Biology subjects

Mullen, C.

Publications and source records attributed to Mullen, C..

7 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

Beyond HCD: complementary fragmentation chemistries expand HLA class I immunopeptidome discovery

Mass spectrometry based immunopeptidomics is limited by peptide identification. HLA class I ligands are non-tryptic, frequently carry low charge states and often fragment incompletely, yet nearly all workflows rely on a single activation method, higher-energy collisional dissociation (HCD). Unlike proteomics, where a protein is inferred from several peptides, in immunopeptidomics the peptide is itself the analyte and the candidate therapeutic target, so a ligand that is not identified is a target that is not found. Here we evaluate whether two alternative activation chemistries, ion trap collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD), recover ligands that HCD does not. To our knowledge this is the first application of either method to HLA class I immunopeptidomics, made practical by the integrated infrared laser of the Orbitrap Tribrid Apex MultiOmics. Four HLA-typed cell lines were analysed in technical triplicate against the canonical human proteome and a Ribo-seq-derived cryptic ORF database. Because fragment-intensity predictors are trained almost exclusively on HCD spectra, AlphaPeptDeep was fine-tuned separately for each method; fine-tuning and Percolator rescoring increased unique peptide identifications in every cell line and method combination (median +17.1% canonical, +54.5% cryptic), and fine-tuned models transferred across cell lines with a median loss of 0.4%, placing the three methods on a common analytical footing. On that footing the three methods yielded immunopeptidomes of similar size and composition, but each recovered peptides the others missed. HCD produced the most complete fragment-ion ladders and recovered more peptides from the basic-anchor alleles A*11:01 and A*33:03, both members of the A3 supertype. CID and IRMPD recovered a larger share of alleles without a basic C-terminal anchor and, for peptides containing no lysine, arginine or histidine, produced higher search scores and more matched b/y ion current in three of four cell lines. Each method therefore contributed exclusive peptides, and at a fixed budget of three LC-MS/MS runs, one run per method yielded on average more unique peptides than triplicate runs of a single method in all four cell lines (+0.4% to +10.9%). Of 239 novel canonical predicted HLA binders in the two cancer cell lines (HCT116 and MDA-MB-231) absent from the Immune Epitope Database, 55% were detected by only one method; nine derive from cancer-associated antigens. CID and IRMPD can therefore provide sequences that may not be identified by HCD alone, or with better spectral quality for a chemically defined subset of ligands. Their value lies alongside HCD rather than in its place, and realising it will require fragment-intensity models and acquisition schemes built for these chemistries rather than adapted from HCD.

immunology↗

bioRxiv2026

Cholesterol and p53 promote senescence and systemic fibrosis in metabolic dysfunction-associated steatohepatitis

Background & aims: TP53 (p53) coordinates diverse cellular stress response programmes including pro-survival activities, senescence, and cell death. During tissue damage, p53 can shape both the local cellular response to injury, including the fibrotic response, and influence distal organ biology. Fibrosis in the liver is a major driver of hepatocellular carcinoma (HCC) risk within metabolic dysfunction-associated steatohepatitis (MASH). It is also an important determinant of dysfunction in multiple distal tissues including the kidneys, lungs, and heart. Despite significant clinical burden, our understanding of the molecular determinants of fibrotic MASH and its relationship to multiorgan fibrosis remain incomplete. Here, we investigate local and systemic effects of hepatocellular p53 activity and cholesterol during MASH development, with implications for disease prevention. Methods: This study utilised a genetic model of stabilised p53, diet-induced MASH models with varying cholesterol compositions, and an in vitro obesogenic system to investigate p53 activity during liver disease development. Non-invasive imaging and histopathological analyses were employed to monitor p53 activity, MASH, and multiorgan fibrosis in vivo. Complementary approaches, including in vitro human multicomponent liver spheroids, cytokine arrays, and analyses of human MASH transcriptomic and proteomic datasets, were used to examine molecular drivers and patient relevance. Results: Using an inducible mouse model of MDM2 E3 ubiquitin ligase deficiency to stabilise p53, we report that hepatocellular MDM2 E3 loss results in progressive fibrotic damage, robust hepatocellular expression of the p53 target gene CDKN1A/p21 (p21),and induces p21 and fibrosis in the kidneys of male mice in a sex-specific manner. In diet-induced MASH, we observe cholesterol and p53-dependent development of liver fibrosis, high expression of hepatocellular p21, and induction of p21 and fibrosis in the kidneys of male mice-reminiscent of features observed in MDM2 E3-deficient mice. We also observe fibrosis in the lungs and heart of male MASH mice. Both a cholesterol-free obesogenic diet and liver-specific loss of p53 mitigate hepatic fibrosis and systemic induction of p21 and fibrosis. Mechanistically, p53 induces hepatic expression of senescence-associated secretory phenotype (SASP) factors, including GDF15, in vivo. A human multicomponent LiverACE spheroid model showed a concordant trend towards increased GDF15 protein abundance under steatotic stress, while in humans, elevated circulating GDF15 levels in advanced MASH correlate with increased TNFRSF1A and EPHA2, circulating markers linked to kidney injury. Conclusions: Our work identifies undue p53 activity within the liver as a driver of multiorgan fibrosis in a sex-specific manner, affecting male but not female mice. We implicate cholesterol in promoting this pro-fibrotic environment in vivo and highlight circulating factors that could identify at-risk patients for multiorgan fibrosis in MASH.

cancer 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

Defining state-selective lipid binding to brain GPCRs - introducing REVEAL

Membrane lipids are central regulators of G protein-coupled receptor (GPCR) function. Defining receptor-specific lipid interactions in native, fully modified mammalian systems remains challenging. Extensive post-translational modifications generate heterogeneous proteoforms that confound conventional mass spectrometry approaches. Here we introduce REVEAL (REceptor enVironment Elucidation by Activated Lipid-release), an automated native top-down mass spectrometry strategy that discriminates specifically bound lipids from background. Applied here to intact, heterogeneous mammalian membrane protein complexes, incubated with a brain polar lipid extract (>1000 components), we define receptor-specific lipid-binding for two neuronal class C GPCRs. We show that agonism remodels lipid occupancy, selectively enriching a reduced repertoire of bound lipids. Plasmalogen lipids emerge as persistent binders across all conformational states of the metabotropic glycine receptor and are preferentially depleted under oxidative stress, implying a protective role at the receptor surface. These findings position lipids as dynamic regulators of both function and response to the cellular redox environment.

biophysics↗

bioRxiv2026

Native Mass Spectrometry-Based Proteomics Reveals the Mechanism of Hemophore Release by Pathogenic Corynebacterium diphtheriae

Iron is an essential micronutrient for nearly all forms of life, including pathogenic microbes that must acquire it from their host during infection. At the host-pathogen interface, humans restrict microbial access to iron through nutritional immunity, which many pathogens overcome by secreting hemophores that scavenge extracellular heme (iron protoporphyrin IX). However, identifying hemophores and other ligand-binding proteins in complex proteomes remains challenging using conventional peptide-based bottom-up mass spectrometry (MS). Here, we introduce ProteoMIX (Proteome Analysis by Mixing), a function-based native top-down proteomics workflow that combines slow-mixing mode native MS with charge reduction to identify ligand-binding proteins directly from complex mixtures. Applying ProteoMIX to the Corynebacterium diphtheriae exoproteome identified ChtA30-314, an abundant soluble hemophore generated by proteolytic processing of the surface-exposed ChtA heme receptor. Using native top-down MS sequencing, cell fractionation, and gene deletion, we show that the protease DIP2069 releases ChtA30-314 by removing ChtAs transmembrane helix, producing a soluble proteoform that delivers heme to support microbial growth. In contrast, the related paralog ChtC is not processed, enabling C. diphtheriae to generate localization-specific heme-binding proteoforms from related gene products. Extending this approach to Staphylococcus aureus, ProteoMIX also revealed soluble IsdA hemophores that coexist with surface-anchored variants, demonstrating the generality of the method and suggesting that protease-mediated hemophore release may operate across gram-positive pathogens.

microbiology↗

bioRxiv2023

Exposing the molecular heterogeneity of glycosylated biotherapeutics

Glycosylated biotherapeutics are an emerging class of drugs with high molecular heterogeneity, which can affect their safety and efficacy. Characterizing this heterogeneity is crucial for drug development and quality assessment, but existing methods are limited in their ability to analyze intact glycoproteins. Here, we present a new approach to glycoform fingerprinting that uses proton-transfer charge-reduction with gas-phase fractionation to analyze intact glycoproteins by mass spectrometry. The method provides a detailed landscape of the intact molecular weights present in biotherapeutic protein preparations in a single experiment and offers insights into glycoform composition when coupled with a suitable bioinformatic strategy. We tested the approach on various biotherapeutic molecules, including Fc-fusion, VHH-fusion, and peptide-bound MHC class II complexes to demonstrate efficacy in measuring the proteoform-level diversity of biotherapeutics. Notably, we inferred the glycoform distribution for hundreds of molecular weights for the eight-times glycosylated fusion drug IL22-Fc, enabling correlations between glycoform sub-populations and the drugs pharmacological properties. Our method is broadly applicable and provides a powerful tool to assess the molecular heterogeneity of emerging biotherapeutics.

biochemistry↗