Biology subjects

Bergen, D.

Publications and source records attributed to Bergen, D..

8 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↗

bioRxiv2025

Co-Substrate Free Valorisation of Lignin Monomers by Assimilation of C1 and C2 By-Products

Lignin is an underutilised global resource with significant potential for the production of chemicals that are currently derived from fossil resources. However, biotechnological lignin valorisation faces various challenges including its recalcitrance and the toxicity of aromatic intermediates, products, and by-products like formaldehyde. While biochemical production from lignin-derived monomers has been demonstrated by disrupting native lignin degradation pathways, this approach required co-feeding additional carbon sources such as glucose for growth. This dependence on additional carbon sources can create competition with the food industry and undermine the economic sustainability of the bioprocess. Here, we report growth of a protocatechuate production strain of Pseudomonas putida EM42 on the by-products from p-coumarate and ferulate valorisation, achieving carbon efficiencies of up to 78 %. Additional flux balance analysis identified C1 assimilation pathways, including two novel pathways, beneficial for the growth on the formaldehyde by-product from ferulate degradation leading to improved carbon utilisation. This study demonstrates how by-product utilisation from lignin conversion can eliminate the need for co-feeding additional carbon sources, thereby potentially improving the efficiency of lignin valorisation.

bioengineering↗

bioRxiv2025

Systematic engineering of synthetic serine cycles in Pseudomonas putida uncovers emergent topologies for methanol assimilation

The urgent need for a circular carbon economy has driven research into sustainable substrates, including one-carbon (C1) compounds. The non-pathogenic soil bacterium Pseudomonas putida is a promising host for exploring synthetic methylotrophy due to its versatile metabolism. In this work, we implemented synthetic serine cycle variants in P. putida for methanol assimilation combining modular engineering and growth-coupled selection, whereby methanol assimilation supported biosynthesis of the essential amino acid serine. The serine cycle forms acetyl-coenzyme A from C1 molecules without carbon loss but has bottlenecks that hinder engineering efforts. We adopted three synthetic variants (serine-threonine cycle, homoserine cycle, and modified serine cycle) that yield serine in a methanol-dependent fashion to overcome these challenges. By dividing these metabolic designs into functional modules, we systematically compared their performance for implementation in vivo. Additionally, we harnessed native pyrroloquinoline quinone-dependent dehydrogenases for engineering methylotrophy. Recursive rewiring of synthetic and native activities revealed novel metabolic topologies for methanol utilization, termed enhanced serine-threonine cycle, providing a blueprint for engineering C1 assimilation in non-model heterotrophic bacteria. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/638773v1_ufig1.gif" ALT="Figure 1"> View larger version (79K): org.highwire.dtl.DTLVardef@1bfccd0org.highwire.dtl.DTLVardef@11f77deorg.highwire.dtl.DTLVardef@f6a904org.highwire.dtl.DTLVardef@1d24a34_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2020

Giantin is required for 1 intracellular N-terminal processing of type I procollagen

Knockout of the golgin giantin leads to skeletal and craniofacial defects driven by poorly studied changes in glycosylation and extracellular matrix deposition. Here, we sought to determine how giantin impacts the production of healthy bone tissue by focussing on the main protein component of the osteoid, type I collagen. Giantin mutant zebrafish accumulate multiple spontaneous fractures in their caudal fin, suggesting their bones may be more brittle. Inducing new experimental fractures revealed defects in the mineralisation of newly deposited collagen as well as diminished procollagen reporter expression in mutant fish. Analysis of giantin knockout cells expressing a GFP-tagged procollagen showed that procollagen trafficking is independent of giantin. However, our data show that intracellular N-propeptide processing of pro-1(I) is defective in the absence of giantin. These data demonstrate a conserved role for giantin in collagen biosynthesis and extracellular matrix assembly. Our work also provides evidence of a giantin-dependent pathway for intracellular procollagen processing.

cell biology↗

bioRxiv2019

A rare SMAD9 mutation identifies the BMP signalling pathway as a potential osteoanabolic target

To identify targets for novel anabolic medicines for osteoporosis, we recruited a large cohort with unexplained high bone mass (HBM). Exome sequencing identified a rare (minor allele frequency 0.0014) missense mutation in SMAD9 (c.65T>C, p.Leu22Pro) segregating with HBM in an autosomal dominant family. The same mutation was identified in another two unrelated individuals with HBM. In-silico protein modelling predicts the mutation severely disrupts the MH1 DNA-binding domain of SMAD9. Affected individuals have bone mineral density [BMD] Z-Scores +3 to +5, with increased volumetric cortical and trabecular BMD, increased cortical thickness, and low/normal bone turnover. Fractures and nerve compressions are not seen. Both genome-wide, and gene-based association testing of heel estimated-BMD in >362,924 UK-Biobank British subjects showed strong associations with SMAD9 (PGWAS=6x10-16; PGENE =8x10-17). Smad9 is highly expressed in murine osteocytes and zebrafish bone tissue. Our findings support SMAD9 as a novel HBM gene, and a potential novel osteoanabolic target.

genetics↗

bioRxiv2017

Regulator of calcineurin-2 is a ciliary protein with a role in cilia length control.

Almost every cell in the human body extends a primary cilium. Defective cilia function leads to a set of disorders known as ciliopathies characterised by debilitating developmental defects affecting many tissues. Here we report a new role for regulator of calcineurin 2, RCAN2, in primary cilia function. It localises to centrioles and the basal body and is required to maintain normal cilia length. RCAN2 was identified as the most strongly upregulated gene from a comparative RNAseq analysis of cells in which expression of the Golgi matrix protein giantin had been abolished by gene editing. In contrast to previous work where we showed that depletion of giantin by RNAi results in defects in ciliogenesis and in cilia length control, giantin knockout cells generate normal cilia on serum withdrawal. Furthermore, giantin knockout zebrafish show increased expression of RCAN2. Importantly, suppression of RCAN2 expression in giantin knockout cells results in the same defects in cilia length control seen on RNAi of giantin itself. Together these data define RCAN2 as a regulator of cilia function that can compensate for loss of giantin function.

cell biology↗

bioRxiv2017

Giantin Knockout Models Reveal The Capacity Of The Golgi To Regulate Its Biochemistry By Controlling Glycosyltransferase Expression

The Golgi is the cellular hub for complex glycosylation, controlling accurate processing of complex proteoglycans, receptors, ligands, and glycolipids. Its structure and organisation is dependent on golgins, which tether cisternal membranes and incoming transport vesicles. Here we show that knockout of the largest golgin, giantin, leads to substantial changes in gene expression despite only limited effects on Golgi structure. Notably, 22 Golgi-resident glycosyltransferases, but not glycan processing enzymes or the ER glycosylation machinery, are differentially expressed following giantin ablation. This includes near-complete loss-of-function of GALNT3 in both mammalian cell and zebrafish models. Giantin knockout zebrafish exhibit hyperostosis and ectopic calcium deposits, recapitulating phenotypes of hyperphosphatemic familial tumoral calcinosis, a disease caused by mutations in GALNT3. These data reveal a new feature of Golgi homeostasis, the ability to regulate glycosyltransferase expression to generate a functional proteoglycome.\n\nSummary statementKnockout of giantin in a genome-engineered cell line and zebrafish models reveals the capacity of the Golgi to control its own biochemistry through changes in gene expression.

cell biology↗