Biology subjects

Viner, R.

Publications and source records attributed to Viner, R..

17 recordsLinked to original sources

bioRxiv2026

Simultaneous single-cell profiling of the transcriptome and proteome

Transcriptomic and proteomic measurements from the same single cell provide complementary information that cannot be inferred from either modality alone, yet methods for the parallel recovery of both analyte classes from a single-cell lysate remain limited. Here, we describe a workflow in which individual cells are isolated by automated dispensing into a minimal, MS-compatible lysis volume, followed by sequential mRNA capture and protein supernatant recovery, prior to independent downstream processing. The method is compatible with standard library preparation and data-independent acquisition proteomics pipelines and requires no dedicated instrumentation beyond a single-cell dispensing platform. We evaluated workflow performance on 67 single cells across 3 iBlastoids. Transcriptomic sequencing detected a median of 5375 genes per cell, and proteomic analysis identified a median of 2123 protein groups per cell across two mass spectrometry platforms. Compared with a standalone single-cell proteomics protocol, incorporating the mRNA extraction step reduced median proteomic depth by approximately 11% (median 1,965 vs. 2,204 protein groups per cell), while mean per-cell identification remained comparable across workflows (1,790 vs. 1,775 protein groups per cell). Direct comparison of paired transcript and protein abundance yielded a median Spearman correlation of {rho} {approx} 0.38; after correction for detection depth, the partial correlation was 0.067.

systems biology↗

bioRxiv2025

Allosteric effects of the coupling cation in melibiose transporter MelB

The major facilitator superfamily (MFS) transporters play significant roles in human health and disease. Salmonella enterica serovar Typhimurium melibiose permease (MelBSt), which catalyzes the symport of galactosides with Na+, H+, or Li+, is a prototype of this important transporter superfamily. We have published the structures of the inward- and outward-facing conformations of MelBSt with galactoside or Na+ bound, determined the binding thermodynamic cycle, and proposed that positive cooperativity between the two co-transported solutes plays a key role in the symport mechanism of MelBSt. The molecular basis for this core mechanism remains unclear. In this study, we determined the molecular basis for this core symport mechanism through analyzing the structural dynamics of MelBSt and effects induced by melibiose, Na+, or both using hydrogen-deuterium exchange mass spectrometry (HDX-MS). We also refined the specific determinants for the sugar recognition in both protein and galactoside molecules by solving the crystal structures of a uniporter D59C MelBSt bound to melibiose and other sugars, and identified a critical water molecule as part of sugar recognition. Our integrated studies from structure, HDX-MS, and molecular dynamics simulations support the conclusion that sugar-binding affinity is directly correlated with protein dynamics. The binding of the coupling cation at a remote site functions as an allosteric activator to restrain the conformational flexibility of dynamic residues in the sugar-binding site and in the cytoplasmic gating salt-bridge network, thereby increasing sugar-binding affinity allosterically. This study provides a molecular-level schematic of the fundamental symport mechanism via positive cooperativity, which may serve as a general mechanism for cation-coupled symporters.

biophysics↗

bioRxiv2025

Membrane phosphoinositides allosterically tune β-arrestin dynamics to facilitate GPCR core engagement

Arrestin proteins bind active G protein-coupled receptors (GPCRs) through coordinated protein-protein, protein-phosphate, and protein-lipid interactions to attenuate G protein signaling and promote GPCR internalization and trafficking. While there are hundreds of diverse GPCRs, only two {beta}-arrestin isoforms ({beta}arrs) must recognize and engage this wide range of receptors with varied phosphorylation patterns. Traditional models suggest that {beta}arr activation requires displacement of its autoinhibitory C-tail by a phosphorylated GPCR C-terminus; however, this paradigm fails to explain how minimally phosphorylated GPCRs still complex with {beta}arrs. Using single-molecule Forster resonance energy transfer imaging and hydrogen-deuterium exchange mass spectrometry, we observe basal dynamics in which the {beta}arr1 C-tail spontaneously releases from the N-domain, transiently adopting an active conformation that can facilitate binding of the phosphorylated GPCR C-terminus. We further demonstrate the importance of an intermediate state of {beta}arr1 arising from spontaneous C-tail release stabilized by the membrane phosphoinositide PI(4,5)P2. Both PI(4,5)P2 and mutations in the proximal or middle regions of the C-tail shift {beta}arr1 towards a partially released state, revealing an allosteric connection that informs a refined model for {beta}arr activation. In this model, membrane engagement conformationally primes {beta}arrs prior to receptor binding, thereby explaining how {beta}arrs are recruited by diverse GPCRs, even those with limited C-terminal phosphorylation.

biophysics↗

bioRxiv2025

Assessment of Reported Error Rates in Crosslinking Mass Spectrometry

We benchmarked eight software tools in crosslinking mass spectrometry (MS) to evaluate their accuracy in detecting protein-protein interactions. Whereas about half performed reliably, others reported up to 31-fold more false interactions than indicated by their reported false discovery rates. During this community-driven effort, we helped correct one of the poorer-performing tools. Our findings demonstrate that, with robust algorithms, crosslinking MS can deliver highly dependable insights into biological systems.

bioinformatics↗

bioRxiv2025

AlphaCross-XL: a seamless tool for automated and proteome-scale map-ping of crosslinked peptides onto three-dimensional protein structures

Crosslinking mass spectrometry (XL-MS) is an exciting proteomics technology to capture native protein conformations in real time within biological systems. Historically, however, implementation of this technology has typically been limited to single purified recombinant proteins or in vitro assembled protein complexes. These limitations are associated with inherent challenges in XL-MS analysis, including extremely low abundance of crosslinked (XL) peptides and complex deconvolution of XL peptide-derived spectral data. However, impressive recent developments in computation and instrumentation have now made it feasible to address biological questions using proteome-wide XL-MS analysis. Although some XL mapping software tools exist, these require manual input of specific Protein Data Bank (PDB) structures at the single protein level, and do not function at the high throughput scale required to analyze datasets derived from thousands of proteins. To address this need, we therefore sought to develop a strategy enabling automated mapping of XL peptides onto the three-dimensional (3D) structures of proteins, at a proteome-wide scale. Herein we describe AlphaCross-XL, a first-in-class seamless computational tool for automated mapping of XL peptides onto the protein structures for intra-protein crosslinks and loop-links. The AlphaCross-XL software first retrieves protein structures from the AlphaFold Protein Structure Database and maps all the identified crosslinks onto the 3D structure. It also calculates the Euclidian distance between the crosslinked residues and reports the violated and satisfied crosslink distances based on a user-defined distance threshold, which is visually discriminated by color in PyMOL. Lastly, the tool also supports further validation of user-submitted protein structures, that can include any computer-predicted protein structure and experimentally derived protein structures (i.e., from PDB). AlphaCross-XL is available at https://github.com/sanjyotshenoy/alphacross-xl.

bioinformatics↗

bioRxiv2025

Recommendations for Automating Hydrogen/Deuterium Exchange Mass Spectrometry Measurements using Data-Independent Acquisition Methods

Hydrogen/deuterium exchange mass spectrometry (HX-MS) is a method used to study solution-phase protein structure and dynamics. Despite its many applications, HX-MS is limited in throughput because manual data analysis is still the norm. We previously developed HX-MS2 technology to add a second dimension of deuteration data and promote automated data processing. Data-independent acquisition (DIA) techniques enable this approach, but we require optimized methods for best performance. Using an Orbitrap Eclipse for illustration, we show that ion optics and collision energy settings typical of a proteomics DIA experiment generate maximal peptide retrieval from the DIA library. As few as three MS2 sequence ions are sufficient to generate a deuteration measurement with a precision that exceeds what is possible in traditional HX-MS. DIA window sizes are based on the chromatographic resolution of the method. An inter-scan window offset method is the recommended default configuration for most HX-DIA applications, but an intra-scan overlap method can be tuned for highest performance and is recommended when maximum peptide retrieval is desired. A fully automated HX-MS2 solution consists of Trajan HDX automation technology, an Orbitrap Eclipse mass spectrometer and AutoHX software. We demonstrate its robustness on an extensive time-course analysis of phosphorylase B and an epitope analysis of single domain antibodies (VHHs, nanobodies) specific to the receptor binding domain of SARS-CoV2 spike protein.

biochemistry↗

bioRxiv2025

Structural basis of K11/K48-branched ubiquitin chain recognition by the human 26S proteasome

Beyond the canonical K48-linked homotypic polyubiquitination for proteasome-targeted proteolysis, K11/K48-branched ubiquitin (Ub) chains are involved in fast-tracking protein turnover during cell cycle progression and proteotoxic stress. Here, we report cryo-EM structures of human 26S proteasome in a complex with a K11/K48-branched Ub chain. The structures revealed a multivalent substrate recognition mechanism involving a hitherto unknown K11-linked Ub binding site at the groove formed by RPN2 and RPN10 in addition to the canonical K48-linkage binding site formed by RPN10 and RPT4/5 coiled-coil. Additionally, RPN2 recognizes an alternating K11-K48 linkage through a conserved motif similar to the K48-specific T1 binding site of RPN1. The insights gleaned from these structures explain the molecular mechanism underlying the recognition of the K11/K48 branched Ub as a priority signal in the ubiquitin-mediated proteasomal degradation.

biochemistry↗

bioRxiv2024

Chemical crosslinking extends and complements UV crosslinking in analysis of RNA/DNA nucleic acid-protein interaction sites by mass spectrometry

UV (ultra-violet) crosslinking with mass spectrometry (XL-MS) has been established for identifying RNA- and DNA-binding proteins along with their domains and amino acids involved. Here, we explore chemical XL-MS for RNA-protein, DNA-protein, and nucleotide-protein complexes in vitro and in vivo. We introduce a specialized nucleotide-protein-crosslink search engine, NuXL, for robust and fast identification of such crosslinks at amino acid resolution. Chemical XL-MS complements UV XL-MS by generating different crosslink species, increasing crosslinked protein yields in vivo almost four-fold, and thus it expands the structural information accessible via XL-MS. Our workflow facilitates integrative structural modelling of nucleic acid-protein complexes and adds spatial information to the described RNA-binding properties of enzymes, for which crosslinking sites are often observed close to their cofactor-binding domains. In vivo UV and chemical XL-MS data from E. coli cells analysed by NuXL establish a comprehensive nucleic acid-protein crosslink inventory with crosslink sites at amino acid level for more than 1500 proteins. Our new workflow combined with the dedicated NuXL search engine identified RNA crosslinks that cover most RNA-binding proteins, with DNA and RNA crosslinks detected in transcriptional repressors and activators.

biochemistry↗

bioRxiv2024

Structural Dynamics of the Ubiquitin Specific Protease USP30 in Complex with a Cyanopyrrolidine-Containing Covalent Inhibitor

Inhibition of the mitochondrial deubiquitinating enzyme USP30 is neuroprotective and presents therapeutic opportunities for the treatment of idiopathic Parkinsons Disease and mitophagy-related disorders. We have integrated structural and quantitative proteomics with biochemical assays to decipher the mode of action of covalent USP30 inhibition by a small molecule containing a cyanopyrrolidine reactive group, USP30-I-1. The inhibitor demonstrated high potency and selectivity for endogenous USP30 in neuroblastoma cells. Enzyme kinetics and Hydrogen Deuterium eXchange mass spectrometry (HDX-MS) infers that the inhibitor binds tightly to regions surrounding the USP30 catalytic cysteine and positions itself to form a binding pocket along the thumb and palm domains of the protein, thereby interfering its interaction with ubiquitin substrates. A comparison to a non-covalent USP30 inhibitor containing a benzosulfonamide scaffold revealed a slightly different binding mode closer to the active site Cys77, which may provide the molecular basis for improved selectivity towards USP30 against other members of the DUB enzyme family. Our results highlight advantages in developing covalent inhibitors, such as USP30-I-1, for targeting USP30 as treatment of disorders with impaired mitophagy.

biochemistry↗

bioRxiv2023

Fast and accurate disulfide bridge detection

Recombinant expression of proteins, propelled by therapeutic antibodies, has evolved into a multi-billion-dollar industry. Essential here is quality control assessment of critical attributes such as sequence fidelity, proper folding, and post-translational modifications (PTMs). Errors can lead to diminished bioactivity and, in the context of therapeutic proteins, an elevated risk for immunogenicity. Over the years, many techniques were developed and applied to validate proteins in a standardized and high-throughput fashion. One parameter has, however, so far been challenging to assess. Disulfide bridges, covalent bonds linking two Cysteine residues, assist in the correct folding and stability of proteins and thus have a major influence on their efficacy. Mass spectrometry promises to be an optimal technique to uncover them in a fast and accurate fashion. In this work, we present a unique combination of sample preparation, data acquisition and analysis facilitating the rapid and accurate assessment of disulfide bridges in purified proteins. Through microwave-assisted acid hydrolysis (MAAH), the proteins are digested rapidly and artifact-free into peptides, with a substantial degree of overlap over the sequence. The nonspecific nature of this procedure, however, introduces chemical background which is efficiently removed by integrating ion mobility preceding the mass spectrometric measurement. The nonspecific nature of the digestion step additionally necessitates new developments in data analysis, for which we extended the XlinkX node in Proteome Discoverer (XlinkX/PD) to efficiently process the data and ensure correctness through effective false discovery rate correction. The entire workflow can be completed within one hour, allowing for high-throughput, high-accuracy disulfide mapping.

biochemistry↗

bioRxiv2023

Mimicking the regulatory state of a major facilitator superfamily sugar transporter

While many 3D structures of cation-coupled transporters have been determined, the mechanistic details governing the obligatory coupling and functional regulations still remain elusive. The bacterial melibiose transporter (MelB) is a prototype of the Na+-coupled major facilitator superfamily transporters. With a conformational nanobody (Nb), we determined a low-sugar affinity inward-facing Na+-bound cryoEM structure. Collectively with the available outward-facing sugar-bound structures, both the outer and inner barriers were localized. The N-and C-terminal residues of the inner barrier contribute to the sugar selectivity pocket. When the inner barrier is broken as shown in the inward-open conformation, the sugar selectivity pocket is also broken. The binding assays by isothermal titration calorimetry revealed that this inward-facing conformation trapped by the conformation-selective Nb exhibited a greatly decreased sugar-binding affinity, suggesting the mechanisms for the substrate intracellular release and accumulation. While the inner/outer barrier shift directly regulates the sugar-binding affinity, it has little or no effect on the cation binding, which is also supported by molecular dynamics simulations. Furthermore, the use of this Nb in combination with the hydron/deuterium exchange mass spectrometry allowed us to identify dynamic regions; some regions are involved in the functionally important inner barrier-specific salt-bridge network, which indicates their critical roles in the barrier switching mechanisms for transport. These complementary results provided structural and dynamic insights into the mobile barrier mechanism for cation-coupled symport.

molecular biology↗

bioRxiv2023

Automating data analysis for hydrogen/deuterium exchange mass spectrometry using data-independent acquisition methodology

We developed a hydrogen/deuterium exchange workflow coupled to tandem mass spectrometry (HX-MS2) that supports the acquisition of peptide fragment ions alongside their peptide precursors. The approach enables true auto-validation of HX data by mining a rich set of deuterated fragments, generated by collisional-induced dissociation (CID), to simultaneously confirm the peptide ID and authenticate MS1-based deuteration calculations. The high redundancy provided by the fragments supports a confidence assessment of deuterium calculations using a combinatorial strategy. The approach requires data-independent acquisition (DIA) methods that are available on most MS platforms, making the switch to HX-MS2 straightforward. Importantly, we find that HX-DIA enables a proteomics-grade approach and wide-spread applications. Considerable time is saved through auto-validation and complex samples can now be characterized and at higher throughput. We illustrate these advantages in a drug binding analysis of the ultra-large protein kinase DNA-PKcs, isolated directly from mammalian cells.

biochemistry↗

bioRxiv2023

Mapping the architecture of the initiating phosphoglycosyl transferase from S. enterica O-antigen biosynthesis in a liponanoparticle.

Bacterial cell surface glycoconjugates are critical for cell survival and for interactions between bacteria and their hosts. Consequently, the pathways responsible for their biosynthesis have untapped potential as therapeutic targets. The localization of many glycoconjugate biosynthesis enzymes to the membrane represents a significant challenge for expressing, purifying, and characterizing these enzymes. Here, we leverage cutting-edge methods to stabilize, purify, and structurally characterize WbaP, a phosphoglycosyl transferase (PGT) from Salmonella enterica (LT2) O-antigen biosynthesis without detergent solubilization from the lipid bilayer. From a functional perspective, these studies establish WbaP as a homodimer, reveal the structural elements responsible for oligomerization, shed light on the regulatory role of a domain of unknown function embedded within WbaP, and identify conserved structural motifs between PGTs and functionally unrelated UDP-sugar dehydratases. From a technological perspective, the strategy developed here is generalizable and provides a toolkit for studying small membrane proteins embedded in liponanoparticles beyond PGTs.

biochemistry↗

bioRxiv2022

Real-time library search increases cross-link identification depth across all levels of sample complexity

Cross-linking mass spectrometry (XL-MS) is a universal tool for probing structural dynamics and protein-protein interactions in vitro and in vivo. Although cross-linked peptides are naturally less abundant than their unlinked counterparts, recent experimental advances improved cross-link identification by enriching the cross-linker modified peptides chemically with the use of enrichable cross-linkers. However, mono-links (i.e., peptides modified with a hydrolyzed cross-linker) still hinder efficient cross-link identification since a large proportion of measurement time is spent on their MS2 acquisition. Currently, cross-links and mono-links cannot be separated by sample preparation techniques or chromatography because they are chemically almost identical. Here, we found that based on the intensity ratios of four diagnostic peaks when using PhoX/tBu-PhoX cross-linkers, cross-links and mono-links can be partially distinguished. Harnessing their characteristic intensity ratios for real-time library search (RTLS)-based triggering of high-resolution MS2 scans increased the number of cross-link identifications from both single protein samples and intact E. coli cells. Specifically, RTLS improves cross-link identification from unenriched samples and short gradients, emphasizing its advantages in high-throughput approaches and when instrument time or sample amount is limited.

systems biology↗

bioRxiv2021

MLC Seq: De novo sequencing of full-length tRNA isoforms by mass ladder complementation

tRNAs can exist in distinct isoforms because of different chemical modifications, which confounds attempts to accurately sequence individual tRNA species using next generation sequencing approaches or to quantify different RNA modifications at specific sites on a tRNA strand. Herein, we develop a mass spectrometric (MS) ladder complementation sequencing (MLC-Seq), allowing for direct and simultaneous sequencing of full-length tRNA molecules, including those with low abundance. MLC-seq is achieved by improved instrumentation, and advanced algorithms that identify each tRNA species and related isoforms in an RNA mixture, and assemble full MS ladders from partial ladders with missing ladder components. Using MLC-Seq, we successfully obtained the sequence of tRNA-Phe from yeast and tRNA-Glu from mouse hepatocytes, and simultaneously revealed new tRNA isoforms derived from nucleotide modifications. Importantly, MLC-Seq pinpointed the location and stoichiometry changes of RNA modifications in tRNA-Glu upon the treatment of dealkylated enzyme AlkB, which confirmed its known enzymatic activity and suggested previously unidentified effects in RNA editing.

biochemistry↗

bioRxiv2021

Evaluation and Optimization of High-Field Asymmetric Waveform Ion Mobility Spectrometry for Multiplexed Quantitative Site-specific N-glycoproteomics

The heterogeneity and complexity of glycosylation hinder the depth of site-specific glycoproteomics analysis. High-field asymmetric-waveform ion-mobility spectrometry (FAIMS) has shown to improve the scope of bottom-up proteomics. The benefits of FAIMS for quantitative N-glycoproteomics have not been investigated yet. In this work, we optimized FAIMS settings for N-glycopeptide identification, with or without the tandem mass tag (TMT) label. The optimized FAIMS approach significantly increased the identification of site-specific N-glycopeptides derived from the purified IgM protein or human lymphoma cells. We explored in detail the changes in FAIMS mobility caused by N-glycopeptides with different characteristics, including TMT labeling, charge state, glycan type, peptide sequence, glycan size and precursor m/z. Importantly, FAIMS also improved multiplexed N-glycopeptide quantification, both with the standard MS2 acquisition method and with our recently developed Glyco-SPS-MS3 method. The combination of FAIMS and Glyco-SPS-MS3 provided the highest quantitative accuracy and precision. Our results demonstrate the advantages of FAIMS for improved mass-spectrometry-based qualitative and quantitative N-glycoproteomics. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/436434v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@126b92forg.highwire.dtl.DTLVardef@147d6e5org.highwire.dtl.DTLVardef@16d6eb0org.highwire.dtl.DTLVardef@17dfe2a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

Expanding the depth and sensitivity of cross-link identification by differential ion mobility using FAIMS

In cross-linking mass spectrometry, the depth and sensitivity is often limited by the low abundance of cross-links compared to non-cross-linked peptides in the digestion mixture. To improve the identification efficiency of low abundant cross-links, here we present a gas-phase separation strategy using high field asymmetric waveform ion mobility spectrometry (FAIMS) coupled to the Orbitrap Tribrid mass spectrometers. By enabling an additional peptide separation step in gas phase using the FAIMS device, we increase the number of cross-link identification by 23% for a medium complex sample and 56% for strong cation exchange-fractionated HEK293 cell lysate. Furthermore, we show that for medium complex samples, FAIMS enables the collection of single-shot cross-linking data with comparable depth to the corresponding sample fractionated by chromatography-based approaches. Altogether, we demonstrate FAIMS is highly beneficial for XL-MS studies by expanding the proteome coverage of cross-links while improving the efficiency and confidence of cross-link identification.

biochemistry↗