Proteomic Analysis of the Characteristic Flavor Components in Bacillus subtilis BSNK-5-Fermented Soymilk.

Fermentation with Bacillus subtilis significantly enhances the physiological activity and bioavailability of soymilk, but the resulting characteristic flavor seriously affects its industrial promotion. The objective of this study was to identify key proteins associated with characteristic flavors in B. subtilis BSNK-5-fermented soymilk using tandem mass tag (TMT) proteomics. The results showed that a total of 765 differentially expressed proteins were identified. Seventy differentially expressed proteins related to characteristic flavor were screened through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. After integrating metabolomics data, fifteen key proteases of characteristic flavor components in BSNK-5-fermented soymilk were further identified, and free ammonia was added. In addition, there were five main formation mechanisms, including the decomposition of urea to produce ammonia; the degradation of glutamate by glutamate dehydrogenase to produce ammonia; the degradation of threonine and non-enzymatic changes to form the derivative 2,5-dimethylpyrazine; the degradation of valine, leucine, and isoleucine to synthesize isovalerate and 2-methylbutyrate; and the metabolism of pyruvate and lactate to synthesize acetate. These results provide a theoretical foundation for the improvement of undesirable flavor in B. subtilis BSNK-5-fermented soy foods.

Proteomic Profiling of Cocos nucifera L. Zygotic Embryos during Maturation of Dwarf and Tall Cultivars: The Dynamics of Carbohydrate and Fatty Acid Metabolism.

There is a limited number of studies analyzing the molecular and biochemical processes regulating the metabolism of the maturation of Cocos nucifera L. zygotic embryos. Our research focused on the regulation of carbohydrate and lipid metabolic pathways occurring at three developmental stages of embryos from the Mexican Pacific tall (MPT) and the Yucatan green dwarf (YGD) cultivars. We used the TMT-synchronous precursor selection (SPS)-MS3 strategy to analyze the dynamics of proteomes from both embryos; 1044 and 540 proteins were determined for the MPT and YGD, respectively. A comparison of the differentially accumulated proteins (DAPs) revealed that the biological processes (BP) enriched in the MPT embryo included the glyoxylate and dicarboxylate metabolism along with fatty acid degradation, while in YGD, the nitrogen metabolism and pentose phosphate pathway were the most enriched BPs. Findings suggest that the MPT embryos use fatty acids to sustain a higher glycolytic/gluconeogenic metabolism than the YGD embryos. Moreover, the YGD proteome was enriched with proteins associated with biotic or abiotic stresses, e.g., peroxidase and catalase. The goal of this study was to highlight the differences in the regulation of carbohydrate and lipid metabolic pathways during the maturation of coconut YGD and MPT zygotic embryos.

Acid cleavable biotin-alkyne improves sensitivity for direct detection of biotin labeled peptides in BONCAT analysis.

BONCAT (Biorthogonal noncanonical amino acid tagging) is a labeling strategy that covalently adds a biotin-alkyne (BA) to methionine analogs via a click reaction. When methionine analogs are incorporated into a proteome, enrichment of the BA-labeled proteins allows the detection of newly synthesized proteins (NSP) by mass spectrometry. We previously reported that using our Direct Detection of Biotin-containing Tags (DidBIT) strategy, protein identifications and confidence are increased by enriching for BA-peptides instead of BA-proteins. We compared cleavable BA (DADPS) and uncleavable BA in the identification and TMT quantification of NSP. More than fifty percent more proteins were identified and quantified using DADPS than with uncleavable BA. Interrogation of the data revealed that multiple factors are responsible for the superior performance of DADPS.

Cognitive screening test for rehabilitation using spatiotemporal data extracted from a digital trail making test part-A.

We investigated a newly developed digitized Trail Making Test using an iPad (iTMT) as a brief cognitive function screening test. We found that the iTMT part-A (iTMT-A) can estimate generalized cognitive function in rehabilitation inpatients examined using the Mini-Mental State Examination (MMSE). Forty-two hospitalized participants undergoing rehabilitation (rehab participants), 30 of whom had cerebral infarction/hemorrhage (stroke participants), performed the iTMT five times (first three times: iTMT-A; fourth: paper version of TMT-A; fifth: the inverse version of iTMT-A) and the MMSE once. Each iTMT-A trial's completion time was divided into the move and dwell times. A linear mixed model following post-hoc tests revealed that the completion time of the third and fourth iTMT-A was faster compared to that of the first iTMT-A, suggesting the presence of a learning effect. In the partial least squares (PLS) regression analysis, the coefficient of determination for estimating the MMSE score was increased by using the dwell and move times extracted from the repeated iTMT-A and the availability of TMT-B, even for subjects with low MMSE scores. These findings indicate that the dwell time of iTMT-A may be important for estimating cognitive function. The iTMT-A extracts significant factors temporally and spatially, and by incorporating the learning effect of repeated trials, it may be possible to screen cognitive and physical functions for rehabilitation patients.

A single exposure to the predator odor 2,4,5-trimethylthiazoline causes long-lasting affective behavioral changes in female mice: Modulation by kappa opioid receptor signaling.

The volatile compound 2,4,5-trimethylthiazoline (TMT, a synthetic predator scent) triggers fear, anxiety, and defensive responses in rodents that can outlast the encounter. The receptor systems underlying the development and persistence of TMT-induced behavioral changes remain poorly characterized, especially in females. Kappa opioid receptors regulate threat generalization and fear conditioning and alter basal anxiety, but their role in unconditioned fear responses in females has not been examined. Here, we investigated the effects of the long-lasting kappa opioid receptor antagonist, nor-binalthorphinmine dihydrochloride (nor-BNI; 10 mg/kg), on TMT-induced freezing and conditioned place aversion in female mice. We also measured anxiety-like behavior in the elevated plus maze three days after TMT and freezing behavior when returned to the TMT-paired context ten days after the single exposure. We found that 35µl of 10 % TMT elicited a robust freezing response during a five-minute exposure in female mice. TMT evoked persistent fear as measured by conditioned place aversion, reduced entries into the open arm of the elevated plus maze, and increased general freezing behavior long after TMT exposure. In line with the known role of kappa-opioid receptors in threat generalization, we found that kappa-opioid receptor antagonism increased basal freezing but reduced freezing during TMT presentation. Together, these findings indicate that a single exposure to TMT causes long-lasting changes in fear-related behavioral responses in female mice and highlights the modulatory role of kappa-opioid receptor signaling on fear-related behavioral patterns in females.

[Mechanism of electroacupuncture for vascular dementia based on proteomics].

OBJECTIVE: To explore the potential mechanism of electroacupuncture (EA) for vascular dementia (VD) using tandem mass tag (TMT) quantitative proteomics technology. METHODS: Among 80 male SPF SD rats, 78 rats which met the selection criteria through the Morris water maze test were selected and randomly divided into a sham surgery group (18 rats) and a surgery group (60 rats). VD model was established by four-vessel occlusion (4-VO) method in the surgery group, and 36 rats with successful modeling were randomly assigned to a model group (18 rats) and an EA group (18 rats). Each group was further divided into three subgroups based on intervention duration, with each subgroup containing 6 rats. Seven days after model establishment, the EA group received EA intervention at left and right "Sishencong" (EX-HN 1) and bilateral "Fengchi" (GB 20), with continuous wave at a frequency of 2 Hz and current intensity of 1 mA, daily for 30 min, with subgroups receiving EA for 7, 14, or 21 d respectively. Cognitive function before and after interventions was assessed using Morris water maze. Proteomic analysis was conducted on the optimal EA subgroup and corresponding sham surgery and model subgroups, identifying differentially expressed proteins and analyzing them through bioinformatics. Differentially expressed target proteins was performed using parallel reaction monitoring (PRM) and Western blot techniques. RESULTS: Compared to the sham surgery group, the model group exhibited prolonged escape latency and reduced number of platform crossings (P<0.01); compared with model group, the EA group showed reductions in escape latency and increased platform crossings after 7, 14, and 21 days of intervention (P<0.01, P<0.05). Compared to the 7 and 14-day intervention, the rats in the EA group of 21-day intervention showed the most significant improvements in reductions of escape latency and increased platform crossings (P<0.01, P<0.05), and was selected for further proteomic, PRM analyses, and Western blot validation. Compared to the sham surgery group, the model group displayed 71 differentially expressed proteins, with 50 up-regulated and 21 down-regulated proteins; compared to the model group, the EA group had 54 differentially expressed proteins, with 30 up-regulated and 24 down-regulated proteins. Functional enrichment and clustering analyses indicated that these proteins were primarily associated with cellular processes, metabolic processes, phagocytosis recognition, immune response, and regulation of extracellular matrix, etc. Enrichment was observed in the mammalian target of rapamycin (mTOR) signaling pathway and neurotrophic factors signaling pathways, involving glycogen synthase kinase 3ß (GSK3ß) and mitogen-activated protein kinase kinase 2 (Map2k2), with PRM and Western blot findings consistent with the proteomic results. Which meant that compared with the model group, the protein expression of GSK3ß and Map2k2 of hippocampus was increased in the EA group (P<0.01, P<0.05). CONCLUSION: EA at "Sishencong" (EX-HN 1) and "Fengchi" (GB 20) could improve cognitive function in VD rats, with the mechanism involving multiple targets and pathways, potentially related to GSK3ß, Map2k2 proteins, and the mTOR and neurotrophic factor signaling pathways.

Comparative proteomics in tall fescue to reveal underlying mechanisms for improving Photosystem II thermotolerance during heat stress memory.

BACKGROUND: The escalating impacts of global warming intensify the detrimental effects of heat stress on crop growth and yield. Among the earliest and most vulnerable sites of damage is Photosystem II (PSII). Plants exposed to recurring high temperatures develop heat stress memory, a phenomenon that enables them to retain information from previous stress events to better cope with subsequent one. Understanding the components and regulatory networks associated with heat stress memory is crucial for the development of heat-resistant crops. RESULTS: Physiological assays revealed that heat priming (HP) enabled tall fescue to possess higher Photosystem II photochemical activity when subjected to trigger stress. To investigate the underlying mechanisms of heat stress memory, we performed comparative proteomic analyses on tall fescue leaves at S0 (control), R4 (primed), and S5 (triggering), using an integrated approach of Tandem Mass Tag (TMT) labeling and Liquid Chromatography-Mass Spectrometry. A total of 3,851 proteins were detected, with quantitative information available for 3,835 proteins. Among these, we identified 1,423 differentially abundant proteins (DAPs), including 526 proteins that were classified as Heat Stress Memory Proteins (HSMPs). GO and KEGG enrichment analyses revealed that the HSMPs were primarily associated with the "autophagy" in R4 and with "PSII repair", "HSP binding", and "peptidase activity" in S5. Notably, we identified 7 chloroplast-localized HSMPs (HSP21, DJC77, EGY3, LHCA4, LQY1, PSBR and DEGP8, R4/S0 > 1.2, S5/S0 > 1.2), which were considered to be effectors linked to PSII heat stress memory, predominantly in cluster 4. Protein-protein interaction (PPI) analysis indicated that the ubiquitin-proteasome system, with key nodes at UPL3, RAD23b, and UCH3, might play a role in the selective retention of memory effectors in the R4 stage. Furthermore, we conducted RT-qPCR validation on 12 genes, and the results showed that in comparison to the S5 stage, the R4 stage exhibited reduced consistency between transcript and protein levels, providing additional evidence for post-transcriptional regulation in R4. CONCLUSIONS: These findings provide valuable insights into the establishment of heat stress memory under recurring high-temperature episodes and offer a conceptual framework for breeding thermotolerant crops with improved PSII functionality.

Trends in co-fractionation mass spectrometry: A new gold-standard in global protein interaction network discovery.

Co-fractionation mass spectrometry (CF-MS) uses biochemical fractionation to isolate and characterize macromolecular complexes from cellular lysates without the need for affinity tagging or capture. In recent years, this has emerged as a powerful technique for elucidating global protein-protein interaction networks in a wide variety of biospecimens. This review highlights the latest advancements in CF-MS experimental workflows including machine learning-guided analyses, for uncovering dynamic and high-resolution protein interaction landscapes with enhanced sensitivity, accuracy and throughput, enabling better biophysical characterization of endogenous protein complexes. By addressing challenges and emergent opportunities in the field, this review underscores the transformative potential of CF-MS in advancing our understanding of functional protein interaction networks in health and disease.

Analysis of Protein Cysteine Acylation Using a Modified Suspension Trap (Acyl-Trap).

Proteins undergo reversible S-acylation via a thioester linkage in vivo. S-palmitoylation, modification by C16:0 fatty acid, is a common S-acylation that mediates critical protein-membrane and protein-protein interactions. The most widely used S-acylation assays, including acyl-biotin exchange and acyl resin-assisted capture, utilize blocking of free Cys thiols, hydroxylamine-dependent cleavage of the thioester and subsequent labeling of nascent thiol. These assays generally require >500 µg of protein input material per sample and numerous reagent removal and washing steps, making them laborious and ill-suited for high throughput and low input applications. To overcome these limitations, we devised "Acyl-Trap", a suspension trap-based assay that utilizes a thiol-reactive quartz to enable buffer exchange and hydroxylamine-mediated S-acyl enrichment. We show that the method is compatible with protein-level detection of S-acylated proteins (e.g., H-Ras) as well as S-acyl site identification and quantification using "on trap" isobaric labeling and LC-MS/MS from as little as 20 µg of protein input. In mouse brain, Acyl-Trap identified 279 reported sites of S-acylation and 1298 previously unreported putative sites. Also described are conditions for long-term hydroxylamine storage, which streamline the assay. More generally, Acyl-Trap serves as a proof-of-concept for PTM-tailored suspension traps suitable for both traditional protein detection and chemoproteomic workflows.

Mass Spectrometry Acquisition and Fractionation Recommendations for TMT11 and TMT16 Labeled Samples.

Proteome coverage and accurate protein quantification are both important for evaluating biological systems; however, compromises between quantification, coverage, and mass spectrometry (MS) resources are often necessary. Consequently, experimental parameters that impact coverage and quantification must be adjusted, depending on experimental goals. Among these parameters is offline prefractionation, which is utilized in MS-based proteomics to decrease sample complexity resulting in higher overall proteome coverage upon MS analysis. Prefractionation leads to increases in required MS analysis time, although this is often mitigated by isobaric labeling using tandem-mass tags (TMT), which allow samples to be multiplexed. Here we evaluate common prefractionation schemes, TMT variants, and MS acquisition methods and their impact on protein quantification and coverage. Furthermore, we provide recommendations for experimental design depending on the experimental goals.

Tandem Mass Tag-based proteomic analysis of protein changes in superchilled crayfish (Procambarus clarkii) presoaked with carrageenan oligosaccharides.

To assess the effectiveness of carrageenan oligosaccharides (COs) in enhancing superchilling storage of crayfish, the physicochemical features of muscle and protein abundance in the refrigerated sample (RS), superchilled sample (SS) and COs soaked superchilled sample (CS) were evaluated. Microstructural and SDS-PAGE analyses suggested that CS exhibited fewer pores, with a microstructure and protein subunits distribution more similar to RS. Tandem Mass Tags quantitative proteomic analysis revealed 66 up-regulated differentially abundant proteins (DAPs) in the CS vs. SS batch, including myosin light chain 2, neural cadherin, integrin beta, lectin-like protein, toll-1, reticulon-1, and moesin/ezrin/radixin homolog 1, which facilitate cells adhesion and maintain membrane/cytoskeleton integrity. Eukaryotic Clusters of Orthologous Groups results confirmed that COs treatment increased the stability of crayfish myofibrillar proteins by up-regulating DAPs, which were concentrated in functional categories such as "posttranslation modification, protein turnover, chaperones", "signal transduction mechanisms", "energy production and conversion", and "cytoskeleton".

GPCR Function in Autophagy Control: A Systematic Approach of Chemical Intervention.

Autophagy facilitates the degradation of cellular content via the lysosome and is involved in cellular homeostasis and stress response pathways. As such, malfunction of autophagy is linked to a variety of diseases ranging from organ-specific illnesses like cardiomyopathy to systemic illnesses such as cancer or metabolic syndromes. Given the variety of autophagic functions within a cell and tissue, regulation of autophagy is complex and contains numerous positive and negative feedback loops. While our knowledge of mechanisms for cargo selectivity has significantly improved over the last decade, our understanding of signaling routes activating individual autophagy pathways remains rather sparse. In this resource study, we report on a well-characterized chemical library containing 77 GPCR-targeting ligands that was used to systematically analyze LC3B-based autophagy as well as ER-phagy flux upon compound treatment. Upon others, compounds TC-G 1004, BAY 60-6583, PSNCBAM-1, TC-G 1008, LPA2 Antagonist 1, ML-154, JTC-801 and ML-290 targeting adenosine receptor A2a (ADORA2A), adenosine receptor A2b (ADORA2B), cannabinoid receptor 1 (CNR1), G-protein coupled receptor 39 (GPR39), lysophosphatidic acid receptor 2 (LPAR2), neuropeptide S receptor 1 (NPSR1), opioid related nociceptin receptor 1 (OPRL1), and relaxin receptor 1 (RXFP1), respectively, were hit compounds for general autophagy flux. From these compounds, only JTC-801 markly increased ER-phagy flux. In addition, the global impact of these selected hit compounds were analyzed by TMT-based mass spectrometry and demonstrated the differential impact of targeting GPCRs on autophagy-associated proteins. This chemical screening exercise indicates to a significant cross-talk between GPCR signaling and regulation of autophagy pathways.

An interim exploratory proteomics biomarker analysis of a phase 2 clinical trial to assess the impact of CT1812 in Alzheimer's disease.

CT1812 is a novel, brain penetrant small molecule modulator of the sigma-2 receptor (S2R) that is currently in clinical development for the treatment of Alzheimer's disease (AD). Preclinical and early clinical data show that, through S2R, CT1812 selectively prevents and displaces binding of amyloid beta (Aß) oligomers from neuronal synapses and improves cognitive function in animal models of AD. SHINE is an ongoing phase 2 randomized, double-blind, placebo-controlled clinical trial (COG0201) in participants with mild to moderate AD, designed to assess the safety and efficacy of 6 months of CT1812 treatment. To elucidate the mechanism of action in AD patients and pharmacodynamic biomarkers of CT1812, the present study reports exploratory cerebrospinal fluid (CSF) biomarker data from 18 participants in an interim analysis of the first set of patients in SHINE (part A). Untargeted mass spectrometry-based discovery proteomics detects >2000 proteins in patient CSF and has documented utility in accelerating the identification of novel AD biomarkers reflective of diverse pathophysiologies beyond amyloid and tau, and enabling identification of pharmacodynamic biomarkers in longitudinal interventional trials. We leveraged this technique to analyze CSF samples taken at baseline and after 6 months of CT1812 treatment. Proteome-wide protein levels were detected using tandem mass tag-mass spectrometry (TMT-MS), change from baseline was calculated for each participant, and differential abundance analysis by treatment group was performed. This analysis revealed a set of proteins significantly impacted by CT1812, including pathway engagement biomarkers (i.e., biomarkers tied to S2R biology) and disease modification biomarkers (i.e., biomarkers with altered levels in AD vs. healthy control CSF but normalized by CT1812, and biomarkers correlated with favorable trends in ADAS-Cog11 scores). Brain network mapping, Gene Ontology, and pathway analyses revealed an impact of CT1812 on synapses, lipoprotein and amyloid beta biology, and neuroinflammation. Collectively, the findings highlight the utility of this method in pharmacodynamic biomarker identification and providing mechanistic insights for CT1812, which may facilitate the clinical development of CT1812 and enable appropriate pre-specification of biomarkers in upcoming clinical trials of CT1812.

Radiographic Assessment of Transverse Tarsometatarsal Instability Complicated by Metatarsus Adductus in Hallux Valgus Patients.

Objective evaluations of transverse tarsometatarsal (TMT) hypermobility/instability are lacking. This study aims to radiographically explore the relationship between transverse TMT instability and metatarsus adductus (MA) in hallux valgus (HV). This study retrospectively analyzed 207 feet with varying degrees of HV, employing the distance between the first and second metatarsals (M1-2 distance) to assess transverse TMT instability of the first ray. Participants were categorized into MA and non-MA groups. It was found that the M1-2 distance significantly increased with the hallux valgus angle (HVA) and metatarsus adductus angle (MAA), demonstrating significant differences between the MA and non-MA groups. The measurement of M1-2 distance showed high reliability, and its cutoff value was determined to be 4.05 mm. Additionally, the results suggest that the widening of the M1-2 distance may be a predisposing factor for MA in HV patients, highlighting its role in the pathogenesis of this foot condition. These findings highlight the need for a comprehensive assessment of TMT instability on both the axial and sagittal planes for the surgical planning of HV, particularly when complicated by a large MAA. Based on these insights, reoriented first-TMT arthrodesis might be recommended for HV with significant MA to address potential multiplanar instability.

Multi-omics in MECP2 duplication syndrome patients and carriers.

MECP2 duplication syndrome (MDS) is an X-linked neurodevelopmental disorder caused by the gain of dose of at least the genes MECP2 and IRAK1 and is characterised by intellectual disability (ID), developmental delay, hypotonia, epilepsy and recurrent infections. It mainly affects males, and females can be affected or asymptomatic carriers. Rett syndrome (RTT) is mainly triggered by loss of function mutations in MECP2 and is a well described syndrome that presents ID, epilepsy, lack of purposeful hand use and impaired speech, among others. As a result of implementing omics technology, altered biological pathways in human RTT samples have been reported, but such molecular characterisation has not been performed in patients with MDS. We gathered human skin fibroblasts from 17 patients with MDS, 10 MECP2 duplication carrier mothers and 21 patients with RTT, and performed multi-omics (RNAseq and proteomics) analysis. Here, we provide a thorough description and compare the shared and specific dysregulated biological processes between the cohorts. We also highlight the genes TMOD2, SRGAP1, COPS2, CNPY2, IGF2BP1, MOB2, VASP, FZD7, ECSIT and KIF3B as biomarker and therapeutic target candidates due to their implication in neuronal functions. Defining the RNA and protein profiles has shown that our four cohorts are less alike than expected by their shared phenotypes.

Specificity profiling of deubiquitylases against endogenously generated ubiquitin-protein conjugates.

Deubiquitylating enzymes (DUBs) remove ubiquitin from proteins thereby regulating their stability or activity. Our understanding of DUB-substrate specificity is limited because DUBs are typically not compared to each other against many physiological substrates. By broadly inhibiting DUBs in Xenopus egg extract, we generated hundreds of ubiquitylated proteins and compared the ability of 30 DUBs to deubiquitylate them using quantitative proteomics. We identified five high-impact DUBs (USP7, USP9X, USP36, USP15, and USP24) that each reduced ubiquitylation of over 10% of the isolated proteins. Candidate substrates of high-impact DUBs showed substantial overlap and were enriched for disordered regions, suggesting this feature may promote substrate recognition. Other DUBs showed lower impact and non-overlapping specificity, targeting distinct non-disordered proteins including complexes such as the ribosome or the proteasome. Altogether our study identifies candidate DUB substrates and defines patterns of functional redundancy and specificity, revealing substrate characteristics that may influence DUB-substrate recognition.

Efficacy of Glycyrrhetinic Acid in the Treatment of Acne Vulgaris Based on Network Pharmacology and Experimental Validation.

Glycyrrhetinic acid (GA) is a saponin compound, isolated from licorice (Glycyrrhiza glabra), which has been wildly explored for its intriguing pharmacological and medicinal effects. GA is a triterpenoid glycoside displaying an array of pharmacological and biological activities, including anti-inflammatory, anti-bacterial, antiviral and antioxidative properties. In this study, we investigated the underlying mechanisms of GA on acne vulgaris through network pharmacology and proteomics. After the intersection of the 154 drug targets and 581 disease targets, 37 therapeutic targets for GA against acne were obtained. A protein-protein interaction (PPI) network analysis highlighted TNF, IL1B, IL6, ESR1, PPARG, NFKB1, STAT3 and TLR4 as key targets of GA against acne, which is further verified by molecular docking. The experimental results showed that GA inhibited lipid synthesis in vitro and in vivo, improved the histopathological damage of skin, prevented mast cell infiltration and decreased the level of pro-inflammatory cytokines, including TNF-α, IL-1ß and IL-6. This study indicates that GA may regulate multiple pathways to improve acne symptoms, and the beneficial effects of GA against acne vulgaris might be through the regulation of sebogenesis and inflammatory responses.

Dysregulation of ferroptosis may participate in the mitigating effect of CoCl2 on contrast-induced nephropathy.

BACKGROUND: Contrast agents can directly or indirectly induce renal tubular ischemia and hypoxic damage. Given that cobalt chloride (CoCl2) can protect renal tubules, the protective effect and potential mechanism of action of CoCl2 on contrast-induced nephropathy (CIN) warrant investigation. METHODS: A CIN mouse model was established to determine the protective effect of CoCl2 on renal injury in vivo. Then, TMT-based proteomics was performed to determine the differentially expressed proteins (DEPs), following which, enrichment analyses of gene ontology and the KEGG pathway were performed. In vitro, a CIN model was constructed with renal tubular epithelial cells (HK-2) to determine the effect of CoCl2 on potential targets and the role of the key protein identified from the in vivo experiments. RESULTS: CoCl2 treatment decreased the levels of BUN and serum creatinine (sCr), while increasing the levels of urea and creatinine (Cr) in the urine of mice after CIN injury. Damage to the renal tubules in the CoCl2 treatment group was significantly less than in the CIN model group. We identified 79 DEPs after treating the in vivo model with CoCl2, and frequently observed ferroptosis-related GO and KEGG pathway terms. Of these, Hp (haptoglobin) was selected and found to have a strong renoprotective effect, even though its expression level in kidney tissue decreased after CoCl2 treatment. In HK-2 cells, overexpression of Hp reduced the ferroptosis caused by erastin, while knocking down Hp negated the attenuation effect of CoCl2 on HK-2 cell ferroptosis. CONCLUSION: CoCl2 attenuated kidney damage in the CIN model, and this effect was associated with the decrease in ferroptosis mediated by Hp.

Evaluation of Protein Identification and Quantification by the diaPASEF Method on timsTOF SCP.

Accurate and precise quantification is crucial in modern proteomics, particularly in the context of exploring low-amount samples. While the innovative 4D-data-independent acquisition (DIA) quantitative proteomics facilitated by timsTOF mass spectrometers gives enhanced sensitivity and selectivity for protein identification, the diaPASEF (parallel accumulation-serial fragmentation combined with data-independent acquisition) parameters have not been systematically optimized, and a comprehensive evaluation of the quantification is currently lacking. In this study, we conducted a thorough optimization of key parameters on a timsTOF SCP instrument, including sample loading amount (50 ng), ramp/accumulation time (140 ms), isolation window width (20 m/z), and gradient time (60 min). To further improve the identification of proteins in low-amount samples, we utilized different column settings and introduced 0.02% n-dodecyl-ß-d-maltoside (DDM) in the sample reconstitution solution, resulting in a remarkable 19-fold increase in protein identification at the single-cell-equivalent level. Moreover, a comprehensive comparison of protein quantification using a tandem mass tag reporter (TMT-reporter), complement TMT ions (TMTc), and diaPASEF revealed a strong correlation between these methods. Both diaPASEF and TMTc have effectively addressed the issue of ratio compression, highlighting the diaPASEF method's effectiveness in achieving accurate quantification data compared to TMT reporter quantification. Additionally, an in-depth analysis of in-group variation positioned diaPASEF between the TMT-reporter and TMTc methods. Therefore, diaPASEF quantification on the timsTOF SCP instrument emerges as a precise and accurate methodology for quantitative proteomics, especially for samples with small amounts.

Integrating Physiological Features and Proteomic Analyses Provides New Insights in Blue/Red Light-Treated Moso Bamboo (Phyllostachys edulis).

Bamboo is one of the most important nontimber forestry products in the world. Light is not only the most critical source of energy for plant photosynthesis but also involved in regulating the biological processes of plants. However, there are few reports on how blue/red light affects Moso bamboo. This study investigated the growth status and physiological responses of Moso bamboo (Phyllostachys edulis) to blue/red light treatments. The growth status of the bamboo plants was evaluated, revealing that both blue- and red-light treatments promoted plant height and overall growth. Gas exchange parameters, chlorophyll fluorescence, and enzyme activity were measured to assess the photosystem response of Moso bamboo to light treatments. Additionally, the blue light treatment led to a higher chlorophyll content and enzyme activities compared to the red light treatment. A tandem mass tag quantitative proteomics approach identified significant changes in protein abundance under different light conditions with specific response proteins associated with distinct pathways, such as photosynthesis and starch metabolism. Overall, this study provides valuable insights into the physiological and proteomic responses of Moso bamboo to blue/red light treatments, highlighting their potential impact on growth and development.