Limitations to many current aqueous-based tryptic digestion methods include lengthy digestion times and both relatively high inter- and intra-day variability for both characteristic peptides identified and sequence coverages. This report describes results from digestion of some complex biomedical samples using the rapid Denaturing Organic Digestion method (DOD), an organic solvent-modified digestion method previously optimized for targeted protein digestion. Advantages of the DOD method included a very rapid digestion only requiring inexpensive solvents and reagents generally available in the laboratory, with no requirement for specialized equipment or expensive, specialized consumables. For this study, samples of E. coli and murine ileum protein extracts, and K562, a mass spectrometry-compatible human protein extract and reference standard routinely used to evaluate methods, were digested. Sequence coverage and characteristic peptide identification results were compared to those from 18 and 24 h conventional aqueous-based digestion methods. Across the samples tested, though the number of characteristic peptides and sequence coverages produced by the 5 min DOD method were very similar to those produced by the aqueous-based digestion methods, the specific characteristic proteins and their corresponding tryptic peptides identified following DOD method digestion included more hydrophilic and less hydrophobic species. In addition, we explored the effect of increasing digestion times with complex samples from 5 to 30 and 90 min for the DOD method. Increasing the digestion time to ≥30 min resulted in improved intra-day precision and the identification of many more peptide products than the currently used aqueous methods to which it was compared. These results suggest that the DOD organic-modified digestion method could, while markedly reducing protein digestion time, also provide more precise analysis and access to a somewhat different area of the proteome than that provided by current aqueous-based digestion methods. SIGNIFICANCE: The DOD tryptic digest method is a very simple and rapid process with no requirement for expensive equipment or consumables. The method markedly reduces tryptic digestion time and cost, and substantially improves within-batch and across-analyst precision for peptide and sequence coverage results over methods to which it was compared. Importantly, it also provides access to a somewhat different subset of the proteome with different peptide products identified as compared to aqueous solvent-based digestion providing potential for increased proteome coverage for bottom-up analysis if used in conjunction with aqueous-based methods.

This study identified potential biomarkers for feed efficiency by blood plasma proteome analysis of a tropically adapted beef cattle breed. Two experimental groups were selected based on residual feed intake (RFI). The proteome was investigated by LC-MS/MS in a data-dependent acquisition mode. After quality control, 123 differentially abundant proteins (DAPs) were identified between the two experimental groups. Among DAPs with the highest absolute log-fold change values, the PRDM2, KRT5, UGGT1, DENND5B, B2M, SLC44A2, SLC7A2, PTPRC, and FETUB were highlighted as potential biomarkers because of their functions that may contribute to RFI. Furthermore, functional enrichment analysis revealed several biological processes, molecular functions and pathways that contributes to RFI, such as cell signaling, cellular responses to stimuli, immune system, calcium, hormones, metabolism and functions of proteins, lipids and carbohydrates. Protein-protein interaction analysis identified 32 and 11 DAPs as important nodes based on their interactions in the high- and low-RFI groups, respectively. This study represents the first comprehensive profiling of the blood plasma proteome of a tropically adapted beef cattle breed and provides valuable insights into the potential roles of these DAPs in key biological processes and pathways, contributing to our understanding of the mechanisms underlying feed efficiency in tropically adapted beef cattle. SIGNIFICANCE: LC-MS/MS analysis was performed to investigate changes in the blood plasma proteome associated with residual feed intake (RFI) in a tropically adapted beef cattle breed (Bos taurus taurus). Some putative biomarkers were identified to distinguish the high-RFI to low-RFI animals, based on their log-fold change value or on their protein-protein interaction network, which provide helpful sources in developing novel selection strategies for breeding programs. Our findings also revealed valuable insights into the metabolic pathways and biological processes that contribute to RFI in beef cattle, such as those closely linked to cell signaling, cellular responses to stimuli, immune system, calcium, hormones, metabolism and functions of proteins, lipids and carbohydrates.

The authenticity of halal meat is a global issue because pork adulteration occurs. Certain religions, such as Islam and Judaism, prohibit the use of pork in food products. The purpose of this study was to evaluate the volume of trypsin with 10, 50 and 100 μL (20 μg/100 μL) and the digestion time from overnight to 30-120 min to establish a fast and straightforward procedure on proteomic analysis for halal authentication of meat and meat products. The method was applied to raw meat and processed pork products. The results show that 30 min digestion time and 50 μL of trypsin could detect specific peptides from pork. The proteins such as L-lactate dehydrogenase (D2SW96), haemoglobin subunit beta (F1RII7), carbonic anhydrase (A0A4XIUCS1), and myosin-1 (A0A481AX92) could be the alternative protein that contains specific peptide from pork that could be used as peptide biomarker in raw pork meat. Two peptides from haemoglobin subunit beta (F1RII7) protein heat stable peptide biomarkers were not previously reported. The method is proven for fast analysis, simple protein extraction, and rapid identification of specific pork peptides in raw meat and processed pork products. SIGNIFICANCE: The authenticity of halal meat is a global issue because pork adulteration occurs. Certain religions, such as Islam and Judaism, prohibit the use of pork in food products. Pork is frequently used as an adulterant in high-quality, high-priced meats such as beef, fish meat slices, lamb, or other meat to increase profits because pork is less expensive than the other meats. The purpose of this study was to evaluate the volume of trypsin and the digestion time to establish a fast and straightforward procedure on proteomic analysis for halal authentication of meat and meat products. The results show that 30 min digestion time and 50 μL of trypsin could detect specific peptides from pork. The proteins such as L-lactate dehydrogenase (D2SW96), haemoglobin subunit beta (F1RII7), carbonic anhydrase (A0A4XIUCS1), and myosin-1 (A0A481AX92) could be the alternative protein that contains specific peptide from pork that could be used as peptide biomarker in raw pork meat. To the best of our knowledge, this is the first report that studied on fast analysis, simple protein extraction, and rapid identification of specific pork peptides in raw meat and processed pork products.

Understanding biological mechanisms underlying anorexia nervosa (AN) is necessary to develop care strategies. Despite many articles dedicated to peptides assessment in AN, there is no systematic review. A scoping review of circulating peptides published in relation to AN, comparing their results with those of controls, was conducted. Embase and PubMed databases were search from 1966 to 2022 (PROSPERO CRD42022323716). All original English articles, assessing peptides in AN (except classical markers) were analyzed. 1151 studies for 207 peptides, in 486 published articles were selected, and evidences/trends in AN were compared to controls. Fifteen clusters of function gathering peptides covering physiopathological aspects of AN were identified. This scoping review revealed a large variety of circulating peptides explored in AN. Some peptides presented with convincing results and helped understanding pathophysiologic aspects. Other peptides presented with nuanced results, partly due to insufficient number of studies, multiple assay techniques, inadequate sampling time, and lack of phenotyping. Conversion from bench-to-bed remains difficult and may explain why peptides evaluations did not currently lead to specific international recommendations or tailored therapeutic/preventive strategies. Peptide evaluation in anorexia nervosa could explore secretion profiles, and test it in well-phenotyped patients with AN, to conclude for potential clinical use, and finally design therapeutic tests.

Inflammatory bowel disease is characterized by severe imbalance of intestinal flora and metabolic disorders. Recent studies have demonstrated that probiotics can effectively alleviate inflammatory bowel disease by restoring the intestinal flora structure and modulating the immune response. However, the role of probiotics in regulating intestinal metabolism disorders is still unclear. This study explores the role of probiotic B. cereus in alleviating DSS-induced colitis. The findings indicated probiotic B. cereus treatment mitigated tissue damage and apoptosis during inflammation. Metabolome and transcriptome analysis revealed B. cereus activated the cholic acid-FXR axis by increasing cholic acid levels, which promoted the gene expression level of NF-κB inhibitor α, reduced the IL-1β, IL-6, IL-18 and TNF-α concentrations. Furthermore, it effectively mitigated the DSS-induced disruption of bile acid metabolism, arginine metabolism, and linoleic acid metabolism. This study explores the effect and mechanisms of probiotic B. cereus on alleviating DSS-induced colitis. It aims to provide a theoretical basis for microbial therapy in inflammatory bowel disease. SIGNIFICANCE: This study used metabolome and transcriptome to reveal the roles and mechanisms, which probiotic Bacillus cereus modulates metabolic disorders and alleviate DSS-induced colitis. We identified the cholic acid-FXR axis as an important target for alleviating DSS-induced colitis. These findings provide new insights into microbial treatment strategies for IBD.

Extreme heterogeneity exists in the hypersensitive stress response exhibited by the dystrophin-deficient mdx mouse model of Duchenne muscular dystrophy. Because stress hypersensitivity can impact dystrophic phenotypes, this research aimed to understand the peripheral pathways driving this inter-individual variability. Male and female mdx mice were phenotypically stratified into "stress-resistant" or "stress-sensitive" groups based on their response to two laboratory stressors. Quantitative proteomics of striated muscle revealed that stress-resistant females were most dissimilar from all other groups, with over 250 proteins differentially regulated with stress hypersensitivity. Males showed less proteomic variation with stress hypersensitivity; however, these changes were associated with pathway enrichment. In the heart, stress-sensitive males had significant enrichment of pathways related to mitochondrial ATP synthesis, suggesting that increased cardio-metabolic capacity is associated with stress hypersensitivity in male mdx mice. In both sexes, stress hypersensitivity was associated with greater expression of beta-actin-like protein 2, indicative of altered cytoskeletal organisation. Despite identifying proteomic signatures associated with stress hypersensitivity, these did not correlate with differences in the serum metabolome acutely after a stressor. These data suggest that the heterogeneity in stress hypersensitivity in mdx mice is partially driven by cytoskeletal organisation, but that sex-specific cardio-metabolic reprogramming may also underpin this phenotype. SIGNIFICANCE: Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease which is associated with a premature loss of ambulation and neurocognitive dysfunction. The hypersensitive stress response in DMD is a heterogeneous phenotype which is poorly understood. This study provided the first investigation of the peripheral mechanisms regulating the hypersensitive stress response by undertaking multi-omics analysis of phenotypically stratified mdx mice. Variations in behaviour and the striated muscle proteomic profiles suggest that cardio-metabolic remodelling and cytoskeletal organisation may contribute to this phenotype. This research offers significant insights into understanding how peripheral dystrophin deficiency relates to the cognitive abnormalities seen in patients with DMD.

The label-free shotgun proteomics analysis carried out in this study aimed to understand the molecular mechanisms that contribute towards tomato susceptibility to Xanthomonas euvesicatoria pv. perforans (Xep). To achieve this, comparative proteomics was performed on susceptible inoculated plants with the bacterium and the control group (saline solution) at 24 and 48 h after inoculation (hai). The results revealed that most of the identified proteins showed increased abundance in the infected group and were classified into different gene ontology groups. Eight of these proteins were related to susceptibility in other pathosystems, suggesting their potential involvement in the development of bacterial spot in tomato. Some of these proteins are involved in the negative regulation of salicylic acid, PR proteins and reactive oxygen species (ROS), as well as contributing to the acquisition of sugars by the pathogen. The results obtained in this study provided us with valuable information for understanding the molecular mechanisms that lead to tomato susceptibility to Xep and will help in developing tomato cultivars resistant to bacterial spot. SIGNIFICANCE: Our proteomic study of tomato plants during infection by Xep allowed for the identification of potential proteins that contribute to bacterial spot tomato disease development. These proteins can act in different ways to favor the pathogen, such as the negative modulation of phytohormones involved in plant defense, the inhibition of PR proteins and reactive oxygen species, as well as to collaborate in the acquisition of sugar for pathogen nutrition.

Inflammation is a complex factor in the pathogenesis of intracranial aneurysms (IA), but its specific cellular inflammatory factors remain uncertain. We collected two cohorts and measured the representation of vascular inflammation-related proteins using the Olink CVD II Vascular Inflammation Panel. We subsequently validated our findings using ELISA and RT-qPCR. Our proteomic analysis identified 11 vascular inflammation-related markers that were significantly differentially represented between the IA and control groups. These markers were implicated in leukocyte migration, immune response, triglyceride and lipoprotein metabolism, acute phase response, T cell regulation, and several key biological pathways, including PPAR, HIF-1, cytokine-cytokine interactions, and PI3K-AKT signaling. Further validation with ELISA and RT-qPCR confirmed the differential representation of IL6, PTX3, LPL, and OLR1 between the two groups. Notably, a combination marker incorporating these four factors demonstrated high diagnostic potential for the early detection of IA. Our study has identified a set of informative biomarkers (IL6, PTX3, LPL, and OLR1) that could be valuable for the early diagnosis of IA. Importantly, this is the first report of significantly elevated OLR1 representation in the plasma of IA patients. Further investigation into the role of OLR1 in the pathogenesis of IA is warranted. SIGNIFICANCE: This study significantly advances our understanding of the molecular mechanisms underlying intracranial aneurysm (IA) pathogenesis. By identifying a panel of novel biomarkers, including the previously unreported elevated expression of OLR1 in IA patients, we provide crucial insights into the inflammatory processes involved in aneurysm formation and development. These findings have important clinical implications, as the identified biomarkers could serve as valuable tools for early diagnosis and potentially targeted therapeutic interventions. Furthermore, the study highlights the complex interplay of inflammatory pathways in IA, suggesting that a multi-faceted approach may be necessary for effective management.

Despite numerous studies on fetal therapy for myelomeningoceles (MMC), the pathophysiology of this malformation remains poorly understood. This study aimed to analyze the biochemical profile and proteome of amniotic fluid (AF) supernatants from MMC fetuses to explore the prenatal pathophysiology. Biochemical analysis of 61 AF samples from MMC fetuses was compared with 45 healthy fetuses' samples. Proteome analysis was conducted in 18 MMC and 18 healthy singleton fetuses, and in 5 dichorionic pregnancies with MMC fetuses and their healthy co-twins. ELISA tests were used to validate proteome results. Biochemical analysis revealed anal incontinence in 37 % of MMC cases, absent in controls (p < 0.0001). Proteomics identified 2453 quantified proteins with 39 significantly up-regulated and 10 down-regulated in the MMC group. Up-regulated proteins included ectodomains of CHL1, APLP1, SEZ6, SEZ6L, known targets of the protease BACE1. We explored the overlap of neonatal cerebrospinal fluid (CSF) and AF proteome and highlighted 411 proteins in common, mostly upregulated in MMC AF compared to controls. Our study thoroughly characterizes the AF proteome and reveals numerous proteins to be changed as a consequence of MMC. Many of these proteins are typical constituents of CSF. No difference in AF inflammation markers were observed between MMC and healthy fetuses. SIGNIFICANCE: This study provides good evidence that neuroepithelial destruction in MMC is independent of inflammation or presumed meconium toxicity.

The ability of axillary meristems to form axillary buds and subsequently develop into branches is influenced by phytohormones, environmental conditions, and genetic factors. The main trunk of Quercus fabri is prone to branching, which not only impacts the appearance and density of the wood and significantly reduces the yield rate. This study conducted transcriptomic, proteomic, and metabolomic analyses on three stages of axillary bud development in Q. fabri. A total of 12,888 differentially expressed genes (DEGs), 8193 differentially accumulated proteins (DAPs), and 1788 differentially accumulated metabolites (DAMs) were identified through comparisons among the stages and subjected to multi-omics joint analysis. Conduct interaction network analysis on DEGs and DAPs to identify the significant transcription factor family (AP2/ERF) involved in the regulation of axillary bud development. Furthermore, KEGG enrichment analysis of DEGs, DAPs and DAMs indicated significant enrichment in plant hormone signaling pathways. The analysis of endogenous hormone levels and qRT-PCR results for pathway genes demonstrated that the expression levels of IAA and tZ significantly increased during late developmental stages, whereas the expression levels of ABA, ACC and JA significantly decreased. In summary, these findings contribute to a comprehensive understanding of the regulatory networks underlying the branching development of Q. fabri. SIGNIFICANCE: Q. fabri exhibits robust vegetative growth, and its primary trunk is prone to branching, significantly influencing the wood yield rate. Through a joint analysis of transcriptomics, proteomics, and metabolomics, we comprehensively examined the regulatory network governing the axillary bud development of Q. fabri. Our findings revealed the crucial roles of the AP2/ERF transcription factor family and plant hormone signal transduction pathways in branch development. These insights contribute to a deeper understanding of the mechanisms regulating branch development.

COVID-19 still spreads worldwide, and repeated infections are hard to avoid. Maternal infection during pregnancy is associated with adverse maternal and neonatal outcomes. Our study used a multi-omics profiling method to explore the proteome and metabolome alteration in early embryonic development after COVID-19 infection. A total of 30 chorionic tissues after artificial abortion (15 infection and 15 no-infection samples) were collected, and the UHPLC-MS/MS and LC-MS/MS were applied in the present study. As a result, 311 significantly differentially expressed proteins were identified. The function annotations revealed that the thermogenesis pathway is the most significantly enriched signaling pathway; PRKAG2, IGF1R, and RPS6KB2 were identified as the hub proteins. There were 359 metabolites significantly altered after infection. The functional annotations revealed that amino acid metabolism was significantly affected, especially beta-alanine metabolism, glutamate metabolism, and histidine metabolism pathways. The metabolites in ovarian steroidogenesis showed a down-regulating trend in the infection group. Finally, we combined the results of proteins and metabolomics analysis. The biosynthesis of the cofactors pathway was identified as significantly enriched in both proteomics and metabolomics datasets. Our findings provide a network of protein regulation and metabolite perturbation during early embryonic development with COVID-19 infection. Our findings can provide valuable insights for further exploration of the complex mechanism of COVID-19-associated pregnancy complications and outcomes. SIGNIFICANCE: COVID-19 has developed into the most prominent and deadliest pandemic respiratory disease in the world, and repeated infections are complicated to avoid. COVID-19 infection during pregnancy increases the risk of adverse maternal and neonatal outcomes, such as preterm birth and stillbirth. However, previous studies mainly focused on its effect on pregnant women, such as the clinical characteristics and gestation outcomes. There is no relevant report about the effects of virus infection on embryos in early pregnancy. The effects of COVID-19 infection changes of the proteins and metabolites during early embryonic development are undefined. Our findings provide an association between protein regulation, metabolite perturbation, and COVID-19 infection, which can provide valuable insights for further exploration of the complex mechanism COVID-19 COVID-19-associated pregnancy complications and adverse pregnancy outcomes.

We analyze the proteome changes during the development of the carnauba palm (Copernicia prunifera) seedlings under skotomorphogenic conditions, by separating the embryo into its two components: haustorium (HA) and cotyledonary petiole (CP) and established the descriptive and quantitative proteomes of these tissues across four developmental stages. 5205 proteins were identified in HA and 6028 in CP. These proteomes are rich in proteins known to maintain the skotomorphogenic state, and in a complete set of proteins involved in cellular respiration and biosynthesis of secondary metabolites. The quantitative analysis employing a label-free approach revealed that 583 proteins in HA and 383 in CP were differentially abundant, with 251 proteins shared between the datasets. The results showed that HA participates in the digestion of food reserves present in HA itself and in the endosperm, acting as a conduit of nitrogen and carbon sources for the growing embryo axis. Among the differentially abundant proteins in the CP, we identified the presence of proteins from the cellular metabolism and proteins involved in the hydrolysis of food reserves such as starch and proteins. This indicates that the CP, in addition to the endosperm and HA, serves as a source of food reserves for the embryo axis. SIGNIFICANCE: Our results also reveal the differential regulation of specific proteins involved in reactive oxygen species scavenging, cell wall remodeling, respiratory metabolism, and protein repair in seeds and seedlings of C. prunifera. These findings have broad implications for understanding the energy metabolism that drives the transition from seed to seedling. For this study, we employed state-of-the-art proteomic techniques, including quantitative mass spectrometry and bioinformatic analysis, that allowed us to create a large dataset that will be a valuable resource for future research on the physiological and biochemical aspects of skotomorphogenesis, photomorphogenesis, and the transition between these states.

Survival of brachyuran crabs is temperature-dependent and thermal stress promotes changes during molting. We aimed to decipher the impact of thermal stresses on the X-organ/sinus gland (XO/SG) complex, a temperature-sensitive neuroendocrine tissue involved in the molting regulation of Callinectes sapidus during the intermolt and premolt phases. We employed a proteogenomic approach using specimens subjected to control (24 °C), cold (19 °C), and heat (29 °C) temperatures. A total of 1463 protein groups with at least two unique peptides were identified and quantified. C. sapidus in the premolt stage exposed to the cold condition exhibited a proteome closely resembling that of the intermolt stage, as evidenced by measurements of circulating ecdysteroid levels. Compared to the intermolt at control temperature, the premolt stage exhibited increased energy metabolism, structural changes in the cuticle mediated by chitin metabolism and glycoproteins, biosynthesis of methyl farnesoate (MF), and elevated tissue levels of molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH), indicating lower secretion rates. Heat temperature (29 °C) seems to induce mitochondrial metabolism in the intermolt XO/SG, while cold temperature elicited a delayed molt cycle in the premolt phase, marked by reduced tissue levels of CHH, indicating increased secretion and Y-organ (YO) inhibition, and decreased MF production (reduced YO stimulation). SIGNIFICANCE STATEMENT: Temperature plays a pivotal role in regulating the metabolism, growth, molting, reproduction, and survival of crabs, such as the blue crab (Callinectes sapidus). Despite the blue crab's significance on both economic and ecological realms, there has been a notable lack of molecular information related to this species and therefore a gap in our knowledge of the blue crab's molecular makeup and genetic diversity. This research established a comprehensive proteome landscape to elucidate the molecular and functional changes in the XO/SG complex involved in the molting process of C. sapidus, and how thermal stresses significantly influence biotransformation processes. Utilizing a proteogenomics approach with multi-round homologous database analysis, we have generated a highly accurate protein repertoire with at least two unique peptide of XO/SG tissue proteome. This resource will be invaluable for future molecular analyses of this species. Our findings demonstrate that thermal stresses induced specific modifications in the XO/SG tissue, depending on the molt cycle phase. Temperature-mediated responses influences the biological processes, enhancing the functional morphogenesis and comprehensive metabolic adaptations on molting cycle supported by a relationship between the XO/SG tissue proteome and circulating ecdysteroid levels.

Periodontal disease affects over 1 billion people globally. This study investigated how periodontitis affects the protein profile of the periodontal ligament (PDL) in rats. Eight Holtzman rats were divided into control and experimental periodontitis groups. The PDL was isolated using laser capture microdissection and protein extracts were analyzed by mass spectrometry. Data analysis utilized specialized software, and Gene Ontology enrichment analysis identified significant protein functions. The data are available via ProteomeXchange with identifier PXD055817. Proteins such as SerpinB1, C5, and Lgals3 were validated through immunohistochemistry, and their gene expression was examined in an in vitro human PDL cell line. This study identified 1326 proteins, with 156 unique to the control group, 294 unique to the periodontitis group, and 876 common to both groups. Enrichment analysis revealed that proteins associated with the regulation of enzyme activity and RNA binding were significantly represented in the periodontitis group. There were increased levels of SerpinB1, C5, and Lgals3 in the periodontitis group based on proteomic and immunohistochemical analyses. Furthermore, these targets showed increased gene expression in stimulated human PDL cells. This study provides insights into the periodontitis-related alterations in the protein composition of the PDL and PDL cells, identifying both novel and previously known disease-associated proteins. SIGNIFICANCE: The periodontal ligament plays a crucial role in oral functions by providing structural support to the tooth. Due to the presence of undifferentiated mesenchymal cells, research into its regenerative capacity is ongoing. Pathological conditions can affect these functions and protein composition. Currently, there is a lack of comprehensive research specifically focusing on evaluating the periodontal ligament in both healthy and diseased states. This pioneering study screened for protein alterations and the mechanisms related to periodontitis. The possibility of using proteomic analysis to evaluate the protein alterations that occur in periodontitis, a disease with a high global incidence, could provide therapeutic targets and new biomarkers for future clinical studies.

In December 2022, China ceased the zero-COVID-19 policy, resulting in an increase in hospitalizations and deaths due to COVID-19, particularly among the elderly population. Predicting non-survivors aims to identify high-risk patients and enable targeted interventions to improve survival rates. Additionally, understanding factors affecting prognosis provides essential insights for further research and optimization of treatment strategies. We applied 4D-DIA mass spectrometry for serum proteome analysis and provided a comprehensive characterization of disease features in elderly patients within the Chinese population. Our study elucidated that immune disorders, lung damage, and cardiovascular disorders are predominant causes of death in these patients. Compared to clinical indices, proteomic analysis is more sensitive in tracing these disorders. We also provided a prediction panel for survival outcomes of elderly patients using levels of CXCL10, CXCL16 and IL1RA, which were validated by ELISA. These biomarkers will help improve predictive efficacy for survival outcomes in elderly patients.

Although the phosphorylation of serine (S), threonine (T), and tyrosine (Y) is well-established, arginine phosphorylation (pR) has recently garnered significant attention due to its crucial role in bacteria pathogenicity and stress response. Mycolicibacterium smegmatis, a nonpathogenic surrogate of Mycobacterium tuberculosis, serves as a model for studying mycobacterial pathogenesis. A recent proteomics study identified six pR proteins in M. smegmatis. To gain a more comprehensive understanding, we performed pR profiling using mass spectrometry in combination with two distinct phosphopeptide enrichment strategies: titanium-immobilized metal ion affinity chromatography (Ti-IMAC) and Fe-NTA cartridge purification. This approach led to the identification of 1192 shared pR peptides with 1553 pR sites in M. smegmatis following both competitive and non-competitive scoring assessments for pR and pS/T/Y. Further stringent filtering through manual verification resulted in 58 high-confident pR sites across 57 proteins. These confirmed pR-proteins are functionally related, particularly in DNA binding and ATP binding. Alterations in the modification of three pR sites during the logarithmic and stationary phases at the phosphorylation level, but not at the total cell protein level, further suggest the role of pR in the bacterium's functional adaptation to its environment. SIGNIFICANCE: Our findings reveal that pR proteins are prevalent and play roles in DNA-binding and ATP-binding activities, providing insights into the broader biological functions of pR peptides in other genetically diverse species. The reliable identification of bacterial pR events in M. smegmatis not only propels the study of pR within the realm of proteomics but also paves the way for exploring its detailed function in bacteria.

The prevalence of chronic kidney disease (CKD) is gradually rising worldwide. Patients often remain asymptomatic for an extended period, leaving them unaware of their condition, which can lead to progressing to end-stage renal disease and cause significant economic burden. Improved understanding of CKD pathogenesis can enhance early detection and facilitate advances in drug development. Here, we performed proteomic and phosphoproteomic analyses of the mouse unilateral ureteral obstruction model to explore the molecular mechanisms of chronic kidney injury. 474 significantly differentially expressed proteins and 96 significantly differentially expressed phosphoproteins were screened, respectively. Chronic kidney injury involves complex metabolic pathways such as citrate cycle and hematopoietic system in proteome, and mitochondrial oxidative phosphorylation suppression is a notable alteration. The phosphoproteomic analysis revealed a significant upregulation in epithelial mesenchymal transition and P53 pathways, with a corresponding increase in the phosphorylation of Jun at serine 73. Utilizing HK2 cells, we observed that the reduction oxidative phosphorylation was consistently associated with an augmentation in oxidative stress, which subsequently activated Jun and induced apoptosis. Proteins that act as hubs in these pathways may be candidate targets for CKD intervention. These findings contribute significantly to the current understanding of CKD and provide valuable insights for future studies. SIGNIFICANCE: Chronic kidney disease (CKD) incidence rising annually with varied etiologies, kidney often irreversibly fibrotic, the treatment options are limited and often ineffective due to deficient understanding of renal fibrosis mechanisms. Despite the extensive efforts and numerous omics studies conducted on renal fibrosis, to date, no study has been undertaken to investigate the role of phosphorylated proteins in UUO models. Previously, we performed a comprehensive transcriptome and proteome analysis based on the CKD model, but the potential alterations in the phosphoproteome were not addressed. Here, an integrated proteomic and phosphoproteomic landscape of CKD was completed, which was the the first phosphoproteomic profiles of UUO model. Phosphoproteomic profile suggests that the epithelial mesenchymal transition and P53 pathways is significantly activated in mouse models of kidney injury, and the core protein Jun played a key role in CKD. And a preliminary correlation between P-Jun and oxidative phosphorylation was found base on HK2 cells. Our work contributes to a deeper understanding of the disease characteristics and molecular mechanisms of CKD. Identifying potential CKD targets from proteome and phosphoproteome may provide valuable insights for early diagnosis and treatment of CKD.

To date, the molecular pathogenic mechanisms between HBsAg and liver metabolic disorders have not been fully understood. To explore the overall effects of HBsAg on liver tissues from HBV transgenic mice, proteome, interactome, and signal pathway analysis were employed to uncover the underlying mechanisms. Bioinformatics analysis of 191 differentially expressed proteins suggested that HBV upregulated the expression of multiple enzymes involved in lipid synthesis, and small HBs (SHBs) caused lipid accumulation in cells. Further studies showed that SHBs bound to binding immunoglobulin protein (Bip), which normally functions in cell homeostasis against the unfolded protein response (UPR) signaling via occupying inositol-requiring enzyme 1 (IRE1). Hijacking Bip by SHBs alleviated the inhibition of post-endoplasmic reticulum (ER) signaling and sequential activation of the IRE1 downstream transcription factors involved in lipid synthesis, such as spliced X-box binding protein 1 (sXBP1) and sterol regulatory element-binding protein 1 (SREBP1), leading to lipid metabolism disorder. The restoration of Bip can alleviate ER stress, and block the sequential post-ER signaling caused by SHBs. This study revealed a new pathway through which SHBs promote lipid disorder, and suggests that Bip may serve as a novel target for intervention in HBV related liver diseases. SIGNIFICANCE: In this study, we found a new pathway promoting the lipid disorder by SHBs through quantitative proteomics studies, and Bip may serve as a novel target for intervention in HBV related liver diseases. These findings highlight a novel role of SHBs in regulating cell lipid metabolism and provide an insight into the relationship between HBV infection and liver fatty disorders, which may serve as a potential therapeutic target for intervention of HBV related liver diseases.

Renal cell carcinoma (RCC) stands among the most lethal urological malignancies. Most RCCs are incidentally diagnosed as initial symptoms are unspecific. Novel, minimally-invasive diagnostic and prognostic methods for RCC are needed, ideally in urine. Using UPLC-Q-ToF MS untargeted metabolomic analysis in urine, we previously revealed p-cresol glucuronide as potential RCC diagnostic marker. Additionally, urine samples one-year post-nephrectomy revealed isobutyryl-l-carnitine and L-proline betaine as potential RCC prognostic markers. Our present aim was to validate these differences in an independent cohort of RCC patients and healthy controls to strengthen their value as non-invasive biomarkers. In an independent cohort of 69 RCC patients and 52 controls we validated an increase in p-cresol glucuronide in urine from patients at diagnosis compared to controls (P = 0.0043). It remained increased one-year post-nephrectomy (P = 0.0288). The value of p-cresol glucuronide for RCC diagnosis was assessed with ROC curves analysis (AUC = 0.66, 95 % Confidence Interval 0.56-0.76). The role of isobutyryl-l-carnitine and L-proline betaine as prognostic markers could not be validated and will require a larger cohort. Our findings confirm the value of p-cresol glucuronide in urine as diagnostic marker for RCC in an independent cohort. This non-invasive method holds promise for enhancing patient care by reducing the need for potentially risky diagnostic procedures. Further metaproteomics-oriented approaches towards the tyrosine oxidation pathway and microbiota metagenomics studies may promote a holistic management of RCC. SIGNIFICANCE: Current imaging techniques available to diagnose and monitor renal cell carcinoma (RCC) are harmful for the patient given the high-radiation dose, and unspecific in low-grade tumors. Thus, novel non-invasive biomarkers with diagnostic and prognostic capabilities are of utmost importance. Herein, we have validated urine p-cresol glucuronide as diagnostic marker for RCC. This novel non-invasive biomarker could improve accurate assessments of tumor behavior, while enhancing patient outcomes by reducing discomfort and detrimental side effects.

This pipeline presents a refined approach for deriving personalized neurobiological insights from iPSC-derived neurospheres. By employing Tandem Mass Tag (TMT) labeling, we optimized sample pooling and multiplexing for robust comparative analysis across experimental conditions, maximizing data yield per sample. Through single-patient-derived neurospheres-composed of neural progenitor cells, early neurons, and radial glia-this study explores proteomic profiling to mirror the cellular complexity of neurodevelopment more accurately than traditional 2D cultures. Given their enhanced relevance, these 3D neurospheres serve as a valuable model for elucidating neurogenesis, differentiation, and neuropathological mechanisms, contributing to the advancement of in vitro neural models and reducing dependency on animal models. SIGNIFICANCE: This study evaluates ten protein extraction protocols using TMT 10-plex labeling to optimize proteomic analysis from single neurospheres. It compares cost, protein yield, and the ability to detect differentially expressed proteins, identifying methods like SPEED and S-Trap as efficient for high-throughput studies, while FASP excels in peptide yield. TMT labeling enhances protein identification, particularly for low-abundance proteins, and allows pre-fractionation to maximize analysis from limited samples. However, challenges such as limited PTM analysis and the potential loss of minor proteins highlight the importance of selecting protocols based on specific research goals. This work contributes to optimizing proteomic workflows for in vitro neural models, advancing single-cell analysis with minimal reliance on animal models.