Search Results (158)

Integral membrane proteins (IMPs), which constitute 50–60% of drug targets, play essential roles in numerous biological processes but remain underrepresented in conventional bottom-up and structural proteomics owing to their hydrophobicity and resistance to proteolysis. Although advances in IMP proteomics have improved global IMP detection, most efforts focus on proteome-scale protein identification rather than targeted structural analysis. Protein footprinting and cross-linking, two approaches in structural proteomics, require high sequence coverage and protein digestion to peptides of suitable length for structural elucidation, necessitating optimized digestion condition for individual IMPs. Here, we report a digestion protocol tailored for structural mass spectrometry and evaluate its performance by using a single amphipathic IMP model featuring distinct extramembrane and transmembrane domains. We evaluated the use of various protease–additive combinations and applied filter-aided sample preparation (FASP) to remove detergents and surfactants efficiently prior to MS analysis. The optimized conditions consistently yielded >90% sequence coverage. Guided by MS retention time calibration and hydrophobic factor simulations, we identified a “sweet spot” for transmembrane peptide detection. Notably, although cleavable surfactants can enhance proteome-wide coverage, our results show that they are not essential for single protein studies as they are in structural proteomics. Instead, detergent removal, protease selection, and generation of suitably sized peptides are critical for enabling reliable bottom-up structural analysis of IMPs. The protocol developed here provides a practical framework for optimizing digestion conditions in IMP characterization. Full article

A clinical strain of the opportunistic pathogen Serratia rubidaea, a known contaminant of healthcare environments and an emerging cause of invasive infections, is described. The studied isolate, recovered from a nurse’s hand skin swab during routine screening, exhibits a broad profile of antibiotic resistance combined with reduced susceptibility to several disinfectants. Phenotypic susceptibility testing using a tablet-based microdilution and disk diffusion method was employed to determine the minimum inhibitory concentrations (MICs) of antimicrobial agents from different classes, while broth microdilution assays with disinfectants revealed high-level tolerance to widely used agents, including 70% C₂H₅OH, 3% H₂O₂, 0.05% polyhexamethylene guanidine (PHMG) and others. Whole-genome sequencing identified multiple resistance-associated determinants, such as chromosome-encoded class C $\beta$-lactamase (ampC), several efflux systems (sdeXY, macAB, and emrAB) combined with multicopy tolC, and specific transferases (fos and arnT). Shotgun bottom-up HPLC-MS/MS proteomics confirmed baseline expression of these and other stress-tolerance-related proteins under non-inducing conditions. Taken together, these data underscore the importance of surveillance for Serratia spp. in healthcare facilities to detect strains that combine intrinsic or acquired multidrug resistance with robust survival traits such as disinfectant tolerance and biofilm formation. The present study provides a reference-level phenotypic, genomic, and proteomic characterization of a S. rubidaea clinical isolate, contributing to the understanding of the adaptive potential of this resilient opportunistic pathogen in clinical environments. Full article

Recent advances in mass spectrometry, data-independent acquisition, proteoform-resolving workflows, and multi-omics integration have significantly expanded the scale and scope of proteomics. However, the reuse and translational application of these datasets are limited by inconsistent standards, insufficient metadata, and inadequate computational interoperability. Proteoform-centric approaches provide higher molecular resolution by capturing intact protein variants and patterns of post-translational modification. Computational methods, including selected applications of machine learning and large language models (LLMs), are increasingly used for tasks such as spectral prediction and pattern discovery in clinical proteomics datasets. Despite these advancements, FAIR (Findable, Accessible, Interoperable, and Reusable) data practices, proteoform biology, and AI analytics are often pursued independently. This work presents an integrated framework for next-generation proteomics in which standardization and FAIR (Findable, Accessible, Interoperable, and Reusable) principles establish machine-actionable foundations for proteoform-resolved analysis and computational inference. It examines community efforts to promote data sharing and interoperability, as well as strategies for characterizing proteoforms using bottom-up, middle-down, and top-down approaches. It also highlights emerging AI and ML applications within the proteomics workflow. The framework emphasizes the importance of treating proteoforms as primary computational entities and adopting FAIR practices during data collection to enable reproducible and interpretable modeling. Finally, it introduces an architectural model that integrates FAIR infrastructures and proteoform resolution. In addition, practical recommendations for making AI-ready proteomics, including a minimal community checklist to support reproducibility, benchmarking, and translational scalability, are provided. Full article

Type 2 diabetes (T2D) causes systemic metabolic and inflammatory changes that affect the oral cavity, but salivary molecular markers remain poorly characterized. A peptide-centric reanalysis of salivary proteomics data was performed using DIA-NN for peptide-level quantification, without collapsing peptide signals into protein-level summaries. Although the qualitative peptide repertoire was largely conserved between T2D and control samples (>96% overlap), T2D showed coordinated quantitative changes in specific peptide subsets. Differentially abundant peptides primarily originated from complement C3, alpha-2-macroglobulin, serotransferrin, mucins, apolipoproteins, and hemoglobin, with a significant enrichment of oxidized cysteine-containing peptides, indicating redox imbalance and low-grade inflammation. Structural analysis with AlphaFold showed that T2D-associated peptides are located in solvent-exposed and conformationally dynamic regions of proteins. These findings suggest that disease specificity in diabetic saliva occurs mainly at the peptide level, offering mechanistic insight into non-invasive biomarker identification and longitudinal disease monitoring. Full article

The saw-scaled viper Echis carinatus, one of the “Big Four” causes of snakebites in India, is found from Sri Lanka to eastern Iraq. To investigate clinical reports regarding the limited efficacy of Indian polyvalent antivenom (IPAV) against envenomation in Echis carinatus sochureki (ECS) in northwestern India, we obtained 22 snakes from three locations in Rajasthan and identified 148–174 toxin isoforms belonging to 21–25 toxin families in their venom using a bottom-up proteomics approach. All samples showed a high abundance of snake venom metalloproteinases (SVMPs), particularly SVMP class III. Other major components were phospholipases A₂, L-amino-acid oxidases, snake venom serine proteases and snaclecs (C-type lectins). Variation in venom composition among locations in Rajasthan, compared to E. c. carinatus (ECC) from southern India, was primarily due to differences in the relative abundance of these toxin families. Recognition of all venom components by IPAV was poor at lower antivenom concentrations. Notably, SVMP classes II and III were poorly recognized at all venom-to-antivenom ratios in all ECS venoms, and a plasma clotting assay revealed poor neutralization of procoagulant activity. This collaborative study highlights the need for the development of regional antivenoms to effectively treat snakebites in northwestern India. Full article

Breeding wheat varieties that are resilient to arid climates, which impart a complex combination of stresses, including excessive Ca, high pH, nutrient deficiency, and aridity, is important. Afghan landrace wheat is assumed to have evolved with a specific prototypical pattern of traits to adapt to its challenging, composite stress environment. Here, a useful semi-hydroponic double cup screen aiding proteomic analysis was exploited to reconstruct the combined excessive Ca²⁺ (100 ppm) and extreme pH (11.0) of the soils and to dissect specific morpho-physiological characteristics and adaptation strategies in Kihara Afghan wheat landrace (KAWLR). When compared to other cultivars and growth habits, several winter-type KAWLR showed lower unused N-K-P and greater rhizosphere pH stability in the bottom cup and higher tolerance in terms of greater root allocation shift, and most of their above ground traits (shoot biomass, chlorophyll content, and stomatal conductance) were strongly correlated with root length and biomass under stress conditions. Quantitative proteomics on the roots of a tolerant winter-type KAWLR, Herat-740 (KU-7449), showed a strong decreasing trend in changed proteins (12 increased/816 decreased). The proteins (such as mitochondrial phosphate carrier protein, cytoskeleton-related α-, and β-tubulin) that increased in abundance were associated with energy transport and cell growth. A metabolism overview revealed that most proteins that were mapped to glycolysis, fermentation, and the TCA cycle decreased in abundance. However, proteins related to cell wall and lipid metabolism pathways remained unchanged. Our results suggest that winter-type KAWLR adopts a homeostatic stress adaptation strategy that globally downshifts metabolic activity, while selectively maintaining root growth machinery. Root allocation shift, rhizosphere pH stabilization (nutrient solubilization), and a selective proteome response maintaining the root growth machinery in winter-type KAWLR could be breeding selection markers for early-stage screening in calcareous-alkaline arid land. Full article

Proteomes are typically analyzed at the level of individual proteins or protein families. In this study, we introduce a bottom-up approach that treats proteomes as holistic entities by examining the properties of k-mers within entire proteomes and protein groups. We performed a comprehensive analysis of short amino acid k-mer (k = 1, 2, 3) distributions across all proteins in a given proteome. Using 86 bacterial proteomes representing 18 clades, we evaluated whether k-mer frequencies characterize uniquely the analyzed organisms. Remarkably, in a post hoc analysis, we found that the k-mer frequency vector unambiguously coevolves with the entire proteome—a pattern not observed even within specific protein groups, such as conserved ribosomal proteins or more variable nucleotide-binding proteins. This finding holds regardless of the k-mer calculation parameters or the distance metrics employed. Our results show that even a simple analysis based on tripeptide frequencies can precisely position proteomes within the k-mer space. Moreover, relationships derived from k-mer comparisons highly correlate with evolutionary relationships derived from phylogenetic trees, reaching up to 99% match with reference classification of the proteomes within major bacterial clades. These findings establish k-mer-based proteomic analysis as an additional robust and powerful feature for characterizing evolutionary relationships, opening new pathways in phylogenetics and evolutionary genomics. Full article

Background: Insect metamorphosis is one of the most fascinating developmental processes in the natural world. Complete metamorphosis requires the breakdown and reorganisation of larval tissues and the coordinated construction and development of adult structures. The molecular events that achieve this transformation are, however, incompletely understood, and there is a particular shortage of data describing changes in protein abundance that occur during the process. Methods: Here, using a label-free quantitative bottom-up approach, we perform a novel whole-organism proteomic analysis of consecutive developmental stages of male Bicyclus anynana butterflies as they develop from caterpillars into adults via pupation. Results: Our analysis generated a dynamic reference dataset representing 2749 detected proteins. Statistical analysis identified 90 proteins changing significantly in abundance during metamorphosis, and functional interpretation highlights cuticle formation, apoptosis and autophagy during the pupal stages, and the up-regulation of respiration and energy metabolism upon completion of the fully formed adult. A preliminary search for potential peptide phosphorylation modifications identified 15 candidates, including three proteins with roles in muscle function. Conclusions: The study provides a basis for future protein-level analysis of butterfly metamorphosis and suggests the importance of dissecting the post-translational regulation associated with this fascinating developmental transformation. Full article

Background: The positive identification of a source of tissue as human plays an important role in various contexts. It is particularly important for investigations concerning tissue and organ trafficking, since unequivocal confirmation is required for legal proceedings involving such cases. While deoxyribonucleic (DNA) methods are considered the gold standard for tissue identification, issues such as degraded DNA or the presence of chemical preservatives can hinder performance and positive identification using DNA techniques. Objectives: The aim of this study was to develop a simple method for presumptive identification of human tissue using standard bottom-up proteomics data. Methods: We identified proteins isolated from human kidney, lung and spleen tissues by bottom-up proteomics and database search using Proteome Discoverer and Sequest HT algorithms. The list of identified proteins was sorted based on liquid chromatography (LC)–mass spectrometry (MS) data metrics such as the number of unique peptides used to identify each protein and the % sequence coverage of an identified protein to determine if any parameter would cluster proteins annotated as human in a distinct category. We found that eliminating proteins identified with fewer than two unique peptides and those with less than 5% sequence coverage resulted in a final list where at least half of the remaining proteins are annotated as human. We applied this data filtration process to blinded LC–MS/MS data from 26 previous experiments to assess accuracy. Results: Using bottom-up proteomics data and the filtration rules established, we identified tissue samples (n = 10), including kidney, spleen, lung, formalin-fixed paraffin-embedded uterus, frozen breast tissue, dry blood and dry saliva as human, and tissue (n = 16) from rat, mouse, bovine, and sheep as non-human, resulting in 100% sensitivity and specificity. Conclusions: The results demonstrate that the list of identified proteins following a standard bottom-up proteomics experiment could be filtered and potentially used as a fast and simple method for presumptive human tissue identification. Full article

Lysine methylation is a regulatory post-translational modification with diverse roles across both histone and non-histone proteins. Despite its biological relevance, comprehensive characterization of lysine methylation remains analytically challenging due to its low stoichiometry, subtle mass changes, and the absence of standardized, robust enrichment strategies. Mass spectrometry (MS) has become the cornerstone of methylation analysis, supporting both targeted and proteome-wide investigations. In this review, we examine the evolution of MS-based workflows for lysine methylation, including advances in ionization and fragmentation techniques, high-resolution mass analyzers, and acquisition strategies such as data-independent acquisition (DIA) and parallel accumulation–serial fragmentation (PASEF). We evaluate bottom-up, middle-down, and top-down proteomic approaches and discuss enrichment methods ranging from immunoaffinity and chromatography to chemical derivatization. Particular attention is given to persistent challenges, including proteolytic constraints and isobaric interference, that complicate confident site-level resolution. Finally, we highlight emerging solutions and future directions aimed at improving the sensitivity, specificity, and reproducibility of lysine methylation profiling. Together, this synthesis provides a forward-looking roadmap for optimizing MS workflows in methyllysine proteomics. Full article

Antibiotic resistance is a major threat to global public health. It is vital to understand the mechanism of antibiotic resistance development to prevent the emergence of new pan-resistant pathogenic bacteria and to develop new antibiotics. Measuring the differences in proteins among single bacterial cells can aid in identifying antibiotic resistance and antibiotic susceptibility due to their regulatory roles in bacterial physiology and homeostasis. Although single-cell proteomics has been successful in mammalian systems, attaining comparable performance in bacteria remains challenging due to the extremely limited proteome content of a single bacterial cell. This review discusses the role of proteomics analysis in determining antibiotic resistance and the various mass spectrometry-based strategies that have been successful in detecting protein biomarkers for antibiotic resistance from bulk proteomics analysis. It highlights the core challenges of bacterial single-cell proteomics in contrast to mammalian systems, explores emerging technologies, and the proteomes beyond the cells in studying antibiotic resistance development and antibiotic susceptibility testing. Full article

Zinc excess (Zn stress) could lead to deleterious effects in plants such as enhanced ROS production, inhibition of photosynthetic machinery, and impairment of nutrient uptake. Hence, we aimed to investigate the complexity of metabolic responses to Zn stress in Amaranthus caudatus young and mature leaves, as well as in roots by means of proteomics. Our previous metabolomics research has indicated potential involvement of gluconate and salicylate in Zn tolerance mechanisms. However, proteomics study of metabolic adjustments underlying Zn stress tolerance can give additional insight to the issue, as a lot of enzymes are known to be affected by the excess of transitional metals. The results obtained through bottom-up proteomics were complementary to our earlier metabolomics data and, furthermore, enlightened other important details in the metabolic response of A. caudatus plants to the applied Zn stress. In particular, the significant involvement of redox-related enzymes was shown, especially for the roots, and their possible interactions with salicylate and jasmonate signaling could be proposed. Furthermore, Zn²⁺-induced changes in roots and young leaves strongly affected sugar metabolism, enhanced protein quality control system, while mature leaves were characterized by remarkable decrease in subunits of photosynthetic electron transport complexes. Thus, this work emphasizes massive metabolic reprogramming aimed to reinforce root defense responses while supporting young leaves with sugar metabolites. Mass spectrometry proteomics data are available via ProteomeXchange with identifier PXD069557. Full article

This research aimed to analyze the proteomic profile of the low-molecular mass fraction of salivary pools from patients with glioblastoma IDH wild type (GBM) to disclose the small protein and peptide components, including protein fragments, cryptides, and tumor-associated peptides, still lacking specific information in the literature, to the best of our knowledge. This fraction, corresponding to the unretained proteome fraction, was obtained by pretreating the acid-soluble fraction of saliva through Filter-Aided Sample Preparation devices with a filter molecular cutoff of 10 kDa. The fraction was analyzed by LC-MS in its entire form, without trypsin pre-digestion, following a top–down approach. Data from the analysis of pre- and post-operative salivary pools from patients with newly diagnosed and recurrent GBM were compared and discussed with data obtained in our previous study on the complementary salivary proteome fraction > 10 kDa analyzed by a bottom–up approach and data from the literature. The results highlighted a panel of GBM-associated peptide fragments from different protein precursors, namely, ANXA1, CFL1, GLUL, PFN1, H2AC12, ACTB, and HBB, which are suggested for further exploration as potential diagnostic and prognostic biomarkers and clinical applications. These findings, although providing only preliminary results on a small scale, offer new insights into the molecular characteristics of GBM tumor and lay the groundwork for further investigations on a large scale using saliva liquid biopsy for biomarker discovery and validation. The aim is to advance precision medicine and improve clinical outcomes in GBM, one of the most aggressive brain tumors with a poor prognosis, for which early diagnosis and monitoring of treatment response remain significant challenges. Full article

Scorpionism is a growing public health concern in Brazil, with the Amazon region presenting the highest mortality rates but remaining understudied, especially regarding local scorpion venoms composition. This study presents the first comprehensive biochemical characterization of venoms from three Amazonian species—Tityus metuendus (TmetuV), Tityus silvestris (TsilvV), and Brotheas amazonicus (BamazV)—using an integrated approach combining Multi-Enzymatic Limited Digestion (MELD)-based bottom-up proteomics, high-resolution LC-MS/MS, chromatography, zymography, and enzymatic assays. Tityus serrulatus venom was included as a reference. Significant biochemical differences were observed: TsilvV was rich in 20–30 kDa proteins and showed strong metalloprotease activity; BamazV exhibited high molecular weight proteins and potent phospholipase A₂ (PLA₂) activity but lacked proteolytic and fibrinogenolytic activities; TmetuV showed the highest hyaluronidase activity and abundance of α-KTx neurotoxins. Zymography revealed a conserved ~45 kDa hyaluronidase in all species. Three novel components were partially characterized: BamazPLA₂ (Group III PLA₂), Tmetu1 (37-residue α-KTx), and TsilvMP_A (a metalloprotease homologous to antarease). This is the first application of MELD-based proteomics to Amazonian scorpion venoms, revealing molecular diversity and functional divergence within Tityus and Brotheas, emphasizing the need for region-specific antivenoms. These findings provide a foundation for future pharmacological studies and the discovery of bioactive peptides with therapeutic potential. Full article

Lymphoma continues to pose a serious challenge to global health, underscoring the urgent need for new therapeutic strategies. Recently, the gut microbiome has been shown to play a potential role in regulating immune responses and influencing cancer progression. However, its molecular mechanisms of action in lymphoma remain poorly understood. This study investigates the antiproliferative and apoptotic activities of gut microbiota-derived metabolites, specifically nisin (N) and urolithin B (UB), individually and in combination 7:3 (5750 μM), against the human lymphoma cell line HKB-11. Comprehensive evaluations were performed using Alamar Blue viability assays, combination index (CI) analyses, reactive oxygen species (ROS) quantification, flow cytometry for apoptosis detection, and advanced bottom-up proteomics analyses. N and UB exhibited potent antiproliferative activity, with the 7:3 combination demonstrating strong synergistic effects (CI < 1), significantly enhancing apoptosis (p < 0.01) and ROS production (p < 0.0001) compared to the untreated control. Proteomics analyses revealed substantial alterations in proteins crucial to ribosomal biogenesis, mitochondrial function, cell cycle control, and apoptosis regulation, including a marked downregulation of ribosomal proteins (RPS27; Log₂FC = −3.47) and UBE2N (Log₂FC = −0.60). These findings highlight the potential of N and UB combinations as a novel and practical therapeutic approach for lymphoma treatment, warranting further in vivo exploration and clinical validation. Full article