IntroductionRheumatoid arthritis (RA) is an autoimmune disease resulting from a response driven by self-reactive CD4+ T cells that recognize autoantigenic peptides presented by antigen-presenting cells (APCs). Recent evidence suggests that CD8+ T cells are also important players in this process. This study aims to define the immunopeptidome of HLA class I molecules from RA synovial tissue (ST)- APCs and synovial fluid (SF)-pulsed monocyte-derived dendritic cells (DCs), and to prove the suitability of this approach to identify peptides recognized by CD8+ T cells from RA patients.MethodsHLA-ABC/peptide complexes were obtained from DCs generated from healthy subjects (HS), which were pulsed with a pool of RA SF (SF-DCs) or left unpulsed (UP-DCs), or obtained directly from RA ST. Isolated peptides were sequenced by mass spectrometry. The autoantigenicity of a set of ten peptides selected from this repertoire was estimated by their ability to activate CD8+ T cells from RA patients, as measured by the induction of intracellular IFN-γ expression and surface exposure of CD107a by flow cytometry.ResultsBetween 107 to 663 peptides were obtained from DC samples, while over 3, 500 class I peptides were identified from each ST sample. The number of peptides was narrowed down based on prioritization steps that included the selection of sequences derived from RA-relevant, immune-related proteins, for further CD8+ T-cell stimulation assays. The frequencies of CD8+ T cells co-stained for IFN-γ and CD107a were significantly higher in RA patients than in HS in response to peptides derived from the proteins MIF, ETS1, USF1, VIM, and AHR.DiscussionIn the present work, we validated the use of an immunopeptidomic strategy to identify a series of novel autoantigenic CD8+ T-cell epitopes for RA, derived from synovial, mostly immune-related proteins, which may be useful for future clinical applications.

While professional antigen-presenting cells drive adaptive immunity, atypical cell types can fulfill this role in the bone marrow. Megakaryocytes (MKs) are canonically recognized for platelet production, but recent studies indicate functional heterogeneity and immune potential. We found that ~20% of bone marrow MKs express Major Histocompatibility Complex (MHC) II and co-stimulatory receptors CD80, CD86, and CD40. These MKs process and present antigen to activate T cells in an MHC II-dependent manner. MK/T cell interactions induced TGF-β1 secretion and promoted induced regulatory T cell differentiation. Immunopeptidomics of MK MHC II receptor confirmed occupancy by exogenous peptides, demonstrating in vivo functionality. Using a model with MK-targeted deletion of MHC II (Pf4-MHCΔ/Δ mice), we observed altered TLR signaling, reduced bone marrow TGF-β1, and decreased numbers of hematopoietic stem cells. Together, these findings identify MHC II+ MKs as noncanonical antigen-presenting cells that modulate adaptive immunity and maintain the hematopoietic niche.

Wastewater analysis has emerged as a powerful tool for wastewater-based epidemiology, environmental surveillance, and monitoring of emerging contaminants. While most studies rely on targeted approaches focusing on predefined compound lists, untargeted metabolomics offers potential to capture a broader and less biased chemical snapshot. However, the complexity of wastewater matrices and the chemical diversity of small molecules pose analytical challenges. In this study, we apply a multi-platform, untargeted metabolomics workflow to profile the influent wastewater metabolome of five wastewater treatment plants in Spain, differing in geographic context, population size, and industrial activity. Twenty-four-hour composite samples were collected in three seasons and analyzed using gas chromatography, reversed-phase liquid chromatography, and hydrophilic interaction liquid chromatography, all coupled to mass spectrometry. A total of 828 unique compounds were annotated across platforms, with minimal overlap, highlighting the complementarity of analytical approaches and extraction strategies. Multivariate analyses revealed reproducible site-associated chemical patterns consistent across seasons and platforms. Dominant contributors included lipids, organic acids, organic oxygen compounds, organoheterocyclic compounds, and benzenoids, reflecting differences in population and human activity. This work demonstrates the feasibility of a multi-platform untargeted workflow for generating complementary and reproducible comparative profiles of wastewater influent. The resulting public dataset is intended as a methodological and exploratory comparative resource, and broader spatial and longitudinal validation is required before generalization or use for source attribution.

Naturally acidic aqueous extracts from Vicia faba L. pod valves are being explored as sustainable, L-DOPA-oriented plant preparations. Pod valves represent an underutilized processing by-product reported to contain L-DOPA, a compound widely used in Parkinson's disease therapy, while acidic aqueous media may help preserve its physicochemical stability. However, the protein macromolecules co-extracted from V. faba pod valves under these conditions remain poorly characterized. This information is relevant because persistent plant proteins may influence extract composition, stability, susceptibility to degradation, and downstream processing requirements. Here, we characterized co-extracted V. faba protein macromolecules in aqueous pod-valve extracts prepared in ultrapure water or naturally acidic media, including 2% Phyllanthus emblica, 5% Punica granatum, and 2% Ribes rubrum. Protein profiles were first evaluated by SDS-PAGE and subsequently analyzed by nanoflow liquid chromatography coupled to high-resolution tandem mass spectrometry (nLC-MS/MS). Protein identifications were complemented with Gene Ontology annotation and a descriptive semi-quantitative assessment of relative protein representation across extraction media. Chitinase was the most represented V. faba-assigned protein macromolecule across the extracts, with additional highly represented proteins including glucan endo-1,3-beta-D-glucosidase, pathogenesis-related proteins, and polyphenol oxidase A1. These co-extracted proteins are mainly associated with plant defense, stress responses, cell-wall remodeling, and oxidative processing, suggesting that they may be relevant for extract quality attributes during handling and storage. This study provides a compositional proteomic reference for the co-extracted protein macromolecules present in acidic aqueous extracts from V. faba pod valves, supporting future studies on extract stability, processing optimization, and the development of standardized plant-based preparations.

Lactated Ringer's solution (LR) has been associated with anti-inflammatory effects in acute pancreatitis, likely attributable to its lactate content, but the underlying mechanisms remain poorly understood. Circulating small extracellular vesicles (sEVs) contribute to systemic inflammation in this condition by activating macrophages, and we investigated whether lactate could modulate their inflammatory activity. sEVs derived from BxPC-3 pancreatic cells were treated with lactate and their inflammatory potential assessed by measuring their capacity to induce pro-inflammatory cytokine expression in macrophages. Circulating sEVs were also isolated from patients with acute pancreatitis receiving either LR or normal saline and analysed using the same approach. Vesicle uptake and protein lactylation levels were evaluated by microscopy and western blot, respectively. Lactate treatment reduced sEV-induced macrophage activation without altering vesicle uptake, and was associated with a modest increase in protein lactylation. A similar reduction in inflammatory activity was observed in circulating sEVs from LR-treated patients, but not in those receiving normal saline, alongside a comparable increase in protein lactylation. These findings suggest that lactate can directly modify sEV activity and that the anti-inflammatory effects of LR in acute pancreatitis may be mediated, at least in part, through lactylation-dependent modulation of sEV inflammatory activity.

While professional antigen-presenting cells drive adaptive immunity, atypical cell types can fulfill this role in the bone marrow. Megakaryocytes (MKs) are canonically recognized for platelet production, but recent studies indicate functional heterogeneity and immune potential. We found that ~20% of bone marrow MKs express Major Histocompatibility Complex (MHC) II and costimulatory receptors CD80, CD86, and CD40. These MKs process and present antigen to activate T cells in an MHC II-dependent manner. MK-T cell interactions induced TGF-β1 secretion and promoted induced regulatory T cell differentiation. Immunopeptidomics of MK MHC II receptor confirmed occupancy by exogenous peptides, demonstrating in vivo functionality. Using a model with MK-targeted deletion of MHC II (Pf4-MHC Δ/Δ mice), we observed altered TLR signaling, reduced bone marrow TGF-β1, and decreased numbers of hematopoietic stem cells. Together, these findings identify MHC II+ MKs as noncanonical antigen-presenting cells that modulate adaptive immunity and maintain the hematopoietic niche.

Translational silence of spermatozoa has long been considered the norm in animals. However, studies in mammals have shown that the mitochondrial ribosomal machinery is selectively activated during capacitation in the female reproductive tract, while cytosolic ribosomes remain inactive. Here, using quantitative proteomics in a piscine model species, we show that proteins involved in mRNA processing and cytoplasmic translation are predominantly accumulated in immature spermatozoa within the extratesticular excurrent ducts, while those related to flagellar motility are enriched in ejaculated (mature) sperm. Based upon in vitro incubation of isolated spermatozoa, motility assays and polysome profiling, we further show that 80S cytoplasmic and 55S mitochondrial ribosomes are actively involved in the translation of motility- and osmoadaptation-related proteins. These findings thus reveal that post-testicular piscine spermatozoa can maintain de novo protein synthesis through both mitochondrial and cytoplasmic ribosomal activity, which is necessary for the acquisition of full sperm function.

Recently, environmental proteomics has revealed its potential to find biomarkers for human health, as part of wastewater-based epidemiology. However, current analytical strategies for conducting environmental proteomics studies face challenges, including high instrumental costs and the time required for sample management. These costs can become even higher when analysis must be repeated due to experimental issues. Therefore, using MALDI-TOF is proposed as a viable and cost-effective method for the initial screening of environmental proteomics samples, allowing for preliminary sample characterization and ensuring sample quality. MALDI-TOF represents a greener alternative compared to other proteomic techniques, as its faster analysis times and reduced reagent and energy consumption contribute to lower environmental impact. However, the signals obtained from MALDI-TOF analysis must be further analyzed to achieve peptide identification and protein inference. In this work, we present Aquasearch, a novel software application designed to identify protein biomarkers from MALDI-TOF raw data. Aquasearch incorporates a comprehensive database of tryptic peptides derived from previously identified biomarkers by our research group. We have validated the effectiveness and accuracy of this tool in identifying protein biomarkers through the analysis of a set of wastewater samples. Additionally, this information was utilized in multisampling, allowing the unsupervised clustering of samples with similar proteomic profiles. Overall, Aquasearch facilitates the initial screening of proteomics samples using a straightforward approach and ensures the effectiveness of subsequent in-depth analyses. Aquasearch and its source code are available at https://github.com/cplqam/IDAEA-IIBB_Aquasearch.

Wastewater-based epidemiology (WBE) offers a unique window into the health and habits of communities through the analysis of pollutants and biomarkers in sewage. Traditionally focused on small molecules, such as pharmaceuticals and illegal drugs, recent advances in environmental proteomics have expanded WBE to include large biomolecules such as proteins. Notably, novel sampling methods using polymeric probes and high-resolution mass spectrometry have facilitated the detection of human and animal proteins, both soluble and in particulate material, linking them to specific populations and industrial activities. An immunological dimension to this approach is fundamental to include the recognition of host immunoglobulins, immune-response proteins, and pathogen antigens in wastewater, potentially serving as indicators of community immune status, infection prevalence, and vaccination coverage. This review consolidates the latest advancements in environmental proteomics as applied to WBE, emphasizing an immunological perspective as a comprehensive tool for assessing population health and environmental conditions to bridge environmental monitoring, public health, and clinical diagnostics.

A considerable number of the physiological functions of extracellular vesicles are conditioned by the protein corona attached to their surface. The composition of this corona is initially defined during their intracellular synthesis, but it can be subsequently modified by interactions with the microenvironment. Here, we evaluated how the corona of small extracellular vesicles exposed to the inflammatory environment generated in acute pancreatitis is modified and what functional changes occur as a result of these modifications. Small extracellular vesicles obtained from a pancreatic cell line were incubated with the ascitic fluid generated in experimental acute pancreatitis in rats. Using proteomic techniques, we detected the appearance of new proteins and an increase the uptake of extracellular vesicles by certain cell types and the response induced in inflammatory cells. The inhibition of different pattern recognition receptors reversed this activation, indicating that some of these effects could be due to binding of damage-associated molecular patterns to the corona. All of this indicates that in pathologies such as acute pancreatitis, characterized by an inflammatory response and intense tissue damage, the microenvironment substantially influences the corona of extracellular vesicles, thus altering their behavior and enhancing their inflammatory activity.

Alternative splicing enhances protein diversity in different ways, including through exonization of transposable elements (TEs). Recent transcriptomic analyses identified thousands of unannotated spliced transcripts with exonizing TEs, but their contribution to the proteome and biological relevance remains unclear. Here, we use transcriptome assembly, ribosome profiling, and proteomics to describe a population of 1,227 unannotated TE exonizing isoforms generated by mRNA splicing and recurrent in human populations. Despite being shorter and lowly expressed, these isoforms are shared between individuals and efficiently translated. Functional analyses show stable expression, specific cellular localization, and, in some cases, modified functions. Exonized TEs are rich in ancient genes, whereas the involved splice sites are recent and can be evolutionarily conserved. In addition, exonized TEs contribute to the secondary structure of the emerging isoforms, supporting their functional relevance. We conclude that TE-spliced isoforms represent a diversity reservoir of functional proteins on which natural selection can act.

Wastewater-based epidemiology (WBE) aims to understand a population’s consumption habits, exposure to chemicals, and the prevalence of specific diseases or pathogens. This is achieved by the chemical or biological/genomic determination of biomarkers (e.g., excreted metabolic products), which are in urban wastewater generated by that population. WBE has been mostly linked to the determination of small molecules of human origin using liquid-chromatography mass spectrometry (LC-MS). In this Perspective, we provide a state-of-the-art and critical evaluation of further developments in the information achieved by determining small molecules as well as the most promising analytical techniques to enlarge the information obtained. By simultaneously monitoring small and large molecules we can comprehensively trace the population’s health by their consumption of prescribed pharmaceuticals and illegal drugs, as well as by the amount of excreted macromolecule biomarkers such as peptides and proteins. Moreover, species-specific protein sequences allow us to monitor animal populations reflecting farming and slaughterhouse activities (poultry, pigs…) or pest occurrences (rats). To this end, the capability of proteomic studies using high-resolution tandem mass spectrometry is highlighted and compared in the context of other advances in the broader field of high-resolution mass spectrometry (HRMS).

Lipoprotein lipase (LPL) is responsible for the intravascular catabolism of triglyceride-rich lipoproteins and plays a central role in whole-body energy balance and lipid homeostasis. As such, LPL is subject to tissue-specific regulation in different physiological conditions, but the mechanisms of this regulation remain incompletely characterized. Previous work revealed that LPL comprises a set of proteoforms with different isoelectric points, but their regulation and functional significance have not been studied thus far. Here we studied the distribution of LPL proteoforms in different rat tissues and their regulation under physiological conditions. First, analysis by two-dimensional electrophoresis and Western blot showed different patterns of LPL proteoforms (i.e., different pI or relative abundance of LPL proteoforms) in different rat tissues under basal conditions, which could be related to the tissue-specific regulation of the enzyme. Next, the comparison of LPL proteoforms from heart and brown adipose tissue between adults and 15-day-old rat pups, two conditions with minimal regulation of LPL in these tissues, yielded virtually the same tissue-specific patterns of LPL proteoforms. In contrast, the pronounced downregulation of LPL activity observed in white adipose tissue during fasting is accompanied by a prominent reconfiguration of the LPL proteoform pattern. Furthermore, refeeding reverts this downregulation of LPL activity and restores the pattern of LPL proteoforms in this tissue. Importantly, this reversible proteoform-specific regulation during fasting and refeeding indicates that LPL proteoforms are functionally diverse. Further investigation of potential differences in the functional properties of LPL proteoforms showed that all proteoforms exhibit lipolytic activity and have similar heparin-binding affinity, although other functional aspects remain to be investigated. Overall, this study demonstrates the ubiquity, differential distribution and specific regulation of LPL proteoforms in rat tissues and underscores the need to consider the existence of LPL proteoforms for a complete understanding of LPL regulation under physiological conditions.

Classically, the characterization of wastewater components has been restricted to the measurement of indirect parameters (chemical and biological oxygen demand, total nitrogen) and small molecules of interest in epidemiology or for environmental control. Despite the fact that metaproteomics has provided important knowledge about the microbial communities in these waters, practically nothing is known about other non-microbial proteins transported in the wastewater. The method described here has allowed us to perform a large-scale characterization of the wastewater proteome. Wastewater protein profiles have shown to be very different in different collection sites probably reflecting their human population and industrial activities. We believe that wastewater proteomics is opening the doors to the discovery of new environmental and health biomarkers and the development of new, more effective monitoring devices for issues like monitorization of population health, pest control, or control of industry discharges. The method developed is relatively simple and combines procedures for the separation of the soluble and particulate fractions of wastewater and their concentration, and conventional shotgun proteomics using high-resolution mass spectrometry for protein identification. •Unprecedented method for wastewater proteome characterization.•Proteins as new potential biomarkers for sewage chemical-information mining, wastewater epidemiology and environmental monitoring.•Wastewater protein profiles reflect human and industrial activities.

Wastewater-based epidemiology has been revealed as a powerful approach for surveying the health and lifestyle of a population. In this context, proteins have been proposed as potential biomarkers that complement the information provided by currently available methods. However, little is known about the range of molecular species and dynamics of proteins in wastewater and the information hidden in these protein profiles is still to be uncovered. In this study, we investigated the protein composition of wastewater from 10 municipalities in Catalonia with diverse populations and industrial activities at three different times of the year. The soluble fraction of this material was analyzed using liquid chromatography high-resolution tandem mass spectrometry using a shotgun proteomics approach. The complete proteomic profile, distribution among different organisms, and semiquantitative analysis of the main constituents are described. Excreta (urine and feces) from humans, and blood and other residues from livestock were identified as the two main protein sources. Our findings provide new insights into the characterization of wastewater proteomics that allow for the proposal of specific bioindicators for wastewater-based environmental monitoring. This includes human and animal population monitoring, most notably for rodent pest control (immunoglobulins (Igs) and amylases) and livestock processing industry monitoring (albumins).

Oncogenesis often implicates epigenetic alterations, including derepression of transposable elements (TEs) and defects in alternative splicing. Here, we explore the possibility that noncanonical splice junctions between exons and TEs represent a source of tumor-specific antigens. We show that mouse normal tissues and tumor cell lines express wide but distinct ranges of mRNA junctions between exons and TEs, some of which are tumor specific. Immunopeptidome analyses in tumor cell lines identified peptides derived from exon-TE splicing junctions associated to MHC-I molecules. Exon-TE junction-derived peptides were immunogenic in tumor-bearing mice. Both prophylactic and therapeutic vaccinations with junction-derived peptides delayed tumor growth in vivo. Inactivation of the TE-silencing histone 3-lysine 9 methyltransferase Setdb1 caused overexpression of new immunogenic junctions in tumor cells. Our results identify exon-TE splicing junctions as epigenetically controlled, immunogenic, and protective tumor antigens in mice, opening possibilities for tumor targeting and vaccination in patients with cancer.

Diabetes mellitus (DM) and calcific aortic stenosis (CAS) are common morbidities in the elderly, which are both chronic, progressive and often concomitant diseases. Several studies revealed that DM increases the risk of developing severe CAS, yet clear information about the relationship between both these diseases and the influence of DM on the progression of CAS is currently lacking. To evaluate the effect of DM on aortic valves and on the process of calcification, and to achieve better patient management in daily clinical practice, we analysed calcified and noncalcified valve tissue from patients with severe CAS, with or without DM. A proteomic strategy using isobaric tags was adopted and the plasma concentrations of nine proteins were studied using 3 orthogonal methods and in a separate cell model. The differentially expressed proteins identified are implicated in biological processes like endopeptidase activity, lipid metabolism, coagulation, and fibrinolysis. The results obtained provide evidence that DM provokes changes in the proteome of aortic valves, affecting valve calcification. This finding may help enhance our understanding of the pathogenesis of CAS and how DM affects the evolution of this condition, an important step in identifying targets to personalize the treatment of these patients.