Biology subjects

Brenes, A. J.

Publications and source records attributed to Brenes, A. J..

13 recordsLinked to original sources

bioRxiv2026

The Single Cell Proteomic blueprint, navigating instrumentation platforms, software tools and high-load libraries in neutrophils, RKO and A549 cells

Mass spectrometry-based single cell proteomics (SCP) is rapidly emerging as a powerful approach for biological research, with applications extending beyond in-vitro cancer cell lines. Recent advances make it possible to apply SCP to ex-vivo human cells from tissues such as the brain and pancreas, as well as to technically challenging immune populations such as neutrophils. However, these analyses remain more challenging and typically result in reduced proteomic coverage. To support the development of robust workflows for SCP data acquisition and analysis, we systematically evaluated multiple DIA search engines, search engine settings, the inclusion of high-load library samples in single-cell search spaces, the impact of contaminants, and the quantitative properties of identified proteins. These comparisons were performed across two major instrumentation platforms, Orbitrap Astral and timsTOF SCP, and across A549, RKO cells and neutrophils, three cell types differing in size and protein content. Our work here provides guidelines on the software parameters to use for SCP, instrument specific results and cell dependent optimizations of high-load libraries, as well as novel evaluation of the quantitative properties of proteins for single cell and low input proteomics.

biochemistry↗

bioRxiv2026

A Context-Aware Single-Cell Proteomics Analysis pipeline.

Single-cell proteomics (SCP) by mass spectrometry can now quantify hundreds to thousands of proteins per cell, but the field still lacks standardised analytical pipelines that accommodate the diversity of instruments, sample preparation workflows and biological contexts encountered in practice. Existing workflows, largely adapted from single-cell transcriptomics, do not account for the informative missingness, pervasive ambient protein contamination and limited feature space that distinguish proteomic from transcriptomic data. In addition, cell type annotation remains a manual bottleneck that is subjective, difficult to reproduce and hard to scale. Here we present an end-to-end pipeline that integrates adaptive quality control, entropy-guided iterative batch correction, multi-modal marker discovery that exploits detection patterns unique to proteomics, and context-aware annotation by large language models (LLMs) coupled to structured contradiction reasoning and orthogonal data-driven validation. Benchmarking on published single-cell proteomic datasets from developing human brain and glioblastoma-associated neutrophils revealed systematic LLM failure modes, including context-insensitive marker vocabulary and misinterpretation of phagocytic or lytic cell states. We addressed these errors using a three-round prompt architecture that combines general biological principles with auto-generated dataset-specific constraints. In held-out validation on a skin tumour dataset acquired, the pipeline showed high concordance with FACS-sorted ground truth. In the caerulein-injured pancreas, orthogonal immunohistochemistry further supported annotations of macrophage, stellate and immune populations. The pipeline is fully automated under fixed settings, and available as Context-Aware Single-Cell Proteomics Analysis (CASPA), providing SCP laboratories and facilities with a reproducible workflow that delivers interpretable, confidence-quantified annotations suitable for downstream expert review.

bioinformatics↗

bioRxiv2025

Single cell proteomic analysis defines discrete neutrophil functional states in human glioblastoma

Neutrophils are vital innate immune cells shown to infiltrate glioblastomas, however we currently lack the molecular understanding of their functional states within the tumour niche. Given that neutrophils are known to display a prominent discordance between mRNA and protein abundance, we developed ultra-sensitive mini-bulk and single cell proteomic (SCP) workflows to study the heterogeneity of peripheral blood and tumour associated neutrophils (TAN) from patients with glioblastoma. Mini-bulk analysis enabled a deeper protein coverage of circulating immature, mature and TAN populations, defining signatures of maturity and demonstrating that TANs resemble mature circulating neutrophils. Analysis of the SCP data resulted in the detection of >1,100 proteins from a single TAN providing a detailed characterization of neutrophil subsets in glioblastoma. Our approach shows evidence of pathogenic and anti-tumorigenic clusters and discovers cell states invisible to scRNAseq, opening new opportunities to selectively target pro-tumoural neutrophil states.

immunology↗

bioRxiv2024

A proteomic map of B cell activation and its shaping by mTORC1, MYC and iron

Using high resolution quantitative mass spectrometry, we have explored how immune activation and the metabolic checkpoint kinase mTORC1 (mammalian target of rapamycin complex 1) regulate the proteome of B lymphocytes. B cell activation via the B cell receptor, CD40 and the IL4 receptor induced considerable re-modelling of the B cell protein landscape, with a 5-fold increase in total cellular protein mass within 24 hours of activation. Analysis of copy numbers per cell of >7,500 proteins revealed increases in the metabolic machinery that supports B cell activation and nutrient and amino acid transporters that fuel B cell biosynthetic capacity. Comparison of T cell-dependent versus T cell-independent B cell stimulation along with an analysis of the synergistic effects of stimuli, identified shared and unique impacts of stimuli and highlighted the drivers of B cell proteome remodelling. In addition, we reveal that mTORC1 controls activation-induced B cell growth and inhibiting mTORC1 impairs the expression of amino acid transporters that fuel B cell protein production. We also show that mTORC1 activity regulates the expression of the transcription factor MYC and the transferrin receptor CD71. Blocking MYC activity phenocopied mTORC1 inhibition in many ways including impaired CD71 expression, while limiting iron availability during B cell activation impaired B cell growth and protein synthesis. This work provides a detailed map of naive and immune activated B cell proteomes and a greater understanding of the cellular machinery that directs B cell phenotypes. This work also provides new insights into the role of mTORC1, MYC and iron in regulating activation-induced proteome remodelling and protein production in B cells.

immunology↗

bioRxiv2024

Calculating and Reporting Coefficients of Variation for DIA-based Proteomics

The Coefficient of Variation (CV) is a measure that is frequently used to assess data dispersion for mass spectrometry-based proteomics. In the current era of burgeoning technical developments, there has been an increased focus on using CVs to measure the quantitative accuracy of the new methods. Thus, it has also become important to define a set of guidelines on how to calculate and report the CVs. This perspective shows the effects that the CV equation, as well as software parameters can have on data dispersion and CVs, highlighting the importance of reporting all these variables within the methods section. It also proposes a set of recommendations to calculate and report CVs for technical studies where the main objective is to benchmark technical developments with a focus on precision. To assist in this process a novel R package to calculate CVs (proteomicsCV) is also included.

biochemistry↗

bioRxiv2024

PROSER1 Modulates DNA Demethylation through Dual Mechanisms to Prevent Syndromic Developmental Malformations

The link between DNA methylation and neurodevelopmental disorders is well established. However, how DNA methylation is fine-tuned - ensuring precise gene expression and developmental fidelity - remains poorly understood. PROSER1, a known TET2 interactor, was recently linked to a severe neurodevelopmental disorder. Here, we demonstrate that PROSER1 interacts with all TET enzymes and stabilizes chromatin-bound TET-OGT-PROSER1-DBHS (TOPD) complexes, which regulate DNA demethylation and developmental gene expression. Surprisingly, we find that PROSER1 also sequesters TET enzymes, preventing widespread demethylation and transposable element de-repression. Our findings identify PROSER1 as a key factor which both positively and negatively regulates DNA demethylation essential for mammalian neurodevelopment.

developmental biology↗

bioRxiv2023

Optimising Spectronaut search parameters to improve data quality with minimal proteome coverage reductions in DIA analyses of heterogeneous samples

Data independent acquisition has seen breakthroughs that enable comprehensive proteome profiling using short gradients. As the proteome coverage continues to increase, the quality of the data generated becomes much more relevant. Using Spectronaut, we show that the default search parameters can be easily optimised to minimise the occurrence of false positives across different samples. Using an immunological infection model system to demonstrate the impact of adjusting search settings we analysed mouse macrophages and compared their proteome to macrophages spiked with Candida albicans. This experimental system enabled the identification of false positives since Candida albicans peptides and proteins should not be present in the mouse only samples. We show that adjusting the search parameters reduced false positive identifications by 89% at the peptide and protein level, thereby considerably increasing the quality of the data. We also show that these optimised parameters incur a moderate cost, only reducing the overall number of true positive identifications across each biological replicate by <6.7% at both the peptide and protein level. We believe the value of our updated search parameters extends beyond a two-organism analysis and would be of great value to any DIA experiment analysing heterogenous populations of cell types or tissues.

biochemistry↗

bioRxiv2023

Quantitative proteomics reveals tissue-specific, infection-induced and species-specific neutrophil protein signatures

Neutrophils are one of the first responders to infection and are a key component of the innate immune system through their ability to phagocytose and kill invading pathogens, secrete antimicrobial molecules and produce extracellular traps. Neutrophils are produced in the bone marrow, circulate within the blood and upon immune challenge migrate to the site of infection. We wanted to understand whether this transition shapes the mouse neutrophil protein landscape, how the mouse neutrophil proteome is impacted by systemic infection and perform a comparative analysis of human and mouse neutrophils. Using quantitative mass spectrometry we reveal tissue-specific, infection-induced and species-specific neutrophil protein signatures. We show a high degree of proteomic conservation between mouse bone marrow, blood and peritoneal neutrophils, but also identify key differences in the molecules that these cells express for sensing and responding to their environment. Systemic infection triggers a change in the bone marrow neutrophil population with considerable impact on the core machinery for protein synthesis and DNA replication along with environmental sensors. We also reveal profound differences in mouse and human blood neutrophils, particularly their granule contents. Our proteomics data provides a valuable resource for understanding neutrophil function and phenotypes across species and model systems.

immunology↗

bioRxiv2022

The Immunological Proteome Resource

The Immunological Proteome Resource (ImmPRes; http://immpres.co.uk/) is an open access public resource integrating proteomic data generated by large-scale mass-spectrometry analysis of murine hematopoietic populations. The initial focus is T lymphocytes and how their proteomes are shaped by immune activation, environment, and intracellular signalling pathways with an aim to expand it to B cells and innate immune cells. It is a multidisciplinary effort between immunology and mass spectrometry-based labs with the objective to help define an in-depth high-quality map of immune cell proteomes. Maintaining data reproducibility and integrity are a priority within the resource, thus there is an in-depth protocols section explaining in detail the sample processing and the mass spectrometry-based analysis. ImmPRes provides open access to proteomic datasets covering a wide range of murine leukocyte populations with analysis of copy numbers per cell of > 10,000 proteins, enabling new understanding of lymphocyte phenotypes. All data is accessible via a simple graphical interface that supports easy interrogation of the data and options to download data summaries and raw data files.

immunology↗

bioRxiv2021

A quantitative comparison of human embryonic and induced stem cell proteomes

Human induced pluripotent stem cells (hiPSCs) have great potential to be used as alternatives to embryonic stem cells (hESCs) in regenerative medicine and disease modelling, thereby avoiding many of the ethical issues arising from the use of embryo-derived cells. However, despite clear similarities between the two cell types, it is likely they are not identical. In this study, we characterise the proteomes of multiple hiPSC and hESC lines derived from independent donors. We find that while hESCs and hiPSCs express a near identical set of proteins, they show consistent quantitative differences in the expression levels of a wide subset of proteins. hiPSCs have increased total protein content, while maintaining a comparable cell cycle profile to hESCs. The proteomic data show hiPSCs have significantly increased abundance of vital cytoplasmic and mitochondrial proteins required to sustain high growth rates, including nutrient transporters and metabolic proteins, which correlated with phenotypic differences between hiPSCs and hESCs. Thus, higher levels of glutamine transporters correlated with increased glutamine uptake, while higher levels of proteins involved in lipid synthesis correlated with increased lipid droplet formation. Some of the biggest metabolic changes were seen in proteins involved in mitochondrial metabolism, with corresponding enhanced mitochondrial potential, shown experimentally using high-resolution respirometry. hiPSCs also produced higher levels of secreted proteins, including ECM components and growth factors, some with known tumorigenic properties, as well as proteins involved in the inhibition of the immune system. Our data indicate that reprogramming of human fibroblasts to iPSCs effectively restores protein expression in cell nuclei to a state comparable to hESCs, but does not similarly restore the profile of cytoplasmic and mitochondrial proteins, with consequences for cell phenotypes affecting growth and metabolism. The data improve understanding of the molecular differences between induced and embryonic stem cells, with implications for potential risks and benefits for their use in future disease modelling and therapeutic applications.

biochemistry↗

bioRxiv2021

Nrf2 is a central regulator of the metabolic landscape in macrophages and finetunes their inflammatory response

To overcome oxidative, inflammatory, and metabolic stress, cells have evolved networks of cytoprotective proteins controlled by nuclear factor erythroid 2 p45-related factor 2 (Nrf2) and its main negative regulator the Kelch-like ECH associated protein 1 (Keap1). Here, we used high-resolution mass-spectrometry to characterize the proteomes of macrophages with genetically altered Nrf2 status. Our analysis revealed significant differences among the genotypes in cellular metabolism and redox homeostasis, which we validated with respirometry and metabolomics, as well as in anti-viral immune pathways and the cell cycle. Nrf2 status significantly affected the proteome following lipopolysaccharide (LPS) stimulation, with alterations in redox, carbohydrate and lipid metabolism, and innate immunity observed. Of note, Nrf2 activation was found to promote mitochondrial fusion in inflammatory macrophages. The Keap1 inhibitor, 4-octyl itaconate (4-OI), a derivative of the mitochondrial immunometabolite itaconate, remodeled the inflammatory macrophage proteome, increasing redox and suppressing anti-viral immune effectors in a Nrf2-dependent manner. These data suggest that Nrf2 activation facilitates metabolic reprogramming and mitochondrial adaptation, and finetunes the innate immune response in macrophages. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/456204v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1f5bbb8org.highwire.dtl.DTLVardef@1f7311dorg.highwire.dtl.DTLVardef@1b9722forg.highwire.dtl.DTLVardef@1f777d4_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIFirst high-resolution proteome of macrophages with genetically altered Nrf2 status C_LIO_LINrf2 is key regulator of macrophage redox and intermediary metabolism C_LIO_LINrf2 finetunes the inflammatory response suppressing anti-viral immune and cytokine effectors, whilst promoting T cell activation factors C_LIO_LINrf2 regulates mitochondrial adaptation in inflammatory macrophages promoting the formation of a fused network C_LIO_LI4-octyl itaconate (4-OI) suppresses anti-viral immune effectors in inflammatory macrophages in a Nrf2-dependent manner C_LI

immunology↗

bioRxiv2021

Tissue environment, not ontogeny, defines intestinal intraepithelial T lymphocytes

Tissue-resident intestinal intraepithelial T lymphocytes (T-IEL) patrol the gut and have important roles in regulating intestinal homeostasis. T-IEL include both induced T-IEL, derived from systemic antigen-experienced lymphocytes, and natural IEL, which are developmentally targeted to the intestine. While the processes driving T-IEL development have been elucidated, the precise roles of the different subsets and the processes driving activation and regulation of these cells remain unclear. To gain functional insights into these enigmatic cells, we used high-resolution, quantitative mass spectrometry to investigate the proteomic landscape of the main T-IEL populations in the gut. Comparing the proteomes of induced T-IEL and natural T-IEL subsets, with naive CD8+ T cells from lymph nodes exposes the dominant effect of the gut environment over ontogeny on T-IEL phenotypes. Analyses of protein copy numbers of >7000 proteins in T-IEL reveal skewing of the cell surface repertoire towards epithelial interactions and checkpoint receptors; strong suppression of the metabolic machinery indicating a high energy barrier to functional activation; and changes in T cell antigen receptor signalling pathways reminiscent of chronically activated T cells. These novel findings illustrate how multiple input signals need to be integrated to regulate T-IEL function.

immunology↗

bioRxiv2020

Erosion of human X chromosome inactivation alters proteome expression from genes across the X chromosome and all autosomes

X chromosome inactivation (XCI) is a dosage compensation mechanism in female mammals whereby genes from one X chromosome are repressed. Analysis of human induced pluripotent stem cell (iPSC) lines using proteomics, RNAseq and polysome profiling showed a major change in the proteome upon XCI erosion. This resulted in amplified RNA and protein expression from X-linked genes. However, increased protein expression was also detected from autosomal genes without a corresponding mRNA increase, altering the protein-RNA correlation between genes on the X chromosome and autosomes. Eroded iPSC lines display ~13% increase in cell protein content, along with increased expression of ribosomal proteins, ribosome biogenesis and translation factors. They also showed significantly increased levels of active polysomes within the eroded lines. We conclude that erosion of XCI causes a major remodelling of the proteome, with translational mechanisms affecting the expression of a much wider range of proteins and disease-linked loci than previously realised.

biochemistry↗