Biology subjects

Multari, D. H.

Publications and source records attributed to Multari, D. H..

5 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↗

bioRxiv2026

If the shoe fits? -- technical nuances of plasma proteomic workflows in clinical and preclinical contexts

The push for new clinical biomarkers has seen rapid innovation in biofluid analysis, particularly for plasma. For mass-spectrometry (MS)-based analysis, achieving depth and quantitative accuracy whilst ensuring throughput continues to shape plasma methods development. Numerous workflows have emerged that mitigate high-abundance suppression and expand dynamic range, especially when paired with next-generation MS instrumentation. Yet systematic evaluations that also consider biological variables (e.g., biofluid type, species) and technical parameters (e.g., MS methods) are limited. Here, we benchmarked eight sample-preparation workflows spanning neat approaches (SP3, STrap), depletion (perchloric acid, PerCA), and corona-enrichment strategies (MagNet HILIC/SAX, Enrich-iST, ProteoNano). We compared their performance across human plasma, human serum, and rat plasma, analysing all samples on an Orbitrap Astral (Thermo) using two plasma-optimised data-independent acquisition (DIA) methods: one discovery-maximised and one throughput-maximised. We identified 2,726 human and 3,767 rat proteins across workflows and methods, including [~]1,000 from neat plasma. Increasing throughput incurred a [~]20-30% reduction in depth, depending on workflow and species. EV-enrichment produced the deepest proteomes but with distinct compositions relative to neat, depleted, and secreted-protein-enriched samples, revealing a unique sub-proteome niche. Several workflows also performed markedly better in rat plasma, supporting improved sensitivity for preclinical analyses. Enrichment or depletion dramatically reshaped the balance of tissue-and cell-specific proteins detectable in plasma, suggesting that workflow choice should be guided by the organs, immune targets, or inflammatory signals most relevant to the study. In this vein, statistical analysis of differentially abundant proteins showed that >90% of detected proteins were significantly altered between workflows, with the largest numbers arising from the corona-enrichment strategies, underscoring how strongly workflow choice shapes the downstream proteome. Taken together, these findings emphasise a rapidly expanding plasma methodological landscape, where the most effective workflow is the one most precisely tailored to a cohorts biology.

systems biology↗

bioRxiv2025

Global analysis of cancer cell responses to USP9X inhibition

The ubiquitin specific protease (USP) enzyme USP9X is amongst the best studied human deubiquitinases (DUBs), with a myriad of described targets and cellular roles. In cancer, USP9X has been touted as both an oncogene and a tumour suppressor in different contexts, which has confounded the field and questioned its viability as a cancer target. We here describe WEHI-092, a novel piperazine-based USP9X specific small molecule inhibitor and map its binding site to a unique region in the USP9X fingers subdomain, distinct from known DUB inhibitor binding sites. Using proteomics and ubiquitinomics, we show that USP9X has a distinct set of substrates compared to USP7 indicating remarkable DUB target specificity, yet the substrate profile of USP9X varies significantly across cancer cell lines. Interestingly, we reveal a core set of 17 proteins commonly regulated by USP9X in most or all cell lines, which we consider as proximal biomarkers for USP9X inhibition. Consistent with our proteomic analyses, we show that WEHI-092 treatment arrests cells in metaphase without inducing cell death, which may account for growth suppression seen in long-term clonogenic assays in most cancer cell lines, and positions USP9X inhibitors as a new potential class of selective mitotic poisons.

cell biology↗

bioRxiv2024

PSKH1 kinase activity is differentially modulated via allosteric binding of Ca2+ sensor proteins

Protein Serine Kinase H1 (PSKH1) was recently identified as a crucial factor in kidney development and is overexpressed in prostate, lung and kidney cancers. However, little is known about PSKH1 regulatory mechanisms, leading to its classification as a "dark" kinase. Here, we used biochemistry and mass spectrometry to define PSKH1s consensus substrate motif, protein interactors, and how interactors, including Ca2+ sensor proteins, promote or suppress activity. Intriguingly, despite the absence of a canonical Calmodulin binding motif, Ca2+-Calmodulin activated PSKH1 while, in contrast, the ER-resident Ca2+ sensor of the CREC family, Reticulocalbin-3, suppressed PSKH1 catalytic activity. In addition to antagonistic regulation of the PSKH1 kinase domain by Ca2+ sensing proteins, we identified UNC119B as a protein interactor that activates PSKH1 via direct engagement of the kinase domain. Our findings identify complementary allosteric mechanisms by which regulatory proteins tune PSKH1s catalytic activity, and raise the possibility that different Ca2+ sensors may act more broadly to tune kinase activities by detecting and decoding extremes of intracellular Ca2+ concentrations.

biochemistry↗

bioRxiv2022

Species identification of early colonial bone artefacts excavated from Pyrmont, Australia, by mass spectrometric identification of collagen peptides

Zooarchaeology by Mass Spectrometry (ZooMS) is a rapidly developing and increasingly utilised peptide mass fingerprinting (PMF) technique that analyses Collagen 1A1 and 1A2 marker peptides for the genus- or species-level identification of fragmentary bones in the archaeological record. Traditionally, this analysis is performed using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-ToF-MS) to identify characteristic m/z values of known marker peptides. Here we present data on the application of a modified ZooMS approach, using nanoflow liquid chromatography - tandem mass spectrometry proteomics, to the analysis of a collection of six early colonial Australian (early to mid-19th Century CE) worked bone artefacts, believed to be mostly knife handles, excavated from a site in Pyrmont, Sydney, Australia in 2017. We were successfully able to identify characteristic marker peptides for bovine COL1A1 and COL1A2 in all six bone artefacts.

biochemistry↗