Biology subjects

Barderas, R.

Publications and source records attributed to Barderas, R..

8 recordsLinked to original sources

bioRxiv2026

A generalizable normalization framework to decouple protocol and instrument effects: Application to high-sensitivity proteomics multicentric study (PME13)

Multicenter studies are essential for benchmarking analytical workflows, yet their interpretation is often confounded by the combined effects of experimental protocols and instrumentation. To address this challenge, we introduce a simple normalization-based analytical framework, the recovery metric ({rho}), designed to decouple protocol driven effects from instrument dependent variability. We applied this framework to the 13th Proteomics Multicentric Experiment (PME13), a large multicentric proteomics dataset generated across 27 laboratories using high sensitivity workflows and varying sample preparation protocols. By leveraging a common digested reference sample, {rho} enables direct cross-comparison of all datasets on a unified scale, effectively minimizing instrument-related biases. Using this approach, we demonstrate that apparent instrument dependent trends are largely removed when evaluated through {rho}, revealing consistent protocol driven effects across laboratories. Statistical modeling identified key variables influencing {rho}, including sample input amount, reduction and alkylation, and the use of n-dodecyl-{beta}-D-maltoside (DDM). While DDM was associated with improved {rho}, reduction and alkylation and additional handling steps led to reduced performance, particularly at low input levels. We further highlight practical considerations for the application of ratio based normalization, including the occurrence of values exceeding theoretical bounds, which reflect deviations from underlying assumptions and require appropriate filtering. Overall, this work establishes a generalizable analytical strategy for disentangling confounding factors in multicentric datasets and provides practical guidelines for optimizing high sensitivity proteomics (HSP) workflows. The proposed framework is broadly applicable to other analytical fields where cross laboratory comparability is required.

bioinformatics↗

bioRxiv2026

Mapping the Human Ghost Proteome: Classification and Experimental Detection Biases in the Identification of Alternative Microproteins

The discovery of alternative proteins (AltProts), translated from non-canonical ORFs, has expanded the human proteome and revealed a hidden layer known as the "ghost proteome". Despite increasing evidence, AltProts detection remains challenging due to their small size, physicochemical heterogeneity, and lack of annotation. Here, we developed an integrated bioinformatic and proteomic workflow to benchmark the detection of reference proteins (RefProts), isoforms, and alternative microproteins (MicroAltProts) in colorectal cancer cells using four extraction protocols--HCl, RIPA buffer, RIPA with chloroform, and RIPA followed by 30 kDa filtration--combined with high-resolution data-independent acquisition mass spectrometry. We identified and quantified using the Orbitrap Astral mass spectrometer a total of 66,438 peptides corresponding to 12,584 different protein groups across methods, with RIPA-based extraction approaches providing the most comprehensive coverage. To reduce redundancy in the OpenProt database and focus on MicroAltProts, we curated the dataset by removing known isoforms and long proteins, yielding a non-redundant set of 183,937 MicroAltProts. K-means clustering based on eight ProtParam-derived features grouped MicroAltProts into four physicochemical clusters. Among them, 43 MicroAltProts (<200 amino acids) were experimentally validated by mass spectrometry and classified into tiers following recent recommended international guidelines. Cluster assignment of detected MicroAltProts revealed that HCl extraction favored disordered, alkaline proteins, while RIPA-based protocols enabled the identification of membrane-associated and amphipathic -helical MicroAltProts. Structural prediction indicated the presence of diverse folding determinants, including transmembrane helices, disordered regions, and nucleic acid-binding-like motifs. Altogether, this study provides a roadmap framework for the unbiased simultaneous detection of RefProts, isoforms, and AltProts, and supports a broader functional role for MicroAltProts.

genomics↗

bioRxiv2025

Beyond One-Size-Fits-All: Tumor Biology Influences Nanoparticle Behaviour in Cancer Models

Nanoparticles (NPs) are a promising tool for cancer therapy, yet few have successfully reached clinical application. Current nanomedicine development pipelines are focused on optimizing physical properties of NPs, overlooking the impact of tumor biology on their behavior. Here, we show that the same NPs exhibit distinct accumulation and penetration patterns in 3D spheroids derived from four tumor models (representative of lung, colon, breast, and cervical cancer). We uncover an inverse relationship between NP uptake and penetration: tumors with slower internalization show deeper NP diffusion. Proteomic analysis revealed that tumor-specific expression of endocytic and extracellular matrix proteins underlies this variability. Our findings challenge the prevailing one-size-fits-all approach and highlight the need to integrate tumor biology into NP design. Tailoring NPs to the unique cellular and extracellular features of each tumor type will be critical for developing more effective and clinically relevant nanotherapies.

cell biology↗

bioRxiv2024

Deciphering stiffness-driven changes in colorectal cancer by proteomics

Tumor stiffening plays a pivotal role in cancer progression. Increased tumor stiffness, resulting from interactions between cancer cells and their surrounding microenvironment, alters the tumors mechanical properties and significantly impacts cancer growth and metastasis, the primary cause of cancer-related deaths. Despite the importance of tumor stiffness, systematic studies exploring its effect on proteomic profiles are limited. In this study, focused on colorectal cancer, we show that matrix stiffness significantly alters the expression of secreted proteins, while intracellular protein levels remain largely unaffected. Functional assays reveal that the changes in the secretome, driven by matrix stiffness, enhance cell migration, angiogenesis, and matrix remodeling, which collectively contribute to a more aggressive cancer phenotype. Our findings emphasize the critical role of matrix stiffness in driving colorectal cancer progression through changes in the secretome, offering valuable insights for the development of biomechanical cancer therapies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/618701v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@fb6b58org.highwire.dtl.DTLVardef@446a31org.highwire.dtl.DTLVardef@197e22forg.highwire.dtl.DTLVardef@82a896_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

Synthesis and mechanical characterization of polyacrylamide (PAAm) hydrogels with different stiffnesses for large-batch cell culture applications

The impact of mechanical cues on cell behavior is increasingly being recognized, rendering hydrogel platforms that mimic the extracellular matrix indispensable in in vitro cell biology research. Here, we present a step-by-step protocol for synthesis and rheological characterization of polyacrylamide (PAAm) hydrogels with varying stiffnesses, produced as large circular unattached gels customizable in shape and size. We outline methods for their use in cell culture and downstream applications involving secretome or cell analysis, and protein visualization by fluorescence microscopy. This protocol is based on the recent work of Shi & Janmey who describe a novel and straightforward method for the production of large PAAm hydrogels for bulk cell culture and mechanobiology studies.1 Their procedure results in one large gel that is not attached to a supporting surface and therefore can be transferred and/or stamped to generate PAAm gels of custom shapes and sizes. The aim of this step-by-step procedure is therefore not to improve the reported protocol, but to create a clearly outlined and repeatable protocol that enables a smooth implementation in any lab for a diverse audience. In addition, our protocol describes besides harvesting of cells also the collection of secretome for downstream biochemical analyses, as well as immunofluorescence labeling using antibodies that can readily be multiplexed for optimization of labeling conditions. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/613503v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@cefcc3org.highwire.dtl.DTLVardef@1348c93org.highwire.dtl.DTLVardef@f935acorg.highwire.dtl.DTLVardef@132dbd0_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

CRISPR targeting of FOXL2 c.402C>G mutation reduces malignant phenotype in granulosa tumor cells and identifies anti-tumoral compounds

FOXL2 is a transcription factor essential for sex determination and ovary development and maintenance. Mutations in this gene are implicated in syndromes involving premature ovarian failure and granulosa cell tumors (GCTs). This rare cancer accounts for less than 5% of diagnosed ovarian cancers and is causally associated with the FOXL2 c.402C>G, p.C134W mutation in 97% of the adult cases (AGCTs). In this study, we employed CRISPR technology to specifically eliminate the FOXL2 c.402C>G mutation in granulosa tumor cells. Our results show that this Cas9-mediated strategy selectively targets the mutation without affecting the wild-type allele. Granulosa cells lacking the FOXL2 c.402C>G exhibit a reduced malignant phenotype, with significant changes in cell proliferation, invasion, and cell death. Furthermore, these modified cells are more susceptible to Dasatinib and Ketoconazole. Transcriptomic and proteomic analyses reveal that CRISPR-modified granulosa tumor cells shift their expression profiles towards a wild-type like phenotype. Additionally, this altered expression signature has led to the identification of new compounds with antiproliferative and pro-apoptotic effects on granulosa tumor cells. Our findings demonstrate the potential of CRISPR technology for the specific targeting and elimination of a mutation causing granulosa cell tumors, highlighting its therapeutic promise for treating this rare ovarian cancer. Simple SummaryAdult granulosa cell tumors (AGCTs), characterized by a specific point mutation (C134W) in the gene FOXL2, are less than 5% of all the diagnosed ovarian cancers. Surgery is the cornerstone treatment for AGCT even at relapse, with systemic therapy showing poor results. The aim of our study is to explore the potential therapeutic effect of the elimination of the C134W mutation. To achieve our goal, we have eliminated the mutant allele using CRISPR technology. Our results demonstrate that CRISPR-mediated elimination of FOXL2-C134W reduces the malignant phenotype of granulosa tumor cells, which change their transcriptional, proteomic, and cellular phenotype to a wild type like, granulosa type. Moreover, the induced changes allowed us to find new compounds with antitumoral activity. This work highlights the therapeutic potential of CRISPR mediated technology for the treatment of AGCT.

cancer biology↗

bioRxiv2023

Flat clathrin lattices are linked to metastatic potential in colorectal cancer

Clathrin assembles at the cells plasma membrane in a multitude of clathrin-coated structures (CCSs). Among these are flat clathrin lattices (FCLs), alternative clathrin structures that have been found in specific cell types, including cancer cells. Here we show that these structures are also present in different colorectal cancer (CRC) cell lines, and that they are extremely stable with lifetimes longer than 8 hours. By combining cell models representative of CRC metastasis with advanced fluorescence imaging and analysis, we discovered that the metastatic potential of CRC is associated with an aberrant membranous clathrin distribution, resulting in a higher prevalence of FCLs in cells with a higher metastatic potential. These findings suggest that clathrin organization might play an important yet unexplored role in cancer metastasis.

cancer biology↗

bioRxiv2021

Targeting the mitochondrial RNA methyltransferase TRMT61B reveals new therapeutic opportunities in aneuploid cancer cells

Chromosomal instability (CIN) is an important source of genetic and phenotypic variation that has been extensively reported as a critical cancer related property that improves tumor cell adaptation and survival. CIN and its immediate consequence, aneuploidy, provoke adverse effects on cellular homeostasis that need to be overcome by developing efficient anti-stress mechanisms. Perturbations in these safeguard responses might be detrimental for cancer cells and represent an important tumor specific Achilles heel since CIN and aneuploidy are very rare events in normal cells. On the other hand, epitranscriptomic marks catalyzed by different RNA modifying enzymes have been found to change under several stress insults. Although CIN and aneuploidy are important intracellular stressors, their biological connection with RNA modifications is pending to be determined. In an in silico search for new cancer biomarkers, we have identified TRMT61B, a mitochondrial RNA methyltransferase enzyme, to be associated with high levels of aneuploidy. In the present work, we study the connection of this molecule with cancer and aneuploidy. First, we show increased protein amounts of TRMT61B in tumor cell lines with imbalanced karyotype as well as in different tumor types compared to unaffected control tissues. In addition, we demonstrate that depletion of TRMT61B in melanoma cells reduces cell proliferation either by fostering apoptosis and inhibiting autophagy in high-aneuploid (ANEhigh) cells or by inducing senescence in the case of low-aneuploid (ANElow) cell lines. Further, TRMT61B elimination compromises mitochondrial function and reduces the expression of several mitochondrial encoded proteins that are part of the electron transport chain. Finally, transwell and xenograft experiments revealed a reduced invasive and tumorigenic capacity upon TRMT61B depletion that strengthen the therapeutic value of this aneuploidy-associated biomarker. These results, which connect tumorigenesis, aneuploidy and mitochondrial RNA methylation, bring to the cancer field a new putative strategy to specifically target high aneuploid tumors.

cancer biology↗