Biology subjects

Brownridge, P.

Publications and source records attributed to Brownridge, P..

13 recordsLinked to original sources

bioRxiv2026

Optimised haemoglobin depletion improves clinical proteomics from dried blood spots

Equitable access to large sample cohorts for robust, high-throughput proteomics for biomarker discovery is a major barrier to widescale clinical implementation. Dried blood spots (DBS) offer a minimally invasive alternative to venous blood draws, enabling at-home microsampling (<50 {micro}L) for centralised analysis, thus enhancing research participation. This approach is particularly relevant for under-represented groups, including children, the elderly, minority backgrounds and those with long-term health conditions such as chronic kidney disease (CKD), where disease fluctuations may occur outside the clinic, and vein preservation is critical. Proteomic analysis has demonstrated great utility in monitoring disease progression, and for biomarker/therapeutic target discovery. However, liquid chromatography-tandem mass spectrometry (LC-MS/MS) of whole blood is hindered by the wide dynamic range and the relatively high abundance of proteins such as haemoglobin, compromising biomarker discovery. Here, we establish an optimised workflow for protein extraction and haemoglobin depletion from microsamples obtained using DBS, enabling sensitive and high-throughput proteomic analysis. We demonstrate that haemoglobin depletion increases protein identifications by [~]50%, mitigating ion suppression and dynamic range effects, enabling the identification of putative biomarkers from patients with stage 5 CKD on dialysis. We also evaluated a commercial cell-free DBS device which yielded a sample more representative of plasma compared to traditional DBS and enabled greater depletion of haemoglobin compared to traditional DBS with haemoglobin depletion methods. Our findings offer a scalable approach for biomarker discovery, facilitating remote, longitudinal clinical studies.

biochemistry↗

bioRxiv2026

MicroAge Mission: Effects of Microgravity and Heat Shock Protein 10 Overexpression on the Proteome of Human Tissue-Engineered Muscle Constructs - Implications for Skeletal Muscle Ageing

Age-related loss of skeletal muscle mass and function, or sarcopenia, presents a growing clinical challenge, mirroring the accelerated muscle atrophy seen in microgravity. This study, part of the UK Space Agencys MicroAge Mission, aimed to investigate microgravity-induced proteomic changes in 3D human skeletal muscle constructs and assess whether mitochondrial Heat Shock Protein 10 (HSP10) overexpression could modulate these responses. Constructs derived from control human AB1167 myoblasts and AB1167 myoblasts that were transduced to overexpress HSP10, were flown to the International Space Station (ISS), with a ground reference experiment (GRE) conducted post-flight. Proteomic analysis using mass spectrometry and bioinformatics revealed significant alterations in metabolic, structural, and mitochondrial protein profiles after microgravity exposure. Microgravity caused downregulation of key proteins involved in energy metabolism, stress responses and structural integrity, while upregulating catabolic and apoptotic enzymes. Many of these modifications parallel previously reported changes in protein composition of muscle with ageing on earth. Overexpression of HSP10 attenuated the effects of microgravity, with fewer proteins showing significant changes and reduced disruption to mitochondrial and cytoskeletal components. Pathway analysis indicated that HSP10 overexpression preserved mitochondrial protein expression, particularly in the matrix, and promoted mitochondrial gene expression and translation under microgravity conditions. Notably, 284 proteins altered by microgravity in unmodified muscle constructs remained stable in HSP10-overexpressing constructs, suggesting a protective effect. MitoCarta 3.0 analysis confirmed that HSP10 expression modulated protein responses at the mitochondrial level, mitigating declines in bioenergetic proteins that are typically associated with microgravity. Collectively, the findings demonstrate that microgravity induces extensive proteomic remodelling in human muscle, which is partially offset by HSP10 overexpression. These results offer insights into muscle atrophy in spaceflight and suggest that targeting mitochondrial stress pathways via chaperone modulation may be a viable strategy to combat sarcopenia and disuse-induced muscle loss on Earth and in space.

physiology↗

bioRxiv2025

From Discovery to Process Control: Positioning DIA Proteomics in Biomanufacturing Pipelines

Gaining control of existing biomanufacturing chassis organisms, such as Escherichia coli K12, and novel isolates, such as the salt tolerant Halomonas bluephagenesis sp TD01 studied here may be facilitated by the investigation and monitoring of their metabolic and regulatory processes, particularly through proteomics. Here we consider the performance of a range of typically available proteomics platforms across a range of price points to map chassis organisms metabolic pathways. A set of model bacterial samples was prepared from E. coli and H. bluephagenesis sp. TD01 in 1:1, 1:2 and 2:1 ratios and analyzed using five LC-MS systems. Of the 8,222 proteins identified across all samples analysed (4,401 proteins from E. coli; 3,821 from Halomonas sp. TD01), the TimsTOF and Exploris were able to achieve extensive proteome coverage quantifying 5.5k and 5k proteins respectively, with the ZenoTOF, Waters MRT and the legacy Waters Vion respectively quantifying 3.5k, 1.3k, and [~]850 proteins at 1% FDR. Proteins comprising core metabolic pathways critical to biomanufacturing in these chassis organisms can be quantified with all instruments. We characterize metabolic adaptation in H. bluephagenesis by showing that replacement of glucose with a carboxylic acid feed stock directs carbon flux towards potential butane precursors as well as how the acquired data permits monitoring of the cobalamin (vitamin B12) production pathway. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/695173v3_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@148fd14org.highwire.dtl.DTLVardef@df4b7borg.highwire.dtl.DTLVardef@1d479b6org.highwire.dtl.DTLVardef@82a4df_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Single-cell phenotypic analysis and multiplet detection through incorporation of microscopy data into cellenONE-based single-cell proteomic data analysis

The reliability of single-cell proteomics (SCP) is intrinsically linked to the fidelity of cell isolation; however, the identification of co-isolated cells (doublets or multiplets) remains a persistent challenge for single-cell (sc-) omics. While single-cell transcriptomics has established probabilistic frameworks for doublet detection, these methods are ill-suited for the sparser throughput of SCP datasets. This work presents scpImaging, a novel computational pipeline that repurposes the latent microscopy data routinely generated during cellenONE-based sample preparation to provide deterministic quality control (QC) and high-content phenotypic profiling. We demonstrate that standard proteomic quality metrics for SCP (e.g., peptide count, signal intensity) paradoxically favour retaining doublets. In contrast, scpImaging applies machine-learning-based cell segmentation (Cellpose-SAM) to identify and exclude these artefacts with high precision. Furthermore, the framework integrates morphological metrics generated using the open-source CellProfiler, with proteomic abundance data, enabling joint analysis that links cell shape and texture to molecular cell state. Provided as an open-source R package, scpImaging offers a scalable, automated solution for enhancing data integrity and adding a phenotypic dimension to SCP experiments without increasing experimental cost or complexity.

systems biology↗

bioRxiv2025

An optimized SP3 sample processing workflow for in-depth and re-producible phosphoproteomics

Protein phosphorylation is a ubiquitous post-translational modification (PTM) found across the kingdoms of life, and is critical for the regulation of protein function in health & disease. Advances in high-throughput mass spectrometry have transformed our ability to interrogate the phosphoproteome. However, sample preparation methodologies optimized for phosphoproteomics have not kept pace, compromising the ability to fully exploit these technological advances. In this study, we present an optimized phosphoproteomics workflow using carboxylated SP3 magnetic beads which have simplified proteomics sample preparation. By employing a washing step with 8 M urea and omitting the conventional C18 SPE clean-up, we demonstrate a significant improvement in phosphopeptide identifications, with application of this refined protocol to HEK-293T cell extracts increasing the number nearly 2-fold compared to standard SP3 techniques (7908 cf. 4129). We also observed a substantial improvement in the detection of multiply phosphorylated peptides. Our findings suggest that the complexity of PTM crosstalk using current peptide-based proteomics workflows is currently under-represented and underscores the necessity of methodological innovations to better capture the intricacies of the phosphoproteome landscape.

biochemistry↗

bioRxiv2024

Rubisco packaging and stoichiometric composition of a native β-carboxysome

Carboxysomes are anabolic bacterial microcompartments that play an essential role in carbon fixation in cyanobacteria. This self-assembling proteinaceous organelle encapsulates the key CO2-fixing enzymes, Rubisco and carbonic anhydrase, using a polyhedral shell constructed by hundreds of shell protein paralogs. Deciphering the precise arrangement and structural organization of Rubisco enzymes within carboxysomes is crucial for understanding the formation process and overall functionality of carboxysomes. Here, we employed cryo-electron tomography and subtomogram averaging to delineate the three-dimensional packaging of Rubiscos within {beta}-carboxysomes in the freshwater cyanobacterium Synechococcus elongatus PCC7942 that were grown under low light. Our results revealed that Rubiscos are arranged in multiple concentric layers parallel to the shell within the {beta}-carboxysome lumen. We also identified the binding of Rubisco with the scaffolding protein CcmM in {beta}-carboxysomes, which is instrumental for Rubisco encapsulation and {beta}-carboxysome assembly. Using QconCAT-based quantitative mass spectrometry, we further determined the absolute stoichiometric composition of the entire {beta}-carboxysome. This study and recent findings on the {beta}-carboxysome structure provide insights into the assembly principles and structural variation of {beta}-carboxysomes, which will aid in the rational design and repurposing of carboxysome nanostructures for diverse bioengineering applications.

biochemistry↗

bioRxiv2024

Activation loop phosphorylation of Cdk11 is restrained by PNUTS-PP1 and regulates Cdk11 activity and function

Organisation of the transcription cycle is facilitated by the reversible phosphorylation of the C-terminal domain of RNA Polymerase II (RNAPII-CTD) and its accessory factors. The PNUTS-PP1 protein phosphatase is crucial for mRNA synthesis and processing, yet the complete spectrum of its physiological targets in these processes remain elusive. Here, using quantitative phosphoproteomics, we discover that Cdk11, in addition to various spliceosomal and RNA processing factors, associates with PNUTS, and that disruption of PP1-binding results in hyperphosphorylation of Cdk11 at an evolutionarily conserved Serine residue, seven amino acid residues C-terminal to DFG residues in the activation loop. In vitro experiments reveal a role for Ser DFG+7 in modulating Cdk11 kinase activity towards RNAPII-CTD Ser5. Making use of a novel technique to conditionally disrupt PP1 binding, we show that PNUTS-PP1 normally serves to restrain Cdk11 phosphorylation in vivo. Mutational analysis shows that cdk11 is not only essential for survival but also plays a widespread role in regulating normal mRNA expression and splicing. Notably, we find that a phosphomimetic mutation in cdk11 exhibits distinct biological effects compared to loss of cdk11 function, including defective processing of intronic small nucleolar RNAs, diminished intronic RNA Pol II velocity, and a decrease in intergenic transcription. These findings underscore physiologically significant roles of Cdk11 dephosphorylation by PNUTS-PP1 in the regulation of mRNA transcription and processing.

molecular biology↗

bioRxiv2021

Probing the biogenesis pathway and dynamics of thylakoid membranes

How thylakoid membranes are generated to form the metabolically active membrane network and how thylakoid membranes orchestrate the insertion and localization of protein complexes for efficient electron flux remain elusive. Here, we develop a method to modulate thylakoid biogenesis in the rod-shaped cyanobacterium Synechococcus elongatus PCC 7942 by modulating light intensity during cell growth, and probe the spatial-temporal stepwise biogenesis process of thylakoid membranes in cells. Our results reveal that the plasma membrane and regularly arranged concentric thylakoid layers have no physical connections. The newly synthesized thylakoid membrane fragments emerge between the plasma membrane and pre-existing thylakoids. Photosystem I monomers appear in the thylakoid membranes earlier than other mature photosystem assemblies, followed by generation of Photosystem I trimers and Photosystem II complexes. Redistribution of photosynthetic complexes during thylakoid biogenesis ensures establishment of the spatial organization of the functional thylakoid network. This study provides insights into the dynamic biogenesis process and maturation of the functional photosynthetic machinery.

plant biology↗

bioRxiv2020

Characterisation of protease activity during SARS-CoV-2 infection identifies novel viral cleavage sites and cellular targets for drug repurposing

SARS-CoV-2 is the causative agent behind the COVID-19 pandemic, and responsible for over 170 million infections, and over 3.7 million deaths worldwide. Efforts to test, treat and vaccinate against this pathogen all benefit from an improved understanding of the basic biology of SARS-CoV-2. Both viral and cellular proteases play a crucial role in SARS-CoV-2 replication, and inhibitors targeting proteases have already shown success at inhibiting SARS-CoV-2 in cell culture models. Here, we study proteolytic cleavage of viral and cellular proteins in two cell line models of SARS-CoV-2 replication using mass spectrometry to identify protein neo-N-termini generated through protease activity. We identify previously unknown cleavage sites in multiple viral proteins, including major antigenic proteins S and N, which are the main targets for vaccine and antibody testing efforts. We discovered significant increases in cellular cleavage events consistent with cleavage by SARS-CoV-2 main protease, and identify 14 potential high-confidence substrates of the main and papain-like proteases, validating a subset with in vitro assays. We showed that siRNA depletion of these cellular proteins inhibits SARS-CoV-2 replication, and that drugs targeting two of these proteins: the tyrosine kinase SRC and Ser/Thr kinase MYLK, showed a dose-dependent reduction in SARS-CoV-2 titres. Overall, our study provides a powerful resource to understand proteolysis in the context of viral infection, and to inform the development of targeted strategies to inhibit SARS-CoV-2 and treat COVID-19.

microbiology↗

bioRxiv2020

Use of the Polo-like kinase 4 (PLK4) inhibitor centrinone to investigate intracellular signaling networks using SILAC-based phosphoproteomics

Polo-like kinase 4 (PLK4) is the master regulator of centriole duplication in metazoan organisms. Catalytic activity and protein turnover of PLK4 are tightly coupled in human cells, since changes in PLK4 concentration and catalysis have profound effects on centriole duplication and supernumerary centrosomes, which are associated with aneuploidy and cancer. Recently, PLK4 has been targeted with a variety of small molecule kinase inhibitors exemplified by centrinone, which rapidly induces inhibitory effects on PLK4 and leads to on-target centrosome depletion. Despite this, relatively few PLK4 substrates have been identified unequivocally in human cells, and PLK4 signaling outside centriolar networks remains poorly characterised. We report an unbiased mass spectrometry (MS)-based quantitative analysis of cellular protein phosphorylation in stable PLK4-expressing U2OS human cells exposed to centrinone. PLK4 phosphorylation was itself sensitive to brief exposure to the compound, resulting in PLK4 stabilization. Analysing asynchronous cell populations, we report hundreds of centrinone-regulated cellular phosphoproteins, including centrosomal and cell cycle proteins and a variety of likely non-canonical substrates. Surprisingly, sequence interrogation of ~300 significantly down-regulated phosphoproteins reveals an extensive network of centrinone-sensitive [Ser/Thr]Pro phosphorylation sequence motifs, which based on our analysis might be either direct or indirect targets of PLK4. In addition, we confirm that NMYC and PTPN12 are PLK4 substrates, both in vitro and in human cells. Our findings suggest that PLK4 catalytic output directly controls the phosphorylation of a diverse set of cellular proteins, including Pro-directed targets that are likely to be important in PLK4-mediated cell signaling.

biochemistry↗

bioRxiv2018

Adding function to the genome of African Salmonella ST313

Salmonella Typhimurium ST313 causes invasive nontyphoidal Salmonella (iNTS) disease in sub-Saharan Africa, targeting susceptible HIV+, malarial or malnourished individuals. An in-depth genomic comparison between the ST313 isolate D23580, and the well-characterized ST19 isolate 4/74 that causes gastroenteritis across the globe, revealed extensive synteny. To understand how the 856 nucleotide variations generated phenotypic differences, we devised a large-scale experimental approach that involved the global gene expression analysis of strains D23580 and 4/74 grown in sixteen infection-relevant growth conditions. Comparison of transcriptional patterns identified virulence and metabolic genes that were differentially expressed between D23580 versus 4/74, many of which were validated by proteomics. We also uncovered the S. Typhimurium D23580 and 4/74 genes that showed expression differences during infection of murine macrophages. Our comparative transcriptomic data are presented in a new enhanced version of the Salmonella expression compendium SalComD23580: bioinf.gen.tcd.ie/cgi-bin/salcom_v2.pl. We discovered that the ablation of melibiose utilization was caused by 3 independent SNP mutations in D23580 that are shared across ST313 lineage 2, suggesting that the ability to catabolise this carbon source has been negatively selected during ST313 evolution. The data revealed a novel plasmid maintenance system involving a plasmid-encoded CysS cysteinyl-tRNA synthetase, highlighting the power of large-scale comparative multi-condition analyses to pinpoint key phenotypic differences between bacterial pathovariants.

microbiology↗

bioRxiv2017

Extensive non-canonical phosphorylation in human cells revealed using strong-anion exchange-mediated phosphoproteomics

Protein phosphorylation is a ubiquitous post-translational modification (PTM) that regulates all aspects of life. To date, investigation of human cell signalling has focussed on canonical phosphorylation of serine (Ser), threonine (Thr) and tyrosine (Tyr) residues. However, mounting evidence suggests that phosphorylation of histidine also plays a central role in regulating cell biology. Phosphoproteomics workflows rely on acidic conditions for phosphopeptide enrichment, which are incompatible with the analysis of acid-labile phosphorylation such as histidine. Consequently, the extent of non-canonical phosphorylation is likely to be under-estimated.\n\nWe report an Unbiased Phosphopeptide enrichment strategy based on Strong Anion Exchange (SAX) chromatography (UPAX), which permits enrichment of acid-labile phosphopeptides for characterisation by mass spectrometry. Using this approach, we identify extensive and positional phosphorylation patterns on histidine, arginine, lysine, aspartate and glutamate in human cell extracts, including 310 phosphohistidine and >1000 phospholysine sites of protein modification. Remarkably, the extent of phosphorylation on individual non-canonical residues vastly exceeds that of basal phosphotyrosine. Our study reveals the previously unappreciated diversity of protein phosphorylation in human cells, and opens up avenues for exploring roles of acid-labile phosphorylation in any proteome using mass spectrometry.

biochemistry↗

bioRxiv2017

Large scale and significant expression from pseudogenes in Sodalis glossinidius - a facultative bacterial endosymbiont

The majority of bacterial genomes have high coding efficiencies, but there are some genomes of intracellular bacteria that have low gene density. The genome of the endosymbiont Sodalis glossinidius contains almost 50% pseudogenes containing mutations that putatively silence them at the genomic level. We have applied multiple omic strategies, combining: Illumina and Pacific Biosciences Single-Molecule Real Time DNA-sequencing and annotation; stranded RNA-sequencing; and proteome analysis to better understand the transcriptional and translational landscape of Sodalis pseudogenes, and potential mechanisms for their control. Between 53% and 74% of the Sodalis transcriptome remains active in cell-free culture. Mean sense transcription from Coding Domain Sequences (CDS) is four-times greater than that from pseudogenes. Comparative genomic analysis of six Illumina-sequenced Sodalis isolates from different host Glossina species shows pseudogenes make up ~40% of the 2,729 genes in the core genome, suggesting that they are stable and/or Sodalis is a recent introduction across the Glossina genus as a facultative symbiont. These data further shed light on the importance of transcriptional and translational control in deciphering host-microbe interactions, and demonstrate that pseudogenes are more complex than a simple degrading DNA sequence. The combination of genomics, transcriptomics and proteomics give a multidimensional perspective for studying prokaryotic genomes with a view to elucidating evolutionary adaptation to novel environmental niches.\n\nImportanceBacterial genomes are generally 1Kb in length, organized efficiently (i.e. with few gaps between genes or operons), and few open reading frames (ORFs) lack any predicted function. Intracellular bacteria have been removed from extracellular selection pressures acting on pathways of declining importance to fitness and thus, these bacteria tend to delete redundant genes in favour of smaller functional repertoires - maintaining genome efficiency. In the genomes of endosymbionts with a recent evolutionary relationship with their host, however, this process of genome reduction is not complete; Genes and pathways may be at an intermediate stage, undergoing mutation linked to reduced selection and small population numbers being vertically transmitted from mother to offspring in their hosts, resulting in an increase in abundance of pseudogenes and reduced coding capacities. A greater knowledge of the genomic architecture of persistent pseudogenes, with respect to their DNA structure, mRNA transcription and even putative translation to protein products, will lead to a better understanding of the evolutionary trajectory of endosymbiont genomes, many of which have important roles in arthropod ecology.

genomics↗