Label-free Shotgun Proteomics Research Articles

Integrated Chemometrics and Data-Independent Acquisition Proteomics for the Discovery of Meat Authenticity Biomarkers: A Study on Early Post-Mortem Pectoralis major Muscle Proteomes of Ross 308 and Ranger Classic Chicken Produced by Organic versus Antibiotic-Free Farming Systems.

Many factors, such as the farming systems and preslaughter rearing practices, can influence the physiological and metabolic functions of poultry with consequent effects on poultry meat quality. In this trial, label-free shotgun proteomics was used to analyze the early post-mortem Pectoralis major muscle proteomes of Ross 308 and Ranger Classic chicken strains raised under two divergent farming systems these being organic and antibiotic-free. The combination of chemometrics using partial-least-square discriminant analysis (PLS-DA) and shotgun proteomics allowed clear discrimination between the different groups. Chicken strains were discriminated by differences in the abundance of 73 and 62 proteins within the antibiotic-free and organic farming systems, respectively. The abundances of 71 and 52 proteins were impacted by the farming system within the Ross 308 and Ranger Classic chicken strains, respectively. The analyses allowed for the proposal of several putative biomarkers of meat authenticity, which were found to be related to muscle structure and energy metabolism pathways. This study is a significant step forward in elucidating the potential of proteomics profiling and chemometrics in chicken meat, which may provide opportunities for the efficient assessment of chicken authenticity.

Proteomic Blueprint of Atlantic Cod (Gadus morhua) Otoliths Revealing Environmental Stress Insights through Label-Free Quantitative Shotgun Proteomics

Otoliths of the fish’s inner ear serve as a natural chronological recorder because of their continuous formation marked by daily, monthly, and annual increments. Despite their importance, the comprehensive protein content of otoliths remains not fully identified. Using the label-free shotgun proteomics method with one-dimensional liquid chromatography coupled to electrospray ionization-orbitrap tandem mass spectrometry, we quantified a broad range of proteins, with individual otoliths containing between 1341 and 1839 proteins. The identified proteins could potentially serve as a blueprint for fish growth from embryo to adult. We quantified eleven heat-shock proteins (HSPs) in both sexes and several proteins impacted by endocrine disruptors, indicating the otolith’s capacity to reflect environmental stress, potentially linked to climate change effects and altering of hormonal and neuroendocrine functions. Our bioinformatic ontology analysis confirmed the presence of proteins critical for various biological processes, including structural and enzymatic proteins. Protein–protein interaction (PPI) mapping also identified key interactions between the identified proteins. These findings significantly advance our understanding of otolith proteomics, offering a solid foundation for future work. Most of the identified proteins deposited daily and influenced by the environment were not implicated in the biomineralization of otolith, raising the potential for the otolith proteome to recreate details of fish life history at previously unrealized levels.

Antibacterial Activities of Phenolic Compounds in Miang Extract: Growth Inhibition and Change in Protein Expression of Extensively Drug-Resistant Klebsiella pneumoniae.

The rising incidence of extensively drug-resistant (XDR) Klebsiella pneumoniae, including carbapenem- and colistin-resistant strains, leads to the limitation of available effective antibiotics. Miang, known as chewing tea, is produced from Camellia sinensis var. assamica or Assam tea leaves fermentation. Previous studies revealed that the extract of Miang contains various phenolic and flavonoid compounds with numerous biological activities including antibacterial activity. However, the antibacterial activity of Miang against XDR bacteria especially colistin-resistant strains had not been investigated. In this study, the compositions of phenolic and flavonoid compounds in fresh, steamed, and fermented Assam tea leaves were examined by HPLC, and their antibacterial activities were evaluated by the determination of the MIC and MBC. Pyrogallol was detected only in the extract from Miang and showed the highest activities with an MIC of 0.25 mg/mL and an MBC of 0.25-0.5 mg/mL against methicillin-susceptible Staphylococcus aureus, methicillin-resistant S. aureus, Escherichia coli ATCC 25922, colistin-resistant E. coli, and colistin-resistant K. pneumoniae. The effects on morphology and proteomic changes in K. pneumoniae NH54 treated with Miang extract were characterized by SEM and label-free quantitative shotgun proteomics analysis. The results revealed that Miang extract caused the decrease in bacterial cell wall integrity and cell lysis. The up- and downregulated expression with approximately a 2 to >5-fold change in proteins involved in peptidoglycan synthesis and outer membrane, carbohydrate, and amino acid metabolism were identified. These findings suggested that Miang containing pyrogallol and other secondary metabolites from fermentation has potential as an alternative candidate with an antibacterial agent or natural active pharmaceutical ingredient against XDR bacteria including colistin-resistant bacteria.

APEX2-based proximity proteomic analysis identifies candidate interactors for Plasmodium falciparum knob-associated histidine-rich protein in infected erythrocytes

The interaction of Plasmodium falciparum—infected red blood cells (iRBCs) with the vascular endothelium plays a crucial role in malaria pathology and disease. KAHRP is an exported P. falciparum protein involved in iRBC remodelling, which is essential for the formation of protrusions or “knobs” on the iRBC surface. These knobs and the proteins that are concentrated within them allow the parasites to escape the immune response and host spleen clearance by mediating cytoadherence of the iRBC to the endothelial wall, but this also slows down blood circulation, leading in some cases to severe cerebral and placental complications. In this work, we have applied genetic and biochemical tools to identify proteins that interact with P. falciparum KAHRP using enhanced ascorbate peroxidase 2 (APEX2) proximity-dependent biotinylation and label-free shotgun proteomics. A total of 30 potential KAHRP-interacting candidates were identified, based on the assigned fragmented biotinylated ions. Several identified proteins have been previously reported to be part of the Maurer’s clefts and knobs, where KAHRP resides. This study may contribute to a broader understanding of P. falciparum protein trafficking and knob architecture and shows for the first time the feasibility of using APEX2-proximity labelling in iRBCs.

Proteomic insight towards key modulating proteins regulated by the aryl hydrocarbon receptor involved in ovarian carcinogenesis and chemoresistance

Gynecological malignancies pose a severe threat to female lives. Ovarian cancer (OC), the most lethal gynecological malignancy, is clinically presented with chemoresistance and a higher relapse rate. Several studies have highly correlated the incidence of OC to exposure to environmental pollutants, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a process mainly mediated through activating the aryl hydrocarbon receptor (AhR). We have previously reported that exposure of OC cells to TCDD, an AhR activator, significantly modulated the expression of several genes that play roles in stemness and chemoresistance. However, the effect of AhR activation on the whole OC cell proteome aiming at identifying novel druggable targets for both prevention and treatment intervention purposes remains unrevealed. For this purpose, we conducted a comparative proteomic analysis of OC cells A2780 untreated/treated with TCDD for 24 h using a mass spectrometry-based label-free shotgun proteomics approach. The most significantly dysregulated proteins were validated by Western blot analysis. Our results showed that upon AhR activation by TCDD, out of 2598 proteins identified, 795 proteins were upregulated, and 611 were downregulated. STRING interaction analysis and KEGG-Reactome pathway analysis approaches identified several significantly dysregulated proteins that were categorized to be involved in chemoresistance, cancer progression, invasion and metastasis, apoptosis, survival, and prognosis in OC. Importantly, selected dysregulated genes identified by the proteomic study were validated at the protein expression levels by Western blot analysis. In conclusion, this study provides a better understanding of the the cross-talk between AhR and several other molecular signaling pathways and the role and involvement of AhR in ovarian carcinogenesis and chemoresistance. Moreover, the study suggests that AhR is a potential therapeutic target for OC prevention and maintenance. SignificanceTo our knowledge, this is the first study that investigates the role and involvement of AhR and its regulated genes in OC by performing a comparative proteomic analysis to identify the critical proteins with a modulated expression upon AhR activation. We found AhR activation to play a tumor-promoting and chemoresistance-inducing role in the pathogenesis of OC. The results of our study help to devise novel therapeutics for better management and prevention and open the doors to finding novel biomarkers for the early detection and prognosis of OC.

A proteomic perspective on the resistance response of Klebsiella pneumoniae to antimicrobial peptide PaDBS1R1.

The synthetic antimicrobial peptide, PaDBS1R1, has been reported as a powerful anti-Klebsiella pneumoniae antimicrobial. However, there is only scarce knowledge about whether K. pneumoniae could develop resistance against PaDBS1R1 and which resistance mechanisms could be involved. Identify via label-free shotgun proteomics the K. pneumoniae resistance mechanisms developed against PaDBS1R1. An adaptive laboratory evolution experiment was performed to obtain a PaDBS1R1-resistant K. pneumoniae lineage. Antimicrobial susceptibility was determined through microdilution assay. Modifications in protein abundances between the resistant and sensitive lineages were measured via label-free quantitative shotgun proteomics. Enriched Gene Ontology terms and KEGG pathways were identified through over-representation analysis. Data are available via ProteomeXchange with identifier PXD033020. K. pneumoniae ATCC 13883 parental strain challenged with increased subinhibitory PaDBS1R1 concentrations allowed the PaDBS1R1-resistant K. pneumoniae lineage to emerge. Proteome comparisons between PaDBS1R1-resistant K. pneumoniae and PaDBS1R1-sensitive K. pneumoniae under PaDBS1R1-induced stress conditions enabled the identification and quantification of 1702 proteins, out of which 201 were differentially abundant proteins (DAPs). The profiled DAPs comprised 103 up-regulated proteins (adjusted P value < 0.05, fold change ≥ 2) and 98 down-regulated proteins (adjusted P value < 0.05, fold change ≤ 0.5). The enrichment analysis suggests that PhoPQ-guided LPS modifications and CpxRA-dependent folding machinery could be relevant resistance mechanisms against PaDBS1R1. Based on experimental evolution and a label-free quantitative shotgun proteomic approach, we showed that K. pneumoniae developed resistance against PaDBS1R1, whereas PhoPQ-guided LPS modifications and CpxRA-dependent folding machinery appear to be relevant resistance mechanisms against PaDBS1R1.

Constitutive proteins of lumpy skin disease virion assessed by next-generation proteomics.

Lumpy skin disease virus (LSDV) is the causative agent of an economically important cattle disease which is notifiable to the World Organisation for Animal Health. Over the past decades, the disease has spread at an alarming rate throughout the African continent, the Middle East, Eastern Europe, the Russian Federation, and many Asian countries. While multiple LDSV whole genomes have made further genetic comparative analyses possible, knowledge on the protein composition of the LSDV particle remains lacking. This study provides for the first time a comprehensive proteomic analysis of an infectious LSDV particle, prompting new efforts toward further proteomic LSDV strain characterization. Furthermore, this first incursion within the capripoxvirus proteome represents one of very few proteomic studies beyond the sole Orthopoxvirus genus, for which most of the proteomics studies have been performed. Providing new information about other chordopoxviruses may contribute to shedding new light on protein composition within the Poxviridae family.

Interactive effects of Pseudomonas entomophila strain 23S and Clavibacter michiganensis subsp. michiganensis on proteome and anti-Cmm compound production

Pseudomonas entomophila strain 23S is an effective biocontrol bacterium for tomato bacterial canker caused by Clavibacter michiganensis subsp. michiganensis (Cmm); it produces an inhibitory compound affecting the growth of Cmm. In this study, the interactions between pure cultures of P. entomophila 23S and Cmm were investigated. First, the population dynamics of each bacterium during the interaction was determined using the selective media. Second, the amount of anti-Cmm compound produced by P. entomophila 23S in the presence of Cmm was quantified using HPLC. Lastly, a label-free shotgun proteomics study of P. entomophila 23S, Cmm, and a co-culture was conducted to understand the effects of the interaction of each bacterium at the proteomic level. Compared with the pure culture grown, the total number of proteins decreased in the interaction for both bacteria. P. entomophila 23S secreted stress-related proteins, such as chaperonins, peptidases, ABC-transporters and elongation factors. The bacterium also produced more proteins related with purine, pyrimidine, carbon and nitrogen metabolisms in the presence of Cmm. The population enumeration study revealed that the Cmm population declined dramatically during the interaction, while the population of P. entomophila 23S maintained. The quantification of anti-Cmm compound indicated that P. entomophila 23S produced significantly higher amount of anti-Cmm compound when it was cultured with Cmm. Overall, the study suggested that P. entomophila 23S, although is cidal to Cmm, was also negatively affected by the presence of Cmm, while trying to adapt to the stress condition, and that such an environment favored increased production of the anti-Cmm compound by P. entomophila 23S. SignificancePseudomonas entomophila strain 23S is an effective biocontrol bacterium for tomato bacterial canker caused by Clavibacter michiganensis subsp. michiganensis (Cmm); it produces an inhibitory compound affecting the growth of Cmm. In this study, secreted proteome of pure cultures of P. entomophila 23S and Cmm, and also of a co-culture was first time identified. Furthermore, the study found that P. entomophila strain 23S produced significantly higher amount of anti-Cmm compound when the bacterium was grown together with Cmm. Co-culture enhancing anti-Cmm compound production by P. entomophila 23S is useful information, particularly from a commercial point of view of biocontrol application, and for scale-up of anti-Cmm compound production.

Molecular mechanisms contributing to the development of beef sensory texture and flavour traits and related biomarkers: Insights from early post-mortem muscle using label-free proteomics

Beef sensory quality comprises a suite of traits, each of which manifests its ultimate phenotype through interaction of muscle physiology with environment, both in vivo and post-mortem. Understanding variability in meat quality remains a persistent challenge, but omics studies to uncover biological connections between natural variability in proteome and phenotype could provide validation for exploratory studies and offer new insights. Multivariate analysis of proteome and meat quality data from Longissimus thoracis et lumborum muscle samples taken early post-mortem from 34 Limousin-sired bulls was conducted. Using for the first-time label-free shotgun proteomics combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS), 85 proteins were found to be related with tenderness, chewiness, stringiness and flavour sensory traits. The putative biomarkers were classified in five interconnected biological pathways; i) muscle contraction, ii) energy metabolism, iii) heat shock proteins, iv) oxidative stress, v) regulation of cellular processes and binding. Among the proteins, PHKA1 and STBD1 correlated with all four traits, as did the GO biological process ‘generation of precursor metabolites and energy’. Optimal regression models explained a high level (58–71%) of phenotypic variability with proteomic data for each quality trait. The results of this study propose several regression equations and biomarkers to explain the variability of multiple beef eating quality traits. Thanks to annotation and network analyses, they further suggest protein interactions and mechanisms underpinning the physiological processes regulating these key quality traits. SignificanceThe proteomic profiles of animals with divergent quality profiles have been compared in numerous studies; however, a wide range of phenotypic variation is required to better understand the mechanisms underpinning the complex biological pathways correlated with beef quality and protein interactions. We used multivariate regression analyses and bioinformatics to analyse shotgun proteomics data to decipher the molecular signatures involved in beef texture and flavour variations with a focus on multiple quality traits. We developed multiple regression equations to explain beef texture and flavour. Additionally, potential candidate biomarkers correlated with multiple beef quality traits are suggested, which could have utility as indicators of beef overall sensory quality. This study explained the biological process responsible for determining key quality traits such as tenderness, chewiness, stringiness, and flavour in beef, which will provide support for future beef proteomics studies.

Label-free shotgun proteomics: Exploiting a reliable and sensitive method to monitor residual host-cell proteins in monoclonal antibody products

The removal of host cell proteins (HCPs) remains a challenge in the downstream processing of monoclonal antibodies (mAbs). It is critical to monitor residual HCPs since they can have an impact on product stability and safety. The enzyme-linked immunosorbent assay (ELISA) only quantifies the total amount of HCPs and does not indicate the identity or quantity of any specific HCP. Mass spectrometry-based proteomics applications demonstrate an advantage in quantitatively profiling individual HCPs. However, technical reproducibility, dynamic range, and ensuring an acceptable statistical significance of scoring measurements are critical hurdles that ought to be overcome. In this paper, we describe a sensitive and reproducible shotgun proteomics approach that includes an efficient mAb depletion strategy and addresses the shortcomings of such workflows. Our methodology is potentially a valuable complement or alternative to ELISA. Additionally, it can facilitate the purification process development and the evaluation of ELISA kits upon process changes.

A new window into fish welfare: A proteomic discovery study of stress biomarkers in the skin mucus of gilthead seabream (Sparus aurata)

Fish skin mucus is a dynamic external mucosal layer that acts as the first line of defense in the innate immune system. Skin mucus' exudation and composition change severely under stress, making it a valuable biofluid to search for minimally invasive stress markers. This study focused on the skin mucus proteome response to repetitive handling, overcrowding, and hypoxia, using Sparus aurata, an important species in the Mediterranean aquaculture, as a model. Biomarker discovery analysis was performed using label-free shotgun proteomics coupled with bioinformatics to unveil the most predictive proteins for the stressed phenotype. A mean of 2166 proteins were identified at a < 0.2% false discovery rate, from which the differentially abundant proteins (DAPs) were mainly involved in the immune system and protein metabolism. A sparse partial least squares regression analysis revealed a high correlation between DAPs and plasma physiological stress indicators. Feature selection, performed by recursive feature elimination followed by logistic regression analysis of the selected proteins, disclosed 28 candidate biomarkers with values of area under the curve >0.75. These minimally invasive biomarkers could be used in forthcoming species-specific stress management protocols to improve fish welfare and promote farmed fish safety, positive societal outcomes, and business sustainability. SignificanceThe fish skin mucus holds a great promise into fish welfare, as a valuable source of minimally invasive biomarkers for stress assessment. In this shotgun proteomics discovery study, we have identified 28 candidate biomarkers by combining a comprehensive functional analysis of the stress regulated proteome with predictive modeling, supported by a significant correlation (p < 0.01) with physiological stress indicators (cortisol, lactate and glucose). The candidate biomarkers showed a good predictive value in the testing set (AUC > 0.75), paving the way for the next step in their validation by targeted proteomics. An early and timely assessment of fish stressful events, by using minimally invasive biomarkers, as those that can be found in the fish skin mucus, can contribute to promote fish health/welfare in the aquaculture sector and its sustainability. The adoption of preventive and surveillance measures based on proteomics approaches can therefore help to avoid unnecessary adverse outcomes with a negative impact on this primordial food sector.

Towards the discovery of goat meat quality biomarkers using label-free proteomics

This study aimed to identify for the first time protein biomarkers of meat quality traits from Longissimus thoracis (LT) muscle of goats (Capra hircus). Male goats of similar age and weight reared under extensive rearing conditions were used to relate the LT muscle proteome with multiple meat quality traits. The early post-mortem muscle proteome analyzed using label-free proteomics was compared among three texture clusters built using hierarchical clustering analysis. Twenty-five proteins were differentially abundant and their mining using bioinformatics revealed three major biological pathways to be involved: 10 muscle structure proteins (MYL1, MYL4, MYLPF, MYL6B, MYH1, MYH2, ACTA1, ACTBL2, FHL1 and MYOZ1); 6 energy metabolism proteins (ALDOA, PGAM2, ATP5F1A, GAPDH, PGM1 and ATP5IF1), and two heat shock proteins: HSPB1 (small) and HSPA8 (large). Seven other miscellaneous proteins belonging to pathways such as regulation, proteolysis, apoptosis, transport and binding, tRNA processing or calmodulin-binding were further identified to play a role in the variability of goat meat quality. The differentially abundant proteins were correlated with the goat meat quality traits in addition to multivariate regression models built to propose the first regression equations of each quality trait. This study is the first to highlight in a multi-trait quality comparison the early post-mortem changes in the goat LT muscle proteome. It also evidenced the mechanisms underpinning the development of several quality traits of interest in goat meat production along the major biochemical pathways at interplay. SignificanceThe discovery of protein biomarkers in the field of meat research is an emerging topic. In the case of goat meat quality, very few studies using proteomics have been conducted with the aim of proposing biomarkers. Therefore, this study is the first to quest for biomarkers of goat meat quality using label-free shotgun proteomics with a focus on multiple quality traits. We identified the molecular signatures underlying goat meat texture variation, which were found to belong to muscle structure and related proteins, energy metabolism and heat shock proteins along with other proteins involved in regulation, proteolysis, apoptosis, transport and binding, tRNA processing or calmodulin-binding. We further evaluated the potential of the candidate biomarkers to explain meat quality using the differentially abundant proteins by means of correlation and regression analyses. The results allowed the explanation of the variation in multiple traits such as pH, color, water-holding capacity, drip and cook losses traits and texture.

A Practical and Analytical Comparative Study of Gel-Based Top-Down and Gel-Free Bottom-Up Proteomics Including Unbiased Proteoform Detection.

Proteomics is an indispensable analytical technique to study the dynamic functioning of biological systems via different proteins and their proteoforms. In recent years, bottom-up shotgun has become more popular than gel-based top-down proteomics. The current study examined the qualitative and quantitative performance of these two fundamentally different methodologies by the parallel measurement of six technical and three biological replicates of the human prostate carcinoma cell line DU145 using its two most common standard techniques, label-free shotgun and two-dimensional differential gel electrophoresis (2D-DIGE). The analytical strengths and limitations were explored, finally focusing on the unbiased detection of proteoforms, exemplified by discovering a prostate cancer-related cleavage product of pyruvate kinase M2. Label-free shotgun proteomics quickly yields an annotated proteome but with reduced robustness, as determined by three times higher technical variation compared to 2D-DIGE. At a glance, only 2D-DIGE top-down analysis provided valuable, direct stoichiometric qualitative and quantitative information from proteins to their proteoforms, even with unexpected post-translational modifications, such as proteolytic cleavage and phosphorylation. However, the 2D-DIGE technology required almost 20 times as much time per protein/proteoform characterization with more manual work. Ultimately, this work should expose both techniques' orthogonality with their different contents of data output to elucidate biological questions.

A Proteomics Data Mining Strategy for the Identification of Quinoa Grain Proteins with Potential Immunonutritional Bioactivities.

Quinoa proteins are attracting global interest for their wide amino acid profile and as a promising source for the development of biomedical treatments, including those against immune-mediated diseases. However, information about the bioactivity of quinoa proteins is scarce. In this study, a quinoa grain proteome map obtained by label-free mass spectrometry-based shotgun proteomics was investigated for the identification of quinoa grain proteins with potential immunonutritional bioactivities, including those related to cancer. After carefully examining the sequence similarities of the 1211 identified quinoa grain proteins against already described bioactive proteins from other plant organisms, 71, 48, and 3 of them were classified as antimicrobial peptides (AMPs), oxidative stress induced peptides (OSIPs), and serine-type protease inhibitors (STPIs), respectively, suggesting their potential as immunomodulatory, anti-inflammatory, and anticancer agents. In addition, data interpretation using Venn diagrams, heat maps, and scatterplots revealed proteome similarities and differences with respect to the AMPs, OSIPs, and STPIs, and the most relevant bioactive proteins in the predominant commercial quinoa grains (i.e., black, red, white (from Peru), and royal (white from Bolivia)). The presented proteomics data mining strategy allows easy screening for potentially relevant quinoa grain proteins and commercial classes for immunonutrition, as a basis for future bioactivity testing.

Proteomic analysis of brain metastatic lung adenocarcinoma reveals intertumoral heterogeneity and specific alterations associated with the timing of brain metastases

Brain metastases are associated with considerable negative effects on patients' outcome in lung adenocarcinoma (LADC). Here, we investigated the proteomic landscape of primary LADCs and their corresponding brain metastases. Proteomic profiling was conducted on 20 surgically resected primary and brain metastatic LADC samples via label-free shotgun proteomics. After sample processing, peptides were analyzed using an Ultimate 3000 pump coupled to a QExactive HF-X mass spectrometer. Raw data were searched using PD 2.4. Further data analyses were carried out using Perseus, RStudio and GraphPad Prism. Proteomic data were correlated with clinical and histopathological parameters and the timing of brain metastases. Mass spectrometry-based proteomic data are available via ProteomeXchange with identifier PXD027259. Out of the 6821 proteins identified and quantified, 1496 proteins were differentially expressed between primary LADCs and corresponding brain metastases. Pathways associated with the immune system, cell-cell/matrix interactions and migration were predominantly activated in the primary tumors, whereas pathways related to metabolism, translation or vesicle formation were overrepresented in the metastatic tumors. When comparing fast- versus slow-progressing patients, we found 454 and 298 differentially expressed proteins in the primary tumors and brain metastases, respectively. Metabolic reprogramming and ribosomal activity were prominently up-regulated in the fast-progressing patients (versus slow-progressing individuals), whereas expression of cell-cell interaction- and immune system-related pathways was reduced in these patients and in those with multiple brain metastases. This is the first comprehensive proteomic analysis of paired primary tumors and brain metastases of LADC patients. Our data suggest a malfunction of cellular attachment and an increase in ribosomal activity in LADC tissue, promoting brain metastasis. The current study provides insights into the biology of LADC brain metastases and, moreover, might contribute to the development of personalized follow-up strategies in LADC.

Gilthead Seabream Liver Integrative Proteomics and Metabolomics Analysis Reveals Regulation by Different Prosurvival Pathways in the Metabolic Adaptation to Stress

The study of the molecular mechanisms of stress appraisal on farmed fish is paramount to ensuring a sustainable aquaculture. Stress exposure can either culminate in the organism's adaptation or aggravate into a metabolic shutdown, characterized by irreversible cellular damage and deleterious effects on fish performance, welfare, and survival. Multiomics can improve our understanding of the complex stressed phenotype in fish and the molecular mediators that regulate the underlying processes of the molecular stress response. We profiled the stress proteome and metabolome of Sparus aurata responding to different challenges common to aquaculture production, characterizing the disturbed pathways in the fish liver, i.e., the central organ in mounting the stress response. Label-free shotgun proteomics and untargeted metabolomics analyses identified 1738 proteins and 120 metabolites, separately. Mass spectrometry data have been made fully accessible via ProteomeXchange, with the identifier PXD036392, and via MetaboLights, with the identifier MTBLS5940. Integrative multivariate statistical analysis, performed with data integration analysis for biomarker discovery using latent components (DIABLO), depicted the 10 most-relevant features. Functional analysis of these selected features revealed an intricate network of regulatory components, modulating different signaling pathways related to cellular stress, e.g., the mTORC1 pathway, the unfolded protein response, endocytosis, and autophagy to different extents according to the stress nature. These results shed light on the dynamics and extent of this species' metabolic reprogramming under chronic stress, supporting future studies on stress markers' discovery and fish welfare research.

A Pilot Study on the Urine Proteome of Cats Fed a High-Protein Complete Diet, Supplemented with or without Arginine, Ornithine or Zeolite.

Proteome analyses can be used to detect biomarkers for the healthy and diseased organism. However, data in cats are scarce, and no information is available on the potential impact of nutritional interventions on the feline urine proteome. In the present study, a label-free shotgun proteomics approach was performed to investigate the urinary proteins of four healthy adult cats. Each animal received a high-protein complete diet without (w/o) or with supplements that could affect the protein metabolism: arginine (+100% compared to the arginine concentration in the w/o diet), ornithine (+200% compared to the arginine concentration in the w/o diet) or zeolite (0.375 g/kg body weight/day). Our results demonstrate a huge number of proteins in the urine of cats (516 ± 49, 512 ± 39, 399 ± 149 and 455 ± 134 in the w/o, arginine, ornithine and zeolite group, respectively), which are associated with several biological processes. In addition, up- and downregulated urinary proteins could be detected in the dietary supplementation periods. Overall, the present pilot study provides basic data on the urine proteome of healthy adult cats. With increasing information, the numerousness of urinary proteins implies the potential to identify biomarkers and metabolic pathways in the feline organism.

Proteomic Changes in Paspalum fasciculatum Leaves Exposed to Cd Stress.

(1) Background: Cadmium is a toxic heavy metal that is widely distributed in water, soil, and air. It is present in agrochemicals, wastewater, battery waste, and volcanic eruptions. Thus, it can be absorbed by plants and enter the trophic chain. P. fasciculatum is a plant with phytoremediation capacity that can tolerate Cd stress, but changes in its proteome related to this tolerance have not yet been identified. (2) Methods: We conducted a quantitative analysis of the proteins present in P. fasciculatum leaves cultivated under greenhouse conditions in mining soils doped with 0 mg kg−1 (control), 30 mg kg−1, or 50 mg kg−1. This was carried out using the label-free shotgun proteomics technique. In this way, we determined the changes in the proteomes of the leaves of these plants, which allowed us to propose some tolerance mechanisms involved in the response to Cd stress. (3) Results: In total, 329 variable proteins were identified between treatments, which were classified into those associated with carbohydrate and energy metabolism; photosynthesis; structure, transport, and metabolism of proteins; antioxidant stress and defense; RNA and DNA processing; and signal transduction. (4) Conclusions: Based on changes in the differences in the leaf protein profiles between treatments, we hypothesize that some proteins associated with signal transduction (Ras-related protein RABA1e), HSPs (heat shock cognate 70 kDa protein 2), growth (actin-7), and cellular development (actin-1) are part of the tolerance response to Cd stress.

In-depth proteomic analysis reveals unique subtype-specific signatures in human small-cell lung cancer.

BackgroundSmall‐cell lung cancer (SCLC) molecular subtypes have been primarily characterized based on the expression pattern of the following key transcription regulators: ASCL1 (SCLC‐A), NEUROD1 (SCLC‐N), POU2F3 (SCLC‐P) and YAP1 (SCLC‐Y). Here, we investigated the proteomic landscape of these molecular subsets with the aim to identify novel subtype‐specific proteins of diagnostic and therapeutic relevance.MethodsPellets and cell media of 26 human SCLC cell lines were subjected to label‐free shotgun proteomics for large‐scale protein identification and quantitation, followed by in‐depth bioinformatic analyses. Proteomic data were correlated with the cell lines’ phenotypic characteristics and with public transcriptomic data of SCLC cell lines and tissues.ResultsOur quantitative proteomic data highlighted that four molecular subtypes are clearly distinguishable at the protein level. The cell lines exhibited diverse neuroendocrine and epithelial–mesenchymal characteristics that varied by subtype. A total of 367 proteins were identified in the cell pellet and 34 in the culture media that showed significant up‐ or downregulation in one subtype, including known druggable proteins and potential blood‐based markers. Pathway enrichment analysis and parallel investigation of transcriptomics from SCLC cell lines outlined unique signatures for each subtype, such as upregulated oxidative phosphorylation in SCLC‐A, DNA replication in SCLC‐N, neurotrophin signalling in SCLC‐P and epithelial–mesenchymal transition in SCLC‐Y. Importantly, we identified the YAP1‐driven subtype as the most distinct SCLC subgroup. Using sparse partial least squares discriminant analysis, we identified proteins that clearly distinguish four SCLC subtypes based on their expression pattern, including potential diagnostic markers for SCLC‐Y (e.g. GPX8, PKD2 and UFO).ConclusionsWe report for the first time, the protein expression differences among SCLC subtypes. By shedding light on potential subtype‐specific therapeutic vulnerabilities and diagnostic biomarkers, our results may contribute to a better understanding of SCLC biology and the development of novel therapies.

The photosynthesis apparatus of European mistletoe (Viscum album).

European mistletoe (Viscum album) is known for its special mode of cellular respiration. It lacks the mitochondrial NADH dehydrogenase complex (Complex I of the respiratory chain) and has restricted capacities to generate mitochondrial adenosine triphosphate (ATP). Here, we present an investigation of the V. album energy metabolism taking place in chloroplasts. Thylakoids were purified from young V. album leaves, and membrane-bound protein complexes were characterized by Blue native polyacrylamide gel electrophoresis as well as by the complexome profiling approach. Proteins were systematically identified by label-free quantitative shotgun proteomics. We identified >1,800 distinct proteins (accessible at https://complexomemap.de/va_leaves), including nearly 100 proteins forming part of the protein complexes involved in the light-dependent part of photosynthesis. The photosynthesis apparatus of V. album has distinct features: (1) comparatively low amounts of Photosystem I; (2) absence of the NDH complex (the chloroplast pendant of mitochondrial Complex I involved in cyclic electron transport (CET) around Photosystem I); (3) reduced levels of the proton gradient regulation 5 (PGR5) and proton gradient regulation 5-like 1 (PGRL1) proteins, which offer an alternative route for CET around Photosystem I; (4) comparable amounts of Photosystem II and the chloroplast ATP synthase complex to other seed plants. Our data suggest a restricted capacity for chloroplast ATP biosynthesis by the photophosphorylation process. This is in addition to the limited ATP supply by the mitochondria. We propose a view on mistletoe's mode of life, according to which its metabolism relies to a greater extent on energy-rich compounds provided by the host trees.