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Background: There is a need for diagnostic tests for screening, triaging and staging of epithelial ovarian cancer (EOC). Glycoproteomics of blood samples has shown promise for biomarker discovery., Methods: We applied glycoproteomics to serum of people with EOC or benign pelvic masses and healthy controls. A total of 653 analytes were quantified and assessed in multivariable models, which were tested in an independent cohort. Additionally, we analyzed glycosylation patterns in serum markers and in tissues., Results: We identified a biomarker panel that distinguished benign lesions from EOC with sensitivity and specificity of 83.5% and 90.1% in the training set, and of 86.7 and 86.7% in the test set, respectively. ROC analysis demonstrated strong performance across a range of cutoffs. Fucosylated multi-antennary glycopeptide markers were higher in late-stage than in early-stage EOC. A comparable pattern was found in late-stage EOC tissues., Conclusions: Blood glycopeptide biomarkers have the potential to distinguish benign from malignant pelvic masses, and early- from late-stage EOC. Glycosylation of circulating and tumor tissue proteins may be related. This study supports the hypothesis that blood glycoproteomic profiling can be used for EOC diagnosis and staging and it warrants further clinical evaluation., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Glycosylation is a key modulator of the functional state of proteins. Recent developments in large-scale analysis of intact glycopeptides have enabled the identification of numerous glycan structures that are relevant in pathophysiological processes. However, one motif found in N -glycans, poly- N -acetyllactosamine (polyLacNAc), still poses a substantial challenge to mass spectrometry-based glycoproteomic analysis due to its relatively low abundance and large size. In this work, we developed approaches for the systematic mapping of polyLacNAc-elongated N -glycans in melanoma cells. We first evaluated five anion exchange-based matrices for enriching intact glycopeptides and selected two materials that provided better overall enrichment efficiency. We then tested the robustness of the methodology by quantifying polyLacNAc-containing glycopeptides as well as changes in protein fucosylation and sialylation. Finally, we applied the optimal enrichment methods to discover glycopeptides containing polyLacNAc motifs in melanoma cells and found that integrins and tetraspanins are substantially modified with these structures. This study demonstrates the feasibility of glycoproteomic approaches for identification of glycoproteins with polyLacNAc motifs.

Monoclonal antibodies targeting the immune checkpoint PD-1 have provided significant clinical benefit across a number of solid tumors, with differences in efficacy and toxicity profiles possibly related to their intrinsic molecular properties. Here, we report that camrelizumab and cemiplimab engage PD-1 through interactions with its fucosylated glycan. Using a combination of protein and cell glycoengineering, we demonstrate that the two antibodies bind preferentially to PD-1 with core fucose at the asparagine N58 residue. We then provide evidence that the concentration of fucosylated PD-1 in the blood of non-small-cell lung cancer patients varies across different stages of disease. This study illustrates how glycoprofiling of surface receptors and related circulating forms can inform the development of differentiated antibodies that discriminate glycosylation variants and achieve enhanced selectivity, and paves the way toward the implementation of personalized therapeutic approaches., (© 2024 Chu et al.)

Cell surface glycosylation has a variety of functions, and its dysregulation in cancer contributes to impaired signaling, metastasis and the evasion of the immune responses. Recently, a number of glycosyltransferases that lead to altered glycosylation have been linked to reduced anti-tumor immune responses: B3GNT3, which is implicated in PD-L1 glycosylation in triple negative breast cancer, FUT8, through fucosylation of B7H3, and B3GNT2, which confers cancer resistance to T cell cytotoxicity. Given the increased appreciation of the relevance of protein glycosylation, there is a critical need for the development of methods that allow for an unbiased interrogation of cell surface glycosylation status. Here we provide an overview of the broad changes in glycosylation at the surface of cancer cell and describe selected examples of receptors with aberrant glycosylation leading to functional changes, with emphasis on immune checkpoint inhibitors, growth-promoting and growth-arresting receptors. Finally, we posit that the field of glycoproteomics has matured to an extent where large-scale profiling of intact glycopeptides from the cell surface is feasible and is poised for discovery of new actionable targets against cancer., Competing Interests: Competing Interests: The authors are employees of InterVenn Biosciences., (© The author(s).)

Currently available enzyme replacement therapies for lysosomal storage diseases are limited in their effectiveness due in part to short circulation times and suboptimal biodistribution of the therapeutic enzymes. We previously engineered Chinese hamster ovary (CHO) cells to produce α-galactosidase A (GLA) with various N-glycan structures and demonstrated that elimination of mannose-6-phosphate (M6P) and conversion to homogeneous sialylated N-glycans prolonged circulation time and improved biodistribution of the enzyme following a single-dose infusion into Fabry mice. Here, we confirmed these findings using repeated infusions of the glycoengineered GLA into Fabry mice and further tested whether this glycoengineering approach, Long-Acting-GlycoDesign (LAGD), could be implemented on other lysosomal enzymes. LAGD-engineered CHO cells stably expressing a panel of lysosomal enzymes [aspartylglucosamine (AGA), beta-glucuronidase (GUSB), cathepsin D (CTSD), tripeptidyl peptidase (TPP1), alpha-glucosidase (GAA) or iduronate 2-sulfatase (IDS)] successfully converted all M6P-containing N-glycans to complex sialylated N-glycans. The resulting homogenous glycodesigns enabled glycoprotein profiling by native mass spectrometry. Notably, LAGD extended the plasma half-life of all three enzymes tested (GLA, GUSB, AGA) in wildtype mice. LAGD may be widely applicable to lysosomal replacement enzymes to improve their circulatory stability and therapeutic efficacy., Competing Interests: A patent application has been filed by the University of Copenhagen. GlycoDisplay ApS has license rights to the patent application. ZY, WT, CK, and HC are named co-inventors, and ZY, CK, and HC have financial interests in GlycoDisplay ApS. Y-HC is an employee of GlycoDisplay ApS. RD is a Consultant to Genzyme-Sanofi and Sangamo Therapeutics, Inc. He owns founder stock in Amicus Therapeutics and options for Sangmo Therapeutics, Inc. and receives royalities from Genzyme-Sanofi. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Chen, Tian, Yasuda, Ye, Song, Mandel, Kristensen, Povolo, Marques, Čaval, Heck, Sampaio, Johannes, Tsukimura, Desnick, Vakhrushev, Yang and Clausen.)

Mucins and glycoproteins with mucin-like regions contain densely O-glycosylated domains often found in tandem repeat (TR) sequences. These O-glycodomains have traditionally been difficult to characterize because of their resistance to proteolytic digestion, and knowledge of the precise positions of O-glycans is particularly limited for these regions. Here, we took advantage of a recently developed glycoengineered cell-based platform for the display and production of mucin TR reporters with custom-designed O-glycosylation to characterize O-glycodomains derived from mucins and mucin-like glycoproteins. We combined intact mass and bottom-up site-specific analysis for mapping O-glycosites in the mucins, MUC2, MUC20, MUC21, protein P-selectin-glycoprotein ligand 1, and proteoglycan syndecan-3. We found that all the potential Ser/Thr positions in these O-glycodomains were O-glycosylated when expressed in human embryonic kidney 293 SimpleCells (Tn-glycoform). Interestingly, we found that all potential Ser/Thr O-glycosites in TRs derived from secreted mucins and most glycosites from transmembrane mucins were almost fully occupied, whereas TRs from a subset of transmembrane mucins were less efficiently processed. We further used the mucin TR reporters to characterize cleavage sites of glycoproteases StcE (secreted protease of C1 esterase inhibitor from EHEC) and BT4244, revealing more restricted substrate specificities than previously reported. Finally, we conducted a bottom-up analysis of isolated ovine submaxillary mucin, which supported our findings that mucin TRs in general are efficiently O-glycosylated at all potential glycosites. This study provides insight into O-glycosylation of mucins and mucin-like domains, and the strategies developed open the field for wider analysis of native mucins., Competing Interests: Conflict of interest The University of Copenhagen has filed a patent application on the cell-based display platform. GlycoDisplay Aps, Copenhagen, Denmark, has obtained a license to the field of the patent application. Y. N. and H. C. are cofounders of GlycoDisplay Aps and hold ownerships in the company. All the other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Mass spectrometry is gaining momentum as a method of choice to de novo sequence antibodies (Abs). Adequate sequence coverage of the hypervariable regions remains one of the toughest identification challenges by either bottom-up or top-down workflows. Methods that efficiently generate mid-size Ab fragments would further facilitate top-down MS and decrease data complexity. Here, we explore the proteases Cathepsins L and D for forming protein fragments from three IgG1s, one IgG2, and one bispecific, knob-and-hole IgG1. We demonstrate that high-resolution native MS provides a sensitive method for the detection of clipping sites. Both Cathepsins produced multiple, albeit specific cleavages. The Abs were cleaved immediately after the CDR3 region, yielding ~ 12 kDa fragments, that is, ideal sequencing-sized. Cathepsin D, but not Cathepsin L, also cleaved directly below the Ab hinge, releasing the F(ab')2. When constrained by the different disulfide bonds found in the IgG2 subtype or by the tertiary structure of the hole-containing bispecific IgG1, the hinge region digest product was not produced. The Cathepsin L and Cathepsin D clipping motifs were related to sequences of neutral amino acids and the tertiary structure of the Ab. A single pot (L + D) digestion protocol was optimized to achieve 100% efficiency. Nine protein fragments, corresponding to the VL, VH, CL, CH1, CH2, CH3, CL + CH1, and F(ab')2, constituted ~ 70% of the summed intensities of all deconvolved proteolytic products. Cleavage sites were confirmed by the Edman degradation and validated with top-down sequencing. The described work offers a complementary method for middle-down analysis that may be applied to top-down Ab sequencing. ENZYMES: Cathepsin L-EC 3.4.22.15, Cathepsin D-EC 3.4.23.5., (© 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Glycosylation represents a critical quality attribute modulating a myriad of physiochemical properties and effector functions of biotherapeutics. Furthermore, a rising landscape of glycosylated biotherapeutics including biosimilars, biobetters, and fusion proteins harboring complicated and dynamic glycosylation profiles requires tailored analytical approaches capable of characterizing their heterogeneous nature. In this work, we perform in-depth evaluation of the glycosylation profiles of three glycoengineered variants of the widely used biotherapeutic erythropoietin. We analyzed these samples in parallel using a glycopeptide-centric liquid chromatography/mass spectrometry approach and high-resolution native mass spectrometry. Although for all of the studied variants the glycopeptide and native mass spectrometry data were in good qualitative agreement, we observed substantial quantitative differences arising from ionization deficiencies and unwanted neutral losses, in particular, for sialylated glycopeptides in the glycoproteomics approach. However, the latter provides direct information about glycosite localization. We conclude that the combined parallel use of native mass spectrometry and bottom-up glycoproteomics offers superior characterization of glycosylated biotherapeutics and thus provides a valuable attribute in the characterization of glycoengineered proteins and other complex biotherapeutics.

O-GalNAc type glycosylation is an abundant and complex protein modification. Recent developments in mass spectrometry resulted in significant success in quantitative analysis of O-GalNAc glycosylation. The analysis of released O-GalNAc type glycans expanded our horizons of understanding the glycome of various biological models. The site-specific analysis of glycosylation micro-heterogeneity of purified proteins opened perspectives for the improved design of glycoprotein therapeutics. Advanced gene editing and chemical technologies applied to O-glycoproteomics enabled to identify O-GalNAc glycosylation at unprecedented depth. Progress in the analysis of intact glycoproteins under native and reduced conditions enabled the monitoring of glycosylation proteoform variants. Despite of the astonishing results in quantitative O-GalNAc glycoproteomics, site-specific mapping of the full O-GalNAc structural repertoire in complex samples is yet a long way off. Here, we summarize the most common quantitative strategies in O-GalNAc glycoproteomics, review recent progress and discuss benefits and limitations of the various approaches in the field., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Sepsis and septic shock remain the leading causes of death in intensive care units (ICUs), yet the pathogenesis originating from the inflammatory response during sepsis remains ambiguous. Acute-phase proteins are typically highly glycosylated, and the nature of the glycans have been linked to the incidence and severity of such inflammatory responses. To further build upon these findings we here monitored, the longitudinal changes in the plasma proteome and, in molecular detail, glycoproteoform profiles of alpha-1-antichymotrypsin (AACT) extracted from plasma of ten individual septic patients. For each patient we included four different time-points, including post-operative (before sepsis) and following discharge from the ICU. We isolated AACT from plasma depleted for albumin, IgG and serotransferrin and used high-resolution native mass spectrometry to qualitatively and quantitatively monitor the multifaceted glycan microheterogeneity of desialylated AACT, which allowed us to monitor how changes in the glycoproteoform profiles reflected the patient's physiological state. Although we observed a general trend in the remodeling of the AACT glycoproteoform profiles, e.g. increased fucosylation and branching/LacNAc elongation, each patient exhibited unique features and responses, providing a resilient proof-of-concept for the importance of personalized longitudinal glycoproteoform profiling. Importantly, we observed that the AACT glycoproteoform changes induced by sepsis did not readily subside after discharge from ICU., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Čaval, Lin, Varkila, Reiding, Bonten, Cremer, Franc and Heck.)

Mass spectrometry-based glycoproteomics has gone through some incredible developments over the last few years. Technological advances in glycopeptide enrichment, fragmentation methods, and data analysis workflows have enabled the transition of glycoproteomics from a niche application, mainly focused on the characterization of isolated glycoproteins, to a mature technology capable of profiling thousands of intact glycopeptides at once. In addition to numerous biological discoveries catalyzed by the technology, we are also observing an increase in studies focusing on global protein glycosylation and the relationship between multiple glycosylation sites on the same protein. It has become apparent that just describing protein glycosylation in terms of micro- and macro-heterogeneity, respectively, the variation and occupancy of glycans at a given site, is not sufficient to describe the observed interactions between sites. In this perspective we propose a new term, meta-heterogeneity, to describe a higher level of glycan regulation: the variation in glycosylation across multiple sites of a given protein. We provide literature examples of extensive meta-heterogeneity on relevant proteins such as antibodies, erythropoietin, myeloperoxidase, and a number of serum and plasma proteins. Furthermore, we postulate on the possible biological reasons and causes behind the intriguing meta-heterogeneity observed in glycoproteins., Competing Interests: Conflict of interest Authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Glycopeptide-centric mass spectrometry has become a popular approach for studying protein glycosylation. However, current approaches still utilize fragmentation schemes and ranges originally optimized and intended for the analysis of typically much smaller unmodified tryptic peptides. Here, we show that by merely increasing the tandem mass spectrometry m / z range from 2000 to 4000 during electron transfer higher energy collisional dissociation (EThcD) fragmentation, a wealth of highly informative c and z ion fragment ions are additionally detected, facilitating improved identification of glycopeptides. We demonstrate the benefit of this extended mass range on various classes of glycopeptides containing phosphorylated, fucosylated, and/or sialylated N-glycans. We conclude that the current software solutions for glycopeptide identification also require further improvements to realize the full potential of extended mass range glycoproteomics. To stimulate further developments, we provide data sets containing all classes of glycopeptides (high mannose, hybrid, and complex) measured with standard (2000) and extended (4000) m / z range that can be used as test cases for future development of software solutions enhancing automated glycopeptide analysis.

Mannose-6-phosphate (M6P) is a distinctive post-translational modification critical for trafficking of lysosomal acid hydrolases into the lysosome. Improper trafficking into the lysosome, and/or lack of certain hydrolases, results in a toxic accumulation of their substrates within the lysosomes. To gain insight into the enzymes destined to the lysosome these glycoproteins can be distinctively enriched and studied using their unique M6P tag. Here we demonstrate, by adapting a protocol optimized for the enrichment of phosphopeptides using Fe 3+ -IMAC chromatography, that proteome-wide M6P glycopeptides can be selectively enriched and subsequently analyzed by mass spectrometry, taking advantage of exclusive phosphomannose oxonium fragment marker ions. As proof-of-concept of this protocol, applying it to HeLa cells, we identified hundreds of M6P-modified glycopeptides on 35 M6P-modified glycoproteins. We next targeted CHO cells, either wild-type or cells deficient in Acp2 and Acp5, which are acid phosphatases targeting M6P. In the KO CHO cells we observed a 20-fold increase of the abundance of the M6P-modification on endogenous CHO glycoproteins but also on the recombinantly over-expressed lysosomal human alpha-galactosidase. We conclude that our approach could thus be of general interest for characterization of M6P glycoproteomes as well as characterization of lysosomal enzymes used as treatment in enzyme replacement therapies targeting lysosomal storage diseases., (© 2019 Čaval et al.)

Recombinant production of glycoprotein therapeutics like erythropoietin (EPO) in mammalian CHO cells rely on the heterogeneous N-glycosylation capacity of the cell. Recently, approaches for engineering the glycosylation capacities of mammalian cells for custom designed glycoforms have been developed. With these opportunities there is an increasing need for fast, sensitive, and global analysis of the glycoproteoform landscape produced to evaluate homogeneity and consistency. Here we use high-resolution native mass spectrometry to measure the glycoproteoform profile of 24 glycoengineered variants of EPO. Based on the unique mass and intensity profiles of each variant, we classify them according to similarities in glycosylation profiles. The classification distinguishes EPO variants with varying levels of glycan branchingand sialylation, which are crucial parameters in biotherapeutic efficacy. We propose that our methods could be of great benefit in the characterization of other glycosylated biopharmaceuticals, ranging from the initial clonal selection to batch-to-batch controls, and the assessment of similarity between biosimilar/biobetter products.