Biology subjects

Veth, T. S.

Publications and source records attributed to Veth, T. S..

9 recordsLinked to original sources

bioRxiv2026

Unifying N- and O-glycopeptide dissociation methods with expedient electron transfer dissociation

The glycoproteome is highly heterogeneous, where each glycosite can be modified by potentially hundreds of glycan compositions. This complexity necessitates methods that can provide glycosite-specific characterization in a high-throughput manner. Electron transfer dissociation (ETD) is particularly valuable for glycopeptide analysis, where it is critical for glycosite localization in O-glycopeptides and often produces higher quality spectra than higher energy collisional dissociation (HCD) for N-glycopeptides. Despite this, lower quality HCD spectra are often favored in high-throughput glycoproteomics experiments because the near instantaneous fragmentation of HCD results in faster MS/MS spectral acquisition relative to ETD. In short, overhead times for ETD reactions limit acquisition rates and sensitivity that can outweigh its advantages. Here we implement changes to ion handling, scan parallelization, and ion-ion reaction conditions to increase ETD MS/MS acquisition rates on the Orbitrap Tribrid MS platform. We explore fundamental parameters of ETD ion-ion reactions, including reaction q-values (i.e., Mathieu q-values) and ion-ion reaction time, showing that we can reduce the overhead of ion-ion reaction scan functions and improve the overall synchronization of data acquisition. These optimizations lead to faster spectral acquisition times for ETD MS/MS spectra while maintaining high spectral quality. Ultimately, these improvements allow the ETD scan functions to be nested within ion accumulation times needed per MS/MS spectrum, enabling ETD spectral acquisition to be parallelized within the duty cycle of the instrument and removing the major time cost to doing ETD-based fragmentation. We argue that this expedient implementation of ETD makes ETD-based fragmentation a universally beneficial dissociation method for both N- and O-glycopeptides.

systems biology↗

bioRxiv2026

Defining glycoproteoform landscapes through an integrated glycoproteomics approach enabled by high-resolving power proton transfer charge reduction tandem mass spectrometry

Glycan heterogeneity is a fundamental property of glycoproteins. A holistic understanding of glycan modification states is critical to translating glycoproteome regulation to biological function, but the high degree of glycosite-level heterogeneity leads to technical challenges in measuring glycoproteoforms. Common bottom-up glycoproteomics provide some insights but cannot recapitulate the full ensemble of glycoproteoforms from glycopeptide measurements alone. Promising efforts to profile masses of intact glycoproteins have recently explored data-independent acquisition (DIA) coupled with proton-transfer charge reduction (PTCR) or electron-capture-induced charge reduction mass spectrometry (MS). While valuable for generating broad glycoproteoform mass distributions, these approaches have remained limited in their ability to generate discrete glycoproteoform mass measurements, largely because they rely on low-resolving power measurements and deconvolution that does not account for isotopic information. Here, we develop a DIA-PTCR workflow that couples high-resolving power (Rp ~240,000 at m/z 200) tandem mass spectra with an open-source processing suite to define glycoproteoform populations within 20 ppm mass accuracy thresholds. We demonstrate the glycoproteoform characterization capabilities of this platform using a collection of glycoproteins with well-described translational interests (EpCAM, TIGIT, CD40, PDL1, and CD24). With a focus on EpCAM, we showcase how intact glycoproteoform masses acquired using our high-resolving power DIA-PTCR (hRp-DIA-PTCR) approach can be integrated with bottom-up intact glycoproteomics and Direct-Mass Technology (i.e., Orbitrap-based charge-detection MS) acquisitions to inform structural and biological insights. Altogether, our hRp-DIA-PTCR method extends the current capabilities of intact glycoprotein analyses by enabling robust characterization of isotopically resolved proteoforms and facilitating deep biological interpretation of glycosylation heterogeneity. Our open-source informatics platform includes a GUI-based tool called PTsliCR to clean PTCR spectra directly from DIA-PTCR raw files and a deconvolution R package called IsoTrac, both of which are freely available on GitHub at https://github.com/riley-research.

systems biology↗

bioRxiv2026

GlycoDiveR: a modular R framework to analyze and visualize highly dimensional glycoproteomics data

Mass spectrometry-based glycoproteomics is a critical platform for understanding the complex roles of protein glycosylation in biological systems, yet visualizing multidimensional glycoproteomics datasets remains a significant bottleneck in data interpretation and communication. Glycan microheterogeneity, i.e., the potential for a glycosite to be modified by multiple glycans, defies the binary presence-absence logic used in analyses of other post-translational modifications. Instead, glycoproteomics necessitates intentionally designed data structures and visualizations that are glycoform-centric, not just site-centric. Additionally, there is a need for complementary degrees of data analysis that alternate between glycoproteome-scale patterns and glycosite-specific regulation. Several bespoke frameworks for visualizing glycoproteomics data have emerged, but they often require advanced programming expertise and are designed for a single study rather than broad application. Here, we present our efforts to harmonize post-search data analysis of glycoproteomics through a modular R framework called GlycoDiveR. This platform streamlines import, transformation, and curation of qualitative and quantitative glycopeptide identifications, including support for raw output from multiple search engines. GlycoDiveR is designed to integrate seamlessly into existing analysis workflows by enabling fast, flexible exploration of highly dimensional glycoproteomics datasets via a consistently formatted data architecture. Our goal is to offer a customizable set of glycosylation-specific visualizations with minimal coding, while keeping data accessible to users who wish to further customize their characterization strategies. It also maintains a modular design that supports the continual addition of visualizations, analyses, and export functions. Ultimately, GlycoDiveR is meant to improve accessibility of glycoproteomic-specific analyses and lower the barrier to exploring biological narratives embedded in rich glycoproteomic datasets. GlycoDiveR is open-source and freely available at https://github.com/riley-research/GlycoDiveR.

systems biology↗

bioRxiv2025

Sialoglycans on human T cells attenuate death programs executed through the Fas pathway

T cells are critical executors of adaptive immune responses and their persistence is tightly regulated. Part of this regulation relies on programmed cell death driven by the Tumor Necrosis Factor (TNF) receptor superfamily. The addition of glycans that terminate in the monosaccharide sialic acid (sialoglycans) to these cell death receptors has been shown to attenuate their apoptotic functions. While this is now understood to be a pro-survival mechanism in settings of cancer pathophysiology, the specific roles of sialoglycans in regulating cell death receptor activity on human T cells remains unexplored. This is of particular importance given the rising interest in T cell glycan editing for therapeutic benefit. Here, we address this gap using both immortalized (Jurkat) and primary human T cells deficient in sialoglycans. We found that T cell sialoglycans suppressed apoptosis induced by the Fas receptor (FasR) but not other TNF receptor superfamily members such as TNFR1 and TRAIL-R1. Dynamic reorganization of FasR was increased on sialoglycan-deficient Jurkat cells, suggesting that these glycans limit receptor clustering. This model was further supported by phosphoproteomics results, which confirmed that loss of sialoglycans negatively regulated the pro-survival MAPK/ERK signalling pathway. Finally, we used a recombinant sialic acid cleaving enzyme (sialidase) to confirm that sialoglycans on primary human T cells are bona fide immunophysiological regulators of FasR-driven programmed cell death. Combined, our results demonstrate that sialoglycan remodelling on T cells influences cell fate driven by the Fas pathway and provide motivation to further characterize the immunoregulatory roles of the glycocalyx in health and disease.

immunology↗

bioRxiv2025

Extracting informative glycan-specific ions from glycopeptide MS/MS spectra with GlyCounter

Glycopeptide tandem mass spectra typically contain numerous glycan-specific fragments that can inform several features of glycan modifications, including glycan class, composition, and structure. While these fragment ions are often straightforward to observe by eye, few tools exist to systemically explore these common glycopeptide spectral features or explore their relationships to each other. Instead, most studies rely on manual inspection to understand glycan-informative ion content in their data, or they are restricted to evaluating the presence of these ions only in the small fraction of spectra that are identified by glycopeptide search algorithms. Here we introduce GlyCounter as a freely available, open-source tool to rapidly extract oxonium, Y-type, and custom ion information from raw data files. We highlight GlyCounters utility by evaluating glycan-specific fragments in a diverse selection of publicly available datasets to demonstrate how others in the field can make immediate use of this software. In several cases, we show how conclusions drawn in these publications are evident simply through GlyCounters extracted ion information without requiring database searches or experiment-specific programs. Although one of our goals is to decouple spectral evaluation from glycopeptide identification, we also show that evaluating oxonium ion content with GlyCounter can supplement a database search as valuable spectral evidence to validate conclusions. In all, we present GlyCounter as a user-friendly platform that can be easily incorporated into most glycoproteomic workflows to refine sample preparation, data acquisition, and post-acquisition identification methods through straightforward evaluation of the glycan content of glycoproteomic data. Software and instructions are available at https://github.com/riley-research/GlyCounter.

bioinformatics↗

bioRxiv2024

Antibodies disrupt bacterial adhesion by ligand mimicry and allosteric interference

A critical step in infections is the attachment of many microorganisms to host cells using lectins that bind surface glycans, making lectins promising antimicrobial targets. Upon binding mannosylated glycans, FimH, the most studied lectin adhesin of type 1 fimbriae in E. coli, undergoes an allosteric transition from an inactive to an active conformation that can act as a catch-bond. Monoclonal antibodies that alter FimH glycan binding in various ways are available, but the mechanisms of these antibodies remain unclear. Here, we use cryoEM, mass spectrometry, binding assays, and molecular dynamics simulations to determine the structure-function relationships underlying antibody-FimH binding. Our study reveals four distinct antibody mechanisms of action: ligand mimicry by an N-linked, high-mannose glycan; stabilization of the ligand pocket in the inactive state; conformational trapping of the active and inactive states; and locking of the ligand pocket through long-range allosteric effects. These structures reveal multiple mechanisms of antibody responses to an allosteric protein and provide blueprints for new antimicrobial that target adhesins.

immunology↗

bioRxiv2023

Multilevel Plasticity and Altered Glycosylation Drive Aggressiveness in Hypoxic and Glucose-Deprived Bladder Cancer Cells

Bladder tumours with aggressive characteristics often present with microenvironmental niches marked by low oxygen levels (hypoxia) and limited glucose supply due to inadequate vascularization. The molecular mechanisms facilitating cellular adaptation to these stimuli remain largely elusive. Employing a multi-omics approach, we discovered that hypoxic and glucose- deprived cancer cells enter a quiescent state supported by mitophagy, fatty acid {beta}-oxidation, and amino acid catabolism, concurrently enhancing their invasive capabilities. Reoxygenation and glucose restoration efficiently reversed cell quiescence without affecting cellular viability, highlighting significant molecular plasticity in adapting to microenvironmental challenges. Furthermore, cancer cells exhibited substantial perturbation of protein O-glycosylation, leading to simplified glycophenotypes with shorter glycosidic chains. Exploiting glycoengineered cell models, we established that immature glycosylation contributes to reduced cell proliferation and increased invasion. Our findings collectively indicate that hypoxia and glucose deprivation trigger cancer aggressiveness, reflecting an adaptive escape mechanism underpinned by altered metabolism and protein glycosylation, providing grounds for clinical intervention.

biochemistry↗

bioRxiv2023

CD5L is a canonical component of circulatory IgM

Immunoglobulin M (IgM) is an evolutionary conserved key component of humoral immunity, and the first antibody isotype to emerge during an immune response. IgM is a large (1 MDa), multimeric protein, for which both hexameric and pentameric structures have been described, the latter additionally containing a joining (J) chain. Using a combination of single-particle mass spectrometry and mass photometry, proteomics and immunochemical assays, we here demonstrate that circulatory (serum) IgM exclusively exists as a complex of J-chain-containing pentamers covalently bound to the small CD5 antigen-like (CD5L, 36 kDa) protein. In sharp contrast, secretory IgM in saliva and milk is principally devoid of CD5L. Unlike IgM itself, CD5L is not produced by B cells, implying that it associates with IgM in the extracellular space. We demonstrate that CD5L integration has functional implications, i.e., it diminishes IgM binding to two of its receptors, the Fc{micro}R and the polymeric Immunoglobulin receptor (pIgR). On the other hand, binding to Fc{micro}R as well as complement activation via C1q seem unaffected by CD5L integration. Taken together, we redefine the composition of circulatory IgM as a J-chain containing pentamer, always in complex with CD5L.

molecular biology↗

bioRxiv2023

Elucidating Fibroblast Growth Factor-induced kinome dynamics using targeted mass spectrometry and dynamic modeling

Fibroblast growth factors (FGFs) are paracrine or endocrine signaling proteins that, activated by their ligands, elicit a wide range of health and disease-related processes, such as cell proliferation and the epithelial-to-mesenchymal transition (EMT). The detailed molecular pathway dynamics that coordinate these responses have remained to be determined. To elucidate these, we stimulated MCF-7 breast cancer cells with either FGF2, FGF3, FGF4, FGF10, or FGF19. Following activation of the receptor, we quantified the kinase activity dynamics of 44 kinases using a targeted mass spectrometry assay. Our system-wide kinase activity data, supplemented with (phospho)proteomics data, reveal ligand-dependent distinct pathway dynamics, elucidate the involvement of not earlier reported kinases such as MARK, and revise some of the pathway effects on biological outcomes. In addition, logic-based dynamic modeling of the kinome dynamics further verifies the biological goodness-of-fit of the predicted models and reveals tight regulation of the RAF kinase family.

systems biology↗