditions of the German Healthcare System. The implementation of physician assistants (PA) in cardiac surgery departments may augment existing human resource concepts in an innovative and sustainable manner, tailored to meet department specific requirements. Long-term experiences from Anglo-American countries prove that the implementation of a PA system may stabilize or potentially even improve medical treatment quality. At the same time, cardiac surgical residents may be relieved from routine tasks, releasing additional time resources for a solid and diverse specialist training. Furthermore, positive effects on economic aspects of an institution may be possible. The required delegation of medical tasks to allied health professionals already has a legal basis in Germany, while a specific legal framework tailored to physician assistants does not exist yet. In this context, it is an important aspect that medical associations define a reliable catalog of tasks that may be delegated to physician assistants. Under evaluation of medical, legal and economic aspects and in a structured manner, this position paper defines medical tasks of physician assistants in cardiac surgery.

[First Insights into Scope of Practice and Salary of Physician Assistants, A New Healthcare Profession, in Germany]. / Eine neue Berufsgruppe kommt in der Praxis an: Erste Erkenntnisse über Einsatzgebiete, Tätigkeiten und Gehalt von Physician Assistants/Arztassistenten in Deutschland.

BACKGROUND: In 2010, the first government-approved physician assistant (PA) program was introduced at the Baden-Wuerttemberg Cooperative State University Karlsruhe (DHBW). There are not sufficient data regarding the scope of practice and salary of our graduates. Therefore, the aim of the present study was to obtain information regarding these. METHODS: The survey included all graduates (2 classes, n=27). A specific questionnaire was developed, including 37 questions e. g. on the current employment status, scope of practice, salary and job satisfaction regarding the PA program and career. A descriptive analysis of the data was carried out using SPSS. RESULTS: 25 graduates participated in the survey (96.1%); the average age of participants was 32.2 years (25-53 years). 88% (n=22) were employed as a PA, most of them worked in internal medicine (n=11) or surgery (n=9). Responsibilities that are often or very often assigned to the PAs are preparing final documents, taking over a coordinating role in the therapeutic team, as well as participation in taking patient medical history and conducting physical examinations. In two-thirds of respondents, the gross monthly base salary (full-time position) was about 3000 euros. 77.3% (n=17) of graduates were generally satisfied or very satisfied with their current situation. CONCLUSIONS: It appears that graduates of the DHBW are well integrated into the staff structure of hospitals, as far as the scope of practice and average salary are concerned. Further studies on the integration of this new profession in Germany and on their extended scope of practice in comparison to established healthcare professions will be conducted.

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.

Site-specific O-Glycosylation Analysis of Human Blood Plasma Proteins.

Site-specific glycosylation analysis is key to investigate structure-function relationships of glycoproteins, e.g. in the context of antigenicity and disease progression. The analysis, though, is quite challenging and time consuming, in particular for O-glycosylated proteins. In consequence, despite their clinical and biopharmaceutical importance, many human blood plasma glycoproteins have not been characterized comprehensively with respect to their O-glycosylation. Here, we report on the site-specific O-glycosylation analysis of human blood plasma glycoproteins. To this end pooled human blood plasma of healthy donors was proteolytically digested using a broad-specific enzyme (Proteinase K), followed by a precipitation step, as well as a glycopeptide enrichment and fractionation step via hydrophilic interaction liquid chromatography, the latter being optimized for intact O-glycopeptides carrying short mucin-type core-1 and -2 O-glycans, which represent the vast majority of O-glycans on human blood plasma proteins. Enriched O-glycopeptide fractions were subjected to mass spectrometric analysis using reversed-phase liquid chromatography coupled online to an ion trap mass spectrometer operated in positive-ion mode. Peptide identity and glycan composition were derived from low-energy collision-induced dissociation fragment spectra acquired in multistage mode. To pinpoint the O-glycosylation sites glycopeptides were fragmented using electron transfer dissociation. Spectra were annotated by database searches as well as manually. Overall, 31 O-glycosylation sites and regions belonging to 22 proteins were identified, the majority being acute-phase proteins. Strikingly, also 11 novel O-glycosylation sites and regions were identified. In total 23 O-glycosylation sites could be pinpointed. Interestingly, the use of Proteinase K proved to be particularly beneficial in this context. The identified O-glycan compositions most probably correspond to mono- and disialylated core-1 mucin-type O-glycans (T-antigen). The developed workflow allows the identification and characterization of the major population of the human blood plasma O-glycoproteome and our results provide new insights, which can help to unravel structure-function relationships. The data were deposited to ProteomeXchange PXD003270.

[Physician assistants in surgery : A young profession through graduates' eyes]. / Physician Assistants in der Chirurgie : Ein junges Berufsbild aus Absolventensicht.

BACKGROUND: In Germany, physician assistant (PA) is a comparatively young profession. The concept paper by the German Medical Association (BÄK) and the Federation of Statutory Health Insurance Physicians (KBV) from 2017 defines the new occupational profile in detail. In contrast, there is hardly any information on the day to day working life of a PA in Germany OBJECTIVE: The aim of this study was to map the employment reality of the PA graduates of the study program of the Baden-Wuerttemberg Cooperative State University Karlsruhe. MATERIAL AND METHODS: Graduates of the PA study program were interviewed using the web-based evaluation system EvaSys V7.1 (Electric Paper Evaluationssysteme GmbH, Lüneburg, Deutschland) in the spring of 2018. The information was evaluated descriptively. RESULTS: The response rate was 70% (48 out of 69 graduates), 44 graduates were employed as a PA and 27 worked in a surgical department. The core tasks of the surgically active PA, which were often performed to varying frequency included assisting surgery and the performance of simple wound closures. In addition, activities in the areas of documentation, communication and information sharing were emphasized. The average salary of a surgical PA was 3718 €. This amount was rated by 44.4% as appropriate or very appropriate. The current occupational situation for 81.5% of the study participants was much better or better than expected before the start of their PA studies. Overall satisfaction was very high: 85.1% of the graduates were satisfied to very satisfied. CONCLUSION: The graduates' level of job satisfaction is remarkable. Many of the activities mentioned in the concept paper of the BÄK and KBV were carried out frequently or very frequently by the PA. Nonetheless, the PA profession has significant development potential, especially in the realm of surgical PAs.

Towards personalized diagnostics via longitudinal study of the human plasma N-glycome.

Facilitated by substantial advances in analytical methods, plasma N-glycans have emerged as potential candidates for biomarkers. In the recent years, several investigations could link aberrant plasma N-glycosylation to numerous diseases. However, due to often limited specificity and sensitivity, only a very limited number of glycan biomarkers were approved by the authorities up to now. The inter-individual heterogeneity of the plasma N-glycomes might mask disease related changes in conventional large cross-sectional cohort studies, with a one-time sampling approach. But, a possible benefit of longitudinal sampling in biomarker discovery could be, that already small changes during disease progression are revealed, by monitoring the plasma N-glycome of individuals over time. To evaluate this, we collected blood plasma samples of five healthy donors over a time period of up to six years (min. 1.5 years). The plasma N-glycome was analyzed by xCGE-LIF, to investigate the intra-individual N-glycome variability over time. It is shown, that the plasma N-glycome of an individual is remarkably stable over a period of several years, and that observed small longitudinal changes are independent from seasons, but significantly correlated with lifestyle and environmental factors. Thus, the potential of future longitudinal biomarker discovery studies could be demonstrated, which is a further step towards personalized diagnostics. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.

Fractionation of biogas plant sludge material improves metaproteomic characterization to investigate metabolic activity of microbial communities.

With the development of high resolving mass spectrometers, metaproteomics evolved as a powerful tool to elucidate metabolic activity of microbial communities derived from full-scale biogas plants. Due to the vast complexity of these microbiomes, application of suitable fractionation methods are indispensable, but often turn out to be time and cost intense, depending on the method used for protein separation. In this study, centrifugal fractionation has been applied for fractionation of two biogas sludge samples to analyze proteins extracted from (i) crude fibers, (ii) suspended microorganisms, and (iii) secreted proteins in the supernatant using a gel-based approach followed by LC-MS/MS identification. This fast and easy method turned out to be beneficial to both the quality of SDS-PAGE and the identification of peptides and proteins compared to untreated samples. Additionally, a high functional metabolic pathway coverage was achieved by combining protein hits found exclusively in distinct fractions. Sample preparation using centrifugal fractionation influenced significantly the number and the types of proteins identified in the microbial metaproteomes. Thereby, comparing results from different proteomic or genomic studies, the impact of sample preparation should be considered. All MS data have been deposited in the ProteomeXchange with identifier PXD001508 (http://proteomecentral.proteomexchange.org/dataset/PXD001508).

The MetaProteomeAnalyzer: a powerful open-source software suite for metaproteomics data analysis and interpretation.

The enormous challenges of mass spectrometry-based metaproteomics are primarily related to the analysis and interpretation of the acquired data. This includes reliable identification of mass spectra and the meaningful integration of taxonomic and functional meta-information from samples containing hundreds of unknown species. To ease these difficulties, we developed a dedicated software suite, the MetaProteomeAnalyzer, an intuitive open-source tool for metaproteomics data analysis and interpretation, which includes multiple search engines and the feature to decrease data redundancy by grouping protein hits to so-called meta-proteins. We also designed a graph database back-end for the MetaProteomeAnalyzer to allow seamless analysis of results. The functionality of the MetaProteomeAnalyzer is demonstrated using a sample of a microbial community taken from a biogas plant.

High-throughput IgG Fc N-glycosylation profiling by mass spectrometry of glycopeptides.

Age and sex dependence of subclass specific immunoglobulin G (IgG) Fc N-glycosylation was evaluated for 1709 individuals from two isolated human populations. IgGs were obtained from plasma by affinity purification using 96-well protein G monolithic plates and digested with trypsin. Fc N-glycopeptides were purified and analyzed by negative-mode MALDI-TOF-MS with 4-chloro-α-cyanocinnamic acid (Cl-CCA) matrix. Age-associated glycosylation changes were more pronounced in younger individuals (<57 years) than in older individuals (>57 years) and in females than in males. Galactosylation and sialylation decreased with increasing age and showed significant sex dependence. Interestingly, the most prominent drop in the levels of galactosylated and sialylated glycoforms in females was observed around the age of 45 to 60 years when females usually enter menopause. The incidence of bisecting N-acetylglucosamine increased in younger individuals and reached a plateau at older age. Furthermore, we compared the results to the total IgG N-glycosylation of the same populations recently analyzed by hydrophilic interaction liquid chromatography (HILIC). Significant differences were observed in the levels of galactosylation, bisecting N-acetylglucosamine and particularly sialylation, which were shown to be higher in HILIC analysis. Age and sex association of glycosylation features was, to a large extent, comparable between MALDI-TOF-MS and HILIC IgG glycosylation profiling.

Cell-free N-glycosylation of peptides using synthetic lipid-linked hybrid and complex N-glycans.

Cell-free, chemoenzymatic platforms are emerging technologies towards generating glycoconjugates with defined and homogeneous glycoforms. Recombinant oligosaccharyltransferases can be applied to glycosylate "empty," i.e., aglycosyalted, peptides and proteins. While bacterial oligosaccharlytransferases have been extensively investigated, only recently a recombinant eukaryotic single-subunit oligosaccharyltransferase has been successfully used to in vitro N-glycosylate peptides. However, its applicability towards synthesizing full-length glycoproteins and utilizing glycans beyond mannose-type glycans for the transfer have not be determined. Here, we show for the first time the synthesis of hybrid- and complex-type glycans using synthetic lipid carriers as substrates for in vitro N-glycosylation reactions. For this purpose, transmembrane-deleted human ß-1,2 N-acetylglucosamintransferase I and II (MGAT1ΔTM and MGAT2ΔTM) and ß-1,4-galactosyltransferase (GalTΔTM) have been expressed in Escherichia coli and used to extend an existing multi-enzyme cascade. Both hybrid and agalactosylated complex structures were transferred to the N-glycosylation consensus sequence of peptides (10 amino acids: G-S-D-A-N-Y-T-Y-T-Q) by the recombinant oligosaccharyltransferase STT3A from Trypanosoma brucei.

Proteomic tracking and analysis of a bacterial mixed culture.

To improve the understanding of microbial behaviors in communities, proteomic tracking, an approach for relative quantification of species-specific population dynamics of mixed cultures, was developed. Therefore, a bacterial mixed culture was analyzed during batch cultivations with and without addition of the antibiotic Ceftazidime. The community was composed of Burkholderia cepacia, Pseudomonas aeruginosa, and Staphylococcus aureus, pathogens causing infections in cystic fibrosis patients. Gel-based proteomics and mass spectrometry were used to obtain qualitative and quantitative proteomic data. During cultivation, P. aeruginosa became dominant within the mixed culture while S. aureus was inhibited in growth. Analysis of samples - taken along cultivation - revealed about 270 differentially expressed proteins. Some of those proteins are related to bacterial interactions, response to antibiotic treatment or metabolic shifts. For instance, the enzymes PhzS(flavin-containing monooxygenase), PhzD (phenazine biosynthesis protein), and PhzG2 (pyridoxamine 5'-phosphate oxidase) indicated the production of the antibiotic pigment pyocyanine by P. aeruginosa that is related to oxidative stress and therefore, might inhibit growth of S. aureus. Overall, the strategy applied not only allows species-specific tracking of the community composition but also provides valuable insights into the behavior of mixed cultures.

MALDI-TOF-MS analysis of sialylated glycans and glycopeptides using 4-chloro-α-cyanocinnamic acid matrix.

For MALDI analysis of glycans and glycopeptides, the choice of matrix is crucial in minimizing desialylation by mass spectrometric in-source and metastable decay. Here, we evaluated the potential of 4-chloro-α-cyanocinnamic acid (Cl-CCA) for MALDI-TOF-MS analysis of labile sialylated tryptic N-glycopeptides and released N- and O-glycans. Similar to DHB, but in contrast to CHCA, the Cl-CCA matrix allowed the analysis of sialylated N-glycans and glycopeptides in negative ion mode MALDI-TOF-MS. Dried droplet preparations of Cl-CCA provided microcrystals with a homogeneous spatial distribution and high shot-to-shot repeatability similar to CHCA, which simplified the automatic measurement and improved the resolution and mass accuracy. Interestingly, reflectron-positive ion mode analysis of 1-phenyl-3-methyl-5-pyrazolone (PMP)-labeled O-glycans with Cl-CCA revealed more complete profiles than with DHB and CHCA. In conclusion, we clearly demonstrate the high potential of this rationally designed matrix for glycomics and glycoproteomics.

Glycoproteomic analysis of human fibrinogen reveals novel regions of O-glycosylation.

Human fibrinogen is a 340 kDa, soluble plasma glycoprotein composed of paired sets of three subunits (α, ß, γ). The protein plays a crucial role in protecting the vascular network against the loss of blood after tissue injury. The beta and gamma subunits each contain one N-glycosylation site, each of which is occupied by a biantennary N-glycan. So far O-linked oligosaccharides have rarely been described. Here, we make use of tryptic- and proteinase K-generated fibrinogen glycopeptides for the detailed analysis of the protein's O-glycosylation by combining information obtained from both one- and two-dimensional nanoLC-ESI-ion trap (IT)-MS approaches. Glycopeptides were analyzed by ion trap-MS/MS which displayed fragmentations of glycosidic linkages and some peptide backbone cleavages. MS3 spectra of the generated O-glycopeptides showed cleavages of the peptide backbone and provided essential information on the peptide sequence. The previously reported N-glycan attachment sites of human fibrinogen could be confirmed. Moreover, we describe seven novel O-glycosylation regions in human fibrinogen, all occupied by a monosialylated T-antigen. Our findings may help to improve the general understanding of human fibrinogen in the blood clotting process.

Glycoproteomics Technologies in Glycobiotechnology.

Glycosylation is a key factor determining the pharmacological properties of biotherapeutics, including their stability, solubility, bioavailability, pharmacokinetics, and immunogenicity. As such, comprehensive information about glycosylation of biotherapeutics is critical to demonstrate similarity. Regulatory agencies also require extensive documentation of the comprehensive analyses of glycosylation-related critical quality attributes (CQAs) during the development, manufacturing, and release of biosimilars. Mass spectrometry has catalysed tremendous advancements in the characterisation of glycosylation CQAs of biotherapeutics. Here we provide a perspective overview on the MS-based technologies relevant for biotherapeutic product characterisation with an emphasis on the recent developments that allow determination of glycosylation features such as site of glycosylation, sialic acid linkage, glycan structure, and content.