Biology subjects

Lucas, N.

Publications and source records attributed to Lucas, N..

4 recordsLinked to original sources

bioRxiv2023

Heat n Beat: A universal high-throughput end-to-end proteomics sample processing platform in under an hour

Proteomic analysis by mass spectrometry (MS) of small ([≤]2 mg) solid tissue samples from diverse formats requires high throughput and comprehensive proteome coverage. We developed a near universal, rapid and robust protocol for sample preparation, suitable for high-throughput projects that encompass most cell or tissue types. This end-to-end workflow extends from original sample to loading the mass spectrometer and is centred on a one tube homogenisation and digestion method called Heat n Beat (HnB). It is applicable to most tissues, regardless of how they were fixed or embedded. Sample preparation was divided to separate challenges. The initial sample washing, and final peptide clean-up steps were adapted to three tissue sources: fresh frozen (FF), optimal cutting temperature (OCT) compound embedded (FF-OCT), and formalin-fixed paraffin-embedded (FFPE). Thirdly, for core processing, tissue disruption and lysis were decreased to a 7 min heat and homogenisation treatment, and reduction, alkylation and proteolysis were optimised into a single step. The refinements produced near doubled peptide yield, delivered consistently high digestion efficiency of 85-90%, and required only 38 minutes for core processing in a single tube, with total processing time being 53-63 minutes. The robustness of HnB was demonstrated on six organ types, a cell line and a cancer biopsy. Its suitability for high throughput applications was demonstrated on a set of 1,171 FF-OCT human cancer biopsies, which were processed for end-to-end completion in 92 hours, producing highly consistent peptide yield and quality for over 3,513 MS runs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=152 SRC="FIGDIR/small/559846v2_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@4b8399org.highwire.dtl.DTLVardef@1acc573org.highwire.dtl.DTLVardef@1d7155eorg.highwire.dtl.DTLVardef@1bbef10_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Proteomic-based stratification of intermediate-risk prostate cancer patients

Gleason grading is an important prognostic indicator for prostate adenocarcinoma and is crucial for patient treatment decisions. However, intermediate-risk patients diagnosed in Gleason Grade Groups (GG) 2 and GG3 can harbour either aggressive or non-aggressive disease, resulting in under- or over-treatment of a significant number of patients. Here, we performed proteomic, differential expression, machine learning, and survival analyses for 1,348 matched tumour and benign sample runs from 278 patients. Three proteins (F5, TMEM126B and EARS2) were identified as candidate biomarkers in patients with biochemical recurrence. Multivariate Cox regression yielded 18 proteins, from which a risk score was constructed to dichotomise prostate cancer patients into low- and high-risk groups. This 18-protein signature is prognostic for the risk of biochemical recurrence and completely independent of the intermediate GG. Our results suggest that markers generated by computational proteomic profiling have the potential for clinical applications including integration into prostate cancer management.

cancer biology↗

bioRxiv2022

SAMHD1 controls innate immunity by regulating condensation of immunogenic self RNA

Recognition of pathogen-derived foreign nucleic acids is central to innate immune defense. This requires discrimination between structurally highly similar self and nonself nucleic acids to avoid aberrant inflammatory responses as in the autoinflammatory disorder Aicardi-Goutieres syndrome (AGS). How vast amounts of self RNA are shielded from immune recognition to prevent autoinflammation is not fully understood. Here we show that SAM domain and HD domain-containing protein 1 (SAMHD1), one of the AGS-causing genes, functions as a single-stranded RNA (ssRNA) 3exonuclease, the lack of which causes cellular RNA accumulation. Increased ssRNA in cells leads to dissolution of RNA-protein condensates, which sequester immunogenic double-stranded RNA (dsRNA). Release of sequestered dsRNA from condensates triggers activation of antiviral type I interferon via retinoic acid-inducible gene I-like receptors. Our results establish SAMHD1 as a key regulator of cellular RNA homeostasis and demonstrate that buffering of immunogenic self RNA by condensates regulates innate immune responses.

immunology↗

bioRxiv2022

Pan-cancer proteomic map of 949 human cell lines reveals principles of cancer vulnerabilities

The proteome provides unique insights into biology and disease beyond the genome and transcriptome. Lack of large proteomic datasets has restricted identification of new cancer biomarkers. Here, proteomes of 949 cancer cell lines across 28 tissue types were analyzed by mass spectrometry. Deploying a clinically-relevant workflow to quantify 8,498 proteins, these data capture evidence of cell type and post-transcriptional modifications. Integrating multi-omics, drug response and CRISPR-Cas9 gene essentiality screens with a deep learning-based pipeline revealed thousands of protein-specific biomarkers of cancer vulnerabilities. Proteomic data had greater power to predict drug response than the equivalent portion of the transcriptome. Further, random downsampling to only 1,500 proteins had limited impact on predictive power, consistent with protein networks being highly connected and co-regulated. This pan-cancer proteomic map (ProCan-DepMapSanger), available at https://cellmodelpassports.sanger.ac.uk, is a comprehensive resource revealing principles of protein regulation with important implications for future clinical studies.

systems biology↗