Protein Profiles Research Articles

Effects of Fungus (Aspergillus niger) Treated Castor seed Cake on the Performance and Egg Quality of Japanese Quail (Coturnix coturnix japonica)

Castor seed cake (CSC) has been recognized as a potential protein source for livestock due to its high Protein and amino acids profile. However, the cake contains some anti-nutrients and toxic compounds that may prevent its full utilization in Livestock feed. The present study was conducted to investigate the effect of partial substitution of Groundnut cake with Fungus (Aspergillus niger) treated Castor seed cake (FCSC) on performance characteristics, egg quality and biochemical blood parameters of Japanese quail. Seventy-two quail chicks used for this experiment were subjected to three dietary treatments: Control diet (A, 0% FCSC), B (30% FCSC) and C (60% FCSC).

Using in vivo intact structure for system-wide quantitative analysis of changes in proteins

Mass spectrometry-based methods can provide a global expression profile and structural readout of proteins in complex systems. Preserving the in vivo conformation of proteins in their innate state is challenging during proteomic experiments. Here, we introduce a whole animal in vivo protein footprinting method using perfusion of reagents to add dimethyl labels to exposed lysine residues on intact proteins which provides information about protein conformation. When this approach is used to measure dynamic structural changes during Alzheimer’s disease (AD) progression in a mouse model, we detect 433 proteins that undergo structural changes attributed to AD, independent of aging, across 7 tissues. We identify structural changes of co-expressed proteins and link the communities of these proteins to their biological functions. Our findings show that structural alterations of proteins precede changes in expression, thereby demonstrating the value of in vivo protein conformation measurement. Our method represents a strategy for untangling mechanisms of proteostasis dysfunction caused by protein misfolding. In vivo whole-animal footprinting should have broad applicability for discovering conformational changes in systemic diseases and for the design of therapeutic interventions.

Serodiagnosis of urogenital schistosomiasis and profiling of immunoreactive protein(s) in Schistosoma haematobium soluble egg and adult worm antigens.

To achieve schistosomiasis eradication plan by 2030, the development of efficient diagnosis is crucial. This study focuses on assessing the immunodiagnostic potential of S. haematobium (Sh) soluble egg antigen (SEA) and worm antigen (SWA) for urogenital schistosomiasis. Urine microscopy identified 50 S. haematobium-positive and 50 negative samples from a total of 500 examined. An additional 50 samples from a non-endemic area were included, bringing the total number of samples used for the assay to 150. Indirect ELISA immunoassays using SEA and SWA as the probing antigens evaluated 50 sera samples each from Sh positive, negative endemic (NE), and non-endemic (NNE) individuals. SDS-PAGE analysis of crude protein extracts was conducted, followed by Western blot analysis using primary antibodies from pooled Sh-infected sera samples. Diagnostic performance was evaluated using the area under the receiver operating characteristic (ROC) curve (AUC), sensitivity, and specificity. The AUC values for Sh SEA and SWA were 0.75 and 0.76 in NE samples, and 0.91 and 0.89 in NNE samples, respectively. Sensitivities 90 (95% CI: 78.64 - 95.65)/ 64.71 (95% CI: 52.17 - 75.92), and specificities 50 (95% CI: 36.64 - 63.36)/ 81.25 (95% CI: 63.56 - 92.79) were recorded for SEA and SWA, respectively in NE samples. In addition, sensitivities 90 (78.64 - 95.65)/ 92 (95% CI: 80.77 - 97.78), and specificities 72 (95% CI: 58.33 - 82.53)/ 72.00 (95% CI: 57.51 - 83.77) were recorded for SEA and SWA, respectively in NNE samples. The mean antibody titer against Sh SEA in infected samples was significantly higher than in non-infected samples (P <0.0001). Eight (8) immunoreactive protein bands; 4 each of SEA and SWA were identified, indicating potential for diagnostic tool development. Sh SEA and SWA demonstrate promise for diagnosing urogenital schistosomiasis in both endemic and non-endemic regions.

Proteomic profiling of extracellular vesicles in takotsubo syndrome

Abstract Background/Purpose Takotsubo syndrome (TS) is an acute form of heart failure characterized by transient regional wall motion abnormalities triggered by a stressful event. The mechanisms underlying its development and recovery are poorly understood, resulting in a lack of disease-specific treatments and diagnostic markers. Extracellular vesicles (EVs) play a significant role in cellular communication and have been shown to be important in various diseases. Their involvement in cardiac conditions like TS is an emerging area of research. The primary objective of this study is to isolate and characterize EVs, as well as profile their proteomic profile, from the heart tissue of rats induced with TS. Methods Rats underwent TS induction through the infusion of 1 mg/kg isoprenaline over 15 minutes (TS24h), or were given saline (control). High-resolution echocardiography verified apical ballooning at 6 hours. After 24 hours, heart tissues from apical and basal segments were collected and processed. EV isolation involved tissue slicing, enzymatic digestion, and differential centrifugation steps, including a final iodixanol cushion separation for large and small EVs (Figure 1). Tandem mass tags and mass spectrometry were utilized for global proteomics and any differentially expressed proteins (DEPs) were identified. Analytical techniques such as volcano plots, heatmaps, enrichment analysis, and protein clustering/networks were employed. The strongest clusters (lowest False Discovery Rate and highest intensity) were selected, and their Gene Ontology (GO) terms/pathways were identified. Results Pure, cup-shaped, vesicles ranging from 50-800nm were successfully isolated (figure 1). EVs from apex of control and TS24h hearts had lower protein concentrations compared to their corresponding basal segments. Global proteomic analysis revealed a distinct protein profile in EVs from the apical segment of TS hearts at 24 hours. A total of 256 (TS24h-apex vs control-apex) and 561 proteins (TS24h-apex vs TS24h-base) were differentially expressed. No DEPs were observed when comparing the basal segments of TS24h and control hearts. Functional enrichment analysis of the DEPs showed an abundant change of biological processes related to metabolic lipid processes, inflammatory response, complement activation, reorganization of extracellular matrix. A downregulation was seen for biological processes related to mitochondrial ATP synthesis coupled electron transport and muscle contraction. Conclusion This study demonstrates a distinct EV protein profile in the apical segments of TS hearts, with marked changes in proteins related to metabolic lipid processes, inflammation, and mitochondrial activity. This extensive catalogue of novel proteins sets the stage for future research aimed at identifying potential therapeutic targets and diagnostic biomarkers, and enabling proof-of-concept studies in TS.

Impact of SGLT2 inhibitor dapagliflozin on myocardial protein profile in rats with long-standing Type 1 diabetes mellitus

Abstract Background Sodium-glucose cotransporter 2 (SGLT2) inhibitors have beneficial effects on the cardiovascular system in diabetes mellitus (DM) patients. However, as most trials have focused on Type 2 DM, the impact of SGLT2 inhibitors on Type 1 DM remains unclear. Objective To investigate the effects of long-term treatment with SGLT2 inhibitor dapagliflozin on the left ventricular (LV) proteome in Type 1 DM rats. Methods Male Wistar rats were divided into three groups: Control (C, n=7); Diabetes (DM, n=6); and Diabetes treated with Dapagliflozin (DM+DAPA, n=8) for 30 weeks. Diabetes was induced by streptozotocin (40 mg/kg). Dapagliflozin was added to chow at 5 mg/kg/day. Label-free mass spectrometry was used to assess LV proteome using a nanoAcquity UPLC-Xevo QTof MS system and ProteinLynx Global Server (PLGS) software. Cytoscape software, Cluster Marker, and Cluego plugins were used for bioinformatic analysis. Statistical analysis: ANOVA and Tukey or Kruskal-Wallis and Dunn. Results Dapagliflozin increased body weight (C 574±43; DM 339±31*; DM+DAPA 413±30*# g; p&amp;lt;0.05: * vs C; # vs DM) and reduced glycemia [C 108 (101–111); DM 554 (529–562)*; DM+DAPA 343 (237–416)*# mg/dL; p&amp;lt;0.05: * vs C; # vs DM]. One rat died in C and two in DM+DAPA. A total of 252 differentially expressed proteins were identified. Most proteins identified in the networks were downregulated in DM vs C and upregulated in DM+DAPA vs DM. Six proteins related to energy metabolism (Atp5j, P21571; CKm, P00564; Ak1, P39069; Atp5h, P31399; Mdh1, O88989; and Idh2, P56574), four involved in myocyte excitation-contraction (Act1, P68065; Casq2, P51868; SERCA1, Q64578; and SERCA2a, P11507), and two associated with oxidative stress (Sod1, P07632; and Sod2, P07895) were upregulated in DM+DAPA x DM. Comparing DM with C, the KEEG Pathway with the highest percentage of gene associations for upregulated proteins was related to gap junction (25.6%), necroptosis (25.6%), and fatty acid degradation (25.6%), and for downregulated proteins was related to Alzheimer disease (27.3%), cardiac muscle contraction (25%) and glycolysis/gluconeogenesis (13.6%). Comparing DM+DAPA with DM, the KEEG Pathway with the highest percentage of gene associations for upregulated proteins was related to Parkinson disease (18.3%), cardiac muscle contraction (14.2%), citrate acid cycle (9.47%), necroptosis (8.88%), and cGMP-PKG signaling (7.10 %), and for downregulated proteins was related to ketone bodies synthesis and degradation (100%). Conclusion Dapagliflozin is safe and prevents changes in the expression of proteins related to energy metabolism, cardiac muscle contraction, and oxidative stress in Type 1 diabetes mellitus rats. These results offer new insights into the cardioprotective mechanisms of dapagliflozin in Type 1 diabetes mellitus.

Sex-biased proteomic regulation in obesity

Abstract Background Obesity is a major risk factor for heart failure with preserved ejection fraction, particularly in women. Obesity-associated inflammation is thought to contribute to this relationship. However, the role of biological sex in the regulation of circulating inflammatory proteins in obesity is not well understood. Purpose We aimed to explore sex differences in the plasma inflammatory protein profile in obese subjects. Methods Clinical data and samples were collected from 450 women and men with a body mass index (BMI) &amp;gt;27 kg/m² without known cardiovascular disease participating in the FAT associated CardiOvasculaR dysfunction study. Obesity was defined as BMI≥30 kg/m². Olink’s Target 96 inflammation panel, which profiles 92 proteins using proximity extension assay technology, was analysed in biobank samples. A comprehensive correlation analysis was performed to describe relationships between protein biomarkers and clinical variables. Gene enrichment analysis was performed to shed light on biological pathways associated with differentially expressed proteins (DEPs). To determine the functional significance of the DEPs, we used the repositories Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. Pearson correlation coefficients were calculated to assess linear associations between the DEPs and relevant clinical variables. Results Women and men did not differ by age (49±9 vs 48±9 years), BMI (32.3±4.6 vs 31.6±3.7 kg/m²) or in prevalences of diabetes (both 11%) or obesity (65% vs 62%) (all p&amp;gt;0.05). However, hypertension was more common in men than women (74% vs 52%, p&amp;lt;0.001). In men (n=188), obesity was associated with downregulation of monocyte chemotactic protein 3, tumor necrosis factor (ligand) superfamily member 12, interleukin 10 and protein S100-A12 and with upregulation of neurotrophin-3 (all p&amp;lt;0.05), thereby suggesting a mechanism for the observed impaired inflammatory response and insulin resistance. In women (n=262), obesity was associated with the downregulation of interleukin 7, interleukin 12 subunit beta, interleukin 6, and C-X-C motif chemokine 11, suggesting a state of persistent low-grade inflammation. In addition, the upregulated sulfotransferase 1A1 and SIR2-like protein 2 proteins indicate a physiologic adaptation to oxidative stress and the metabolic excess endemic to obesity. Conclusion We found that the abundance of several circulating inflammatory proteins was altered in presence of obesity, and that the plasma proteomic profile differed by sex. Interestingly, there was no overlap in the DEPs between women and men in the presence of obesity. These findings support the hypothesis that the pathophysiological mechanisms driving obesity-associated inflammation and immune dysregulation differ by sex. In turn, this may aid in the pursuit of discovering sex-specific biomarkers to identify obese individuals at particular risk of heart failure.

New insights into the pathophysiology of rheumatic valve disease based on different genetic and proteomics profiles

Abstract Introduction Rheumatic heart disease (RHD) pathogenesis is not fully understood. Purpose The protein profile in rheumatic heart disease may have a different fingerprint in the aortic and mitral valves, which results in different evolution in terms of valve damage. Methods To evaluate the genetic profile in symptomatic RHD, 4 simultaneous mitral and aortic valves were obtained during valve replacement, as well as 4 "normal" valves from cadavers (Control Group). The expression of 90 genes was evaluated using a customized RT-PCR array kit. A second experiment was carried out to evaluate proteomics. Then, 7 mitral and aortic valves, from the same patient, were also collected during valve surgery. The Control Group consisted of 6 mitral and aortic valves obtained from explanted hearts during heart transplantation due to non-valvular pathologies. Additionally, histopathological and sectoral proteomic analysis of amyloid deposits, found in some mitral and aortic valves, from the second experiment, were also reported. Results Mitral rheumatic valves exhibited higher expression of genes linked to calcification and immuno-inflammation compared to aortic rheumatic valves (Figure 1). Proteomic analysis, visualized through PCA graphs, highlighted differences in proteomic patterns. Despite having the same genetic pattern, control valves exhibited different proteomic profiles (Figure 1) Rheumatic mitral and aortic valves, even from the same individual, showed distinct proteomic fingerprint (Figure 1). A total of 49 significant proteins were identified in distinguishing healthy mitral and aortic valves from the same individual (Figure 1). The aortic valve displayed a richer extracellular matrix and higher levels of housekeeping proteins that maintain valve homeostasis compared to the mitral valve (Figure 1). The dominant proteomic pattern in valvular rheumatic disease was identified as alterations in critical mitochondrial metabolic activities related to proteostasis. (Figure 2). Particularly notable was the upregulation of pathways associated with lipid metabolism (Figure 2). Finally, amyloid deposits suggested APOA1 as a potential deposit protein in RHD (Figure 2). Conclusion The study confirms distinct constitutional differences between mitral and aortic valves affecting rheumatic disease progression. It emphasizes the role of altered proteostasis and altered proteins deposit, mitochondrial metabolic changes, and inflammatory responses in the disease's pathophysiology, highlighting the complexity of valvular rheumatic disease involvement.For the first time amyloid deposition has been reported as a likely consequence of significant disruption in valvular proteostasis and the proteins potentially comprising this deposit within a rheumatic valve.Genomic and proteomic overviewproteomic fingerprint of RHD and amyloid

Plasma protein profile associated with a family history of early-onset coronary heart disease

Abstract Introduction Although family history of coronary heart disease (CHD) is an established risk factor for subsequent CHD, its relation to the circulating proteome is unknown. Proteins linked to coronary atherosclerosis in subjects with a family history could uncover new pathophysiological mechanisms of heritable CHD. Purpose Firstly, we aimed to study the protein profile associated with a family history of early-onset CHD. Secondly, we aimed to identify proteins among which family history was an effect modifier for the association between each protein and coronary atherosclerosis. Methods Plasma levels of proteins were assessed with Olink panels CVD-II and -III in the population-based Swedish CArdioPulmonary bioImage Study (SCAPIS) cohort. The coronary segment involvement score (SIS) was assessed from computed tomography angiography. Subjects without known CHD were cross-referenced with national registers to identify records of myocardial infarction or coronary revascularization in any parent before 55 or 65 years of age in males and females, respectively. The associations between a family history of CHD and each protein were studied with linear regression adjusted for age, sex and study site, using a False Discovery Rate (FDR) of 5%. Associations were subsequently adjusted for cardiovascular risk factors. Similarly, associations between each protein and SIS were modelled linearly with an FDR of 5%. For significant proteins, the statistical interactions between each protein and family history for the association between proteins and SIS were studied. Results Of 4,251 subjects, 9.5% had a family history CHD. 43 proteins were significantly associated with family history, and when adjusted for risk factors 29 protein associations remained significant (p&amp;lt;0,05) [Figure 1]. The strongest associations were observed for cathepsin D, paraoxonase 3, interleukin-1 receptor antagonist protein and renin. 79 proteins were associated with SIS at FDR&amp;lt;5%, and family history was a significant effect modifier 18 for proteins [Figure 2]. The strongest modifying effects were observed for transferrin receptor protein 1 (β 0.64, 95% CI 0.25–1.04 for those with family history vs β -0.04, 95% CI -0.14–0.06 in those without, p for interaction=0.001), LDL-receptor (β 0.68, 95% CI 0.36–1.00 vs β 0.18, 95% CI 0.08–0.26) and tissue-type plasminogen activator (β 0.45, 95% CI 0.19–0.70 vs β 0.13, 95% CI 0.05–0.21). Conclusion We found that 43 proteins, with biological features of inflammation and vascular function, were associated with a family history of early-onset CHD. Most were also linked to the coronary atherosclerosis burden. Notably, 18 biomarkers, among them transferrin receptor protein 1, LDL-receptor and tissue-type plasminogen activator, exhibited strong statistical interactions with family history in the association with SIS, indicating that they are of particular importance for the genetic susceptibility and the pathophysiology of heritable CHD.

Collagen VI and endotrophin: potential extracellular matrix targets to attenuate adverse cardiac remodeling post-infarction

Abstract Background The extracellular matrix (ECM) is a key component of cardiac repair after myocardial infarction (post-MI). After tissue damage following myocardial ischaemia, the ECM becomes enriched in proteases that are known to induce partial enzymatic cleavage and fragmentation of native structural and fibrillar macromolecules such as collagens. While some of the resulting protein fragments (matrikines) may have beneficial effects by mediating an active scar formation, other such as the collagen VI (COL6) proteolytic component "endothrophin" has been related to deleterious evolution and fibrosis, particularly in the presence of cardiovascular risk factors. Purpose We aimed to investigate the collagen profile in the structural fraction of ECM (enriched in fibrillar and structural proteins) of post-MI hearts at late evolution stages after MI. Methods The ECM protein profile was investigated in the ischemic (ISCH) and non-ischemic (NISCH) zones of the post-MI myocardium in pigs. MI was induced by closed-chest balloon occlusion of the LAD for 90 minutes. Studies were performed in normo- and hypercholesterolemic (diet induced) animals. Myocardial tissue was obtained 30d after MI. ECM proteins were selectively extracted, identified by LC/MS-MS and validated by Western Blot. Plasma levels of endothrophin were quantified by ELISA. Myocardial tissue from the same areas was characterized by immunohistochemistry. Results The ECM in the ISCH zone showed a differential signature of 39 proteins (14 upregulated / 25 downregulated) 30 days after MI compared to the NISCH area of the same animals. In the ISCH region, significant changes in collagen referring to COL6A3 and COL6A1 (increase) and COL6A2 and COL6A6 (decrease) were detected. COL6A3 protein content was 1.8fold higher (p=0.01) in the ISCH-ECM. A similar increase was found in normo- and hypercholesterolemic animals. A higher trend in COL6A3 levels was found in the ISCH- vs, the NISCH-zone in the myocardial cellular fraction suggesting that the COL6 ECM-related increase in ISCH is produced at a transcript level. Importantly, the COL6A3 found in the ECM did not contain the protein peptide (C5 domain) corresponding to endotrophin matrikine, suggesting its cleavage and release. Using a specific ELISA, hypercholesterolemic animals showed significantly higher endothrophin plasma levels than normolipemic controls (p=0.003) even in the absence of MI. Conclusion Our results evidence an increase in COL6A3 content in the ECM of the ischemic myocardium at 30 days post-MI. COL6A3 is depleted in a peptide of the C5 domain corresponding to endotrophin, found to be enhanced in hypercholesterolemia. These findings support a role for collagen type VI in adverse cardiac remodeling post-MI.

Differential expression of hemolymph proteins in wild bumblebees provides insights into species‐specific impacts of heat stress

AbstractWildlife faces an increasing threat from extreme climatic events, such as heatwaves, which can have a severe impact on various species, including crucial pollinators like bumblebees. Bumblebees are cold‐adapted and heterothermic, possessing the ability to regulate their internal temperature. The impact of heat stress seems species specific in bumblebees. While most species are impacted, some bumblebee species manage to survive, potentially by employing physiological mechanisms, including the modulation of their protein profile (e.g. Heat Shock Proteins). However, there is limited understanding of how their protein profiles are associated with heat exposure. In this study, we examined the global variation in the protein profile of males from two bumblebee species sampled in the wild: the heat‐tolerant Bombus terrestris and the heat‐sensitive Bombus magnus. After subjecting them to heat stupor at 40°C in controlled condition, it was observed that nearly all B. terrestris survived the stress, while over 50% of B. magnus individuals succumbed to the heat exposure. Through off‐gel bottom‐up proteomics and LC–MS/MS analysis of the hemolymph proteome, we identified 164 proteins in both species with a large part of differentially expressed proteins after heat exposure. Additionally, quantitative analysis of fat bodies revealed that the relative mass was stable in B. terrestris, while it was significantly lower in B. magnus exposed to heat stress. Our data suggest that compared with B. magnus, B. terrestris displays a higher adaptability of its hemolymph proteome in response to heat stress. This adaptability could be a key factor contributing to the high physiological resistance of B. terrestris and its ability to adapt to new, stressful environments expected due to climate change. Understanding these mechanisms of protein regulation in bumblebees could provide valuable insights into their resilience and vulnerability facing environmental stresses.

Pulses as Sustainable Protein Sources: Benefits, Drawbacks, and Gaps

Abstract: Pulses have redeemed the interest in terms of a sustainable and healthy diet due to their high protein content. Furthermore, the vitamins, minerals, and phytochemicals they contain also make them more valuable nutritionally. A sustainable diet should improve public health and food security and has a low environmental footprint. Pulses have been considered one of the emerging plant food proteins. Although they have beneficial components such as vitamins, minerals, phytochemicals, and antioxidants, many drawbacks limit their use to consumers and the food industry. One of the drawbacks is their incomplete proteins due to the lack of sulfur-containing amino acids in their protein profile. Furthermore, the protein digestibility of pulses is low due to the antinutritional compounds they contain. The other drawback of pulses is their beany flavor and bitter taste that limits the acceptability of consumers. From the consumer's point of view, the other disadvantage is the laborious and timeconsuming preparation period. : This article provides a concise overview of the current state of knowledge on pulses as sustainable protein sources, underlining the gaps that limit their extensive application in the food industry. Based on the available scientific facts on pulses, finding an efficient method for removing beany flavor and overall sensorial acceptability and antinutritional factors, thus increasing protein digestibility is crucial. The most promising option to increase pulse protein digestibility seems to combine conventional and novel technologies that can modulate digestibility by different mechanisms, such as the removal of antinutritional factors, protein denaturation, and the design of the food matrix.

Proteomic analysis of extracellular vesicles derived from canine mammary tumour cell lines identifies protein signatures specific for disease state

BackgroundCanine mammary tumours (CMT) are among the most common types of tumours in female dogs. Diagnosis currently requires invasive tissue biopsies and histological analysis. Tumour cells shed extracellular vesicles (EVs) containing RNAs and proteins with potential for liquid biopsy diagnostics. We aimed to identify CMT subtype-specific proteome profiles by comparing the proteomes of EVs isolated from epithelial cell lines derived from morphologically normal canine mammary tissue, adenomas, and carcinomas.MethodsWhole-cell protein lysates (WCLs) and EV-lysates were obtained from five canine mammary cell lines: MTH53A (non-neoplastic); ZMTH3 (adenoma); MTH52C (simple carcinoma); 1305, DT1406TB (complex carcinoma); and their proteins identified by LC-MS/MS analyses. Gene Ontology analysis was performed on differentially abundant proteins from each group to identify up- and down-regulated biological processes. To establish CMT subtype-specific proteomic profiles, weighted gene correlation network analysis (WGCNA) was carried out.ResultsWCL and EVs displayed distinct protein abundance signatures while still showing the same increase in adhesion, migration, and motility-related proteins in carcinoma-derived cell lines, and of RNA processing and RNA splicing factors in the adenoma cell line. WGCNA identified CMT stage-specific co-abundant EV proteins, allowing the identification of adenoma and carcinoma EV signatures not seen in WCLs.ConclusionsEVs from CMT cell lines exhibit distinct protein profiles reflecting malignancy state, allowing us to identify potential biomarkers for canine mammary carcinomas, such as biglycan. Our dataset could therefore potentially serve as a basis for the development of a less invasive clinical diagnostic tool for the characterisation of CMT.

A Non-targeted Proteomics Newborn Screening Platform for Inborn Errors of Immunity

PurposeNewborn screening using dried blood spot (DBS) samples for the targeted measurement of metabolites and nucleic acids has made a substantial contribution to public healthcare by facilitating the detection of neonates with genetic disorders. Here, we investigated the applicability of non-targeted quantitative proteomics analysis to newborn screening for inborn errors of immunity (IEIs).MethodsDBS samples from 40 healthy newborns and eight healthy adults were subjected to non-targeted proteomics analysis using liquid chromatography-mass spectrometry after removal of the hydrophilic fraction. Subsequently, DBS samples from 43 IEI patients were analyzed to determine whether patients can be identified by reduced expression of disease-associated proteins.ResultsDBS protein profiling allowed monitoring of levels of proteins encoded by 2912 genes, including 1110 listed in the Online Mendelian Inheritance in Man database, in healthy newborn samples, and was useful in identifying patients with IEIs by detecting reduced levels of disease causative proteins and their interacting proteins, as well as cell-phenotypical alterations.ConclusionOur results indicate that non-targeted quantitative protein profiling of DBS samples can be used to identify patients with IEIs and develop a novel newborn screening platform for genetic disorders.

Predicting protein synergistic effect in Arabidopsis using epigenome profiling

Histone modifications can regulate transcription epigenetically by marking specific genomic loci, which can be mapped using chromatin immunoprecipitation sequencing (ChIP-seq). Here we present QHistone, a predictive database of 1534 ChIP-seqs from 27 histone modifications in Arabidopsis, offering three key functionalities. Firstly, QHistone employs machine learning to predict the epigenomic profile of a query protein, characterized by its most associated histone modifications, and uses these modifications to infer the protein’s role in transcriptional regulation. Secondly, it predicts synergistic regulatory activities between two proteins by comparing their profiles. Lastly, it detects previously unexplored co-regulating protein pairs by screening all known proteins. QHistone accurately identifies histone modifications associated with specific known proteins, and allows users to computationally validate their results using gene expression data from various plant tissues. These functions demonstrate an useful approach to utilizing epigenome data for gene regulation analysis, making QHistone a valuable resource for the scientific community (https://qhistone.paoyang.ipmb.sinica.edu.tw).

CG17192 is a Phospholipase That Regulates Signaling Lipids in the Drosophila Gut upon Infection.

The chemoproteomics technique, activity-based protein profiling (ABPP), has proven to be an invaluable tool in assigning functions to enzymes. The serine hydrolase (SH) enzyme superfamily, in particular, has served as an excellent example in displaying the versatility of various ABPP platforms and has resulted in a comprehensive cataloging of the biochemical activities associated within this superfamily. Besides SHs, in mammals, several other enzyme classes have been thoroughly investigated using ABPP platforms. However, the utility of ABPP platforms in fly models remains underexplored. Realizing this knowledge gap, leveraging complementary ABPP platforms, we reported the full array of SH activities during various developmental stages and adult tissues in the fruit fly (Drosophila melanogaster). Following up on this study, using ABPP, we mapped SH activities in adult fruit flies in an infection model and found that a gut-resident lipase CG17192 showed increased activity during infection. To assign a biological function to this uncharacterized lipase, we performed an untargeted lipidomics analysis and found that phosphatidylinositols were significantly elevated when CG17192 was depleted in the adult fruit fly gut. Next, we overexpressed this lipase in insect cells, and using biochemical assays, we show that CG17192 is a secreted enzyme that has phospholipase C (PLC) type activity, with phosphatidylinositol being a preferred substrate. Finally, we show during infection that heightened CG17192 regulates phosphatidylinositol levels and, by doing so, likely modulates signaling pathways in the adult fruit fly gut that might be involved in the resolution of this pathophysiological condition.

Impact of different food-grade protein extraction methods on the proteomic profile and potential allergenicity of Spirulina (Arthrospira platensis)

Various optimized methods for cell disintegration and protein extraction of algae have been reported, but there is a lack of information regarding the proteomic characterization of the extracted proteins; thus, systematic verification of the protein profiles to find potentially exploitable proteins, while minimizing allergenicity risks, is profitable. The method described in this study was developed to thoroughly investigate the proteome characterization of A. platensis following aqueous protein extraction by high-shear homogenization and pH shift. Liquid chromatography-tandem mass spectrometry was applied to assess the proteome obtained from four protein extraction methods using data-dependent proteome analysis. Overall, 699 proteins were identified; however, each extraction method identified unique proteins. The maximum number of exclusive proteins was observed using a combination of high-shear homogenization and acidic pH. The evaluation of protein abundance revealed a clear distinction between proteins isolated from pH 2 treatment compared to those obtained via neutral or basic pH treatment. The applied extraction methods affected the in silico computed physicochemical properties of the proteins. Using the AllerCatPro database, 12 putative allergenic proteins were detected, the most predominant of which were related to the C-phycocyanin beta subunit (P72508). This study demonstrated that extraction methods affect the detection, physicochemical properties, and potential allergenicity of the obtained proteins. Hence, the extraction methods used to obtain novel proteins must consider these aspects before using them as functional foods.

Microbial Protein and Metabolite Profiles of Klebsiella oxytoca M5A1 in a Bubble Column Bioreactor.

The production of microbial proteins (MPs) has emerged as a critical focus in biotechnology, driven by the need for sustainable and scalable alternatives to traditional protein sources. This study investigates the efficacy of two experimental setups in producing MPs using the nitrogen-fixing bacterium Klebsiella oxytoca M5A1. K. oxytoca M5A1, known for its facultative anaerobic growth and capability to fix atmospheric nitrogen, offers a promising avenue for environmentally friendly protein production. This research compares the performance of a simple bubble column (BC) bioreactor, which promotes efficient mixing and cross-membrane gas transfer, with static fermentation, a traditional method lacking agitation and aeration. The study involved the parallel cultivation of K. oxytoca M5A1 in both systems, with key parameters such as microbial growth, glucose utilization, protein concentration, and metabolite profiles monitored over a 48 h period. The results indicate that the BC bioreactor consistently outperformed static fermentation regarding the growth rate, protein yield, and glucose utilization efficiency. The BC exhibited a significant increase in protein production, reaching 299.90 µg/mL at 48 h, compared to 219.44 µg/mL in static fermentation. The organic acid profile reveals both synthesis and utilization regimes of varying patterns. These findings highlight the advantages of the BC bioreactor for MP production, particularly its ability to maintain aerobic conditions that support higher growth and yield.

Vascular endothelial growth factor/connective tissue growth factor and proteomic analysis of aqueous humor after intravitreal conbercept for proliferative diabetes retinopathy.

To investigate the role of connective tissue growth factor (CTGF) and vascular endothelial growth factor (VEGF) in the protein profile of the aqueous humor in patients with proliferative diabetic retinopathy (PDR) following intravitreal injection of conbercept. This study included 72 PDR patients and 8 cataract patients as controls. PDR patients were divided into 3 groups according to the intervals of 3, 5, and 7d between intravitreal conbercept (IVC, 0.5 mg/0.05 mL) injection and pars plana vitrectomy (PPV) performed. Aqueous humor samples were collected before and after IVC and PPV for VEGF and CTGF levels detected with enzyme-linked immunosorbent assay (ELISA). The differential proteomics of 10 patients who underwent PPV surgery 5d after IVC and 8 normal controls was studied, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed on the data, and the protein interaction network of 23 differential proteins was studied. Post-IVC, VEGF levels decreased and CTGF levels increased significantly in aqueous humor, with the CTGF/VEGF ratio rising significantly at all intervals. Liquid chromatography tandem mass spectrometry (LC-MS/MS) identified differentially expressed proteins between pre- and post-IVC samples. GO and KEGG analyses revealed involvement in immune response, stress response, complement and coagulation cascades, ferroptosis, and PPAR signaling pathways. PPI analysis highlighted key proteins like APOA1, C3, and transferrin (TF). ELISA assay confirmed the differential expression of proteins such as HBA1, SERPINA1, COL1A1, and ACTB, with significant changes in the IVC groups. The study demonstrates that IVC effectively reduces VEGF levels while increasing CTGF levels, thereby modifying the CTGF/VEGF ratio, and IVC significantly alters the protein profile in the aqueous humor of patients with PDR. Proteomic analysis reveals that these changes are associated with critical biological pathways and protein interactions involved in immune response, stress response, and cellular metabolism.

PhoXplex: Combining Phospho-enrichable Cross-Linking with Isobaric Labeling for Quantitative Proteome-Wide Mapping of Protein Interfaces.

Integrating cross-linking mass spectrometry (XL-MS) into structural biology workflows provides valuable information about the spatial arrangement of amino acid stretches, which can guide elucidation of protein assembly architecture. Additionally, the combination of XL-MS with peptide quantitation techniques is a powerful approach to delineate protein interface dynamics across diverse conditions. While XL-MS is increasingly effective with isolated proteins or small complexes, its application to whole-cell samples poses technical challenges related to analysis depth and throughput. The use of enrichable cross-linkers has greatly improved the detectability of protein interfaces in a proteome-wide scale, facilitating global protein-protein interaction mapping. Therefore, bringing together enrichable cross-linking and multiplexed peptide quantification is an appealing approach to enable comparative characterization of structural attributes of proteins and protein interactions. Here, we combined phospho-enrichable cross-linking with TMT labeling to develop a streamline workflow (PhoXplex) for the detection of differential structural features across a panel of cell lines in a global scale. We achieved deep coverage with quantification of over 9000 cross-links and long loop-links in total including potentially novel interactions. Overlaying AlphaFold predictions and disorder protein annotations enables exploration of the quantitative cross-linking data set, to reveal possible associations between mutations and protein structures. Lastly, we discuss current shortcomings and perspectives for deep whole-cell profiling of protein interfaces at large-scale.

Protein fractionation by microfiltration of goat milk: Influence on proteolysis, metabolites and sensory characteristics of cheeses produced throughout lactation

This study addresses seasonal variations in goat milk by examining the effects of microfiltration (MF) and micro- and diafiltration (MDF) in standardising goat milk composition for cheese production. The research focuses on MF and MDF treatments at two lactation stages, analysing their impact on the quality of a hard goat milk cheese. Key parameters such as total solids, pH, organic acids, free amino acids, protein profiles, and sensory properties during cheese ripening were evaluated. Sensory attributes were assessed using Quantitative Descriptive Analysis (QDA), revealing different profiles between treatments. The MF cheeses showed a balanced profile, while MDF cheeses varied more in sourness, acidity, and texture, also influenced by lactation stage.