Lysine 2-hydroxyisobutyrylation (Khib) is one of the newly discovered post-translational modifications (PTMs) through protein acylation. It has been reported to be widely distributed in both eukaryotes and prokaryotes, and plays an important role in chromatin conformation change, gene transcription, protein subcellular localization, protein-protein interaction, signal transduction, and cellular proliferation. In this study, the Khib modification proteome of siliques from A. thaliana under salt stress (Ss) and those in the control (Cs) were compared. The results showed that Khib modification was abundant in siliques. Totally 3810 normalized Khib sites on 1254 proteins were identified, and the Khib modification showed a downregulation trend dramatically: it was down-regulated at 282 sites on 205 proteins while was up-regulated at 96 sites on 78 proteins in Ss siliques (Data are available via ProteomeXchange with identifier PXD028116 and PXD026643). Among them, 13 proteins, including F4IVN6, Q9M1P5, and Q9LF33, had sites with the most significant regulation of Khib modification. Bioinformatics analysis suggested that the differentially Khib-regulated proteins mainly participated in glycolysis/gluconeogenesis and endocytosis. In particular, there were differentially117 Khib-regulated proteins that were mapped to the protein-protein interaction database. In the KEGG pathway enrichment analysis, Khib-modified proteins were enriched in several pathways related to energy metabolism, including gluconeogenesis pathway, pentose phosphate pathway, and pyruvate metabolism. Overall, our work reveals the first systematic analysis of Khib proteome in Arabidopsis siliques under salt stress, and sheds a light on the future studies on the regulatory mechanisms of Khib during the salt stress response of plants. SIGNIFICANCE: In this study, we found the Khib-modified proteins in silique under salt stress and described the enrichment of Khib-modified proteins involved in the biological processes and cellular localization. Proteins undergoing 2-hydroxyisobutylation were mainly involved in the gluconeogenesis pathway, pentose phosphate pathway, and pyruvate metabolism, suggesting that 2-hydroxyisobutylation affects the energy metabolic pathway, and thus the development of the plant. In addition, specific candidate proteins that may affect plant development under salt stress were selected. This study will provide a theoretical basis for revealing the function and mechanism of these proteins and their 2-hydroxyisobutyryl modifications during the development of silique under salt stress.

Quantitative label-free mass spectrometry (MS) is an increasingly powerful technology for profiling thousands of proteins from complex biological samples. One of the primary goals of analyses performed on such proteomics data is to detect differentially expressed proteins (DEPs) under different experimental conditions. Many statistical methods have been developed and assessed for DEP detection in various proteomics studies. However, it remains a challenge for many proteomics scientists to choose an appropriate statistical procedure. Therefore, in this study, we organized 12 common testing algorithms and 6 P-value combination methods and further provided Cohen's d effect size for every protein and three evaluation criteria to help proteomics scientists investigate their influence on DEP detection in a systematic manner. To promote the widespread use of these methods, we developed a user-friendly web tool, StatsPro, and presented two case studies involving label-free quantitative proteomics data obtained using data-dependent acquisition and data-independent acquisition to illustrate its practicability. This tool is freely available in our GitHub repository (https://github.com/YanglabWCH/StatsPro/). SIGNIFICANCE: One of the primary goals of analyses performed on liquid chromatography-mass spectrometry (LC-MS) based proteomics data is to detect differentially expressed proteins (DEPs) under different experimental conditions. Despite of many research efforts have been proposed to detect DEPs, to date, there is a scarcity of efficient, systematic, and easy-to-handle tools that are tailored for proteomics scientists to choose an appropriate statistical procedure. Herein, we present a new tool, StatsPro, to enable implementation and evaluation of different statistical methods for proteomics scientists. This tool has two significant advances compared to existing software: a) It integrates up to 18 common statistical approaches (12 statistical tests and 6 P-value combination strategies) and performs Cohen's d effect size systematically for users, moreover, it provides a web-based interface and can be quite conveniently operated by users, even those with less profound computational background. b) It supports three performance evaluation criteria (e.g. number of DEPs, correlation coefficient between P-values and effect sizes, Area under the ROC curve) for users to review the final statistical results, which may guide the method selection for DEPs detection.

The collection of blood plasma is minimally invasive, and the fluid is a rich source of proteins for biomarker studies in both humans and animals. Plasma protein analysis by mass spectrometry (MS) can be challenging, though modern data acquisition strategies, such as sequential window acquisition of all theoretical fragment ion spectra (SWATH), enable reproducible quantitation of hundreds of proteins in non-depleted plasma from humans and laboratory model animals. Although there is strong potential to enhance veterinary and translational research, SWATH-based plasma proteomics in non-laboratory animals is virtually non-existent. One limitation to date is the lack of comprehensively annotated genomes to aid protein identification. The current study established plasma peptide spectral repositories for sheep and cattle that enabled quantification of over 200 proteins in non-depleted plasma using SWATH approach. Moreover, bioinformatics pipeline was developed to leverage inter-species homologies to enhance the depth of baseline libraries and plasma protein quantification in bovids. Finally, the practical utility of using bovid libraries for SWATH data extraction in taxonomically related non-domestic ungulate species (giraffe) has been demonstrated. SIGNIFICANCE: Ability to quickly generate comprehensive spectral libraries is limiting the applicability of data-independent acquisition, such as SWATH, to study proteomes of non-laboratory animals. We describe an approach to obtain relatively shallow foundational plasma repositories from domestic ruminants and employ homology searches to increase the depth of data, which we subsequently extend to unsequenced ungulates using SWATH method. When applied to cross-species proteomics, the number of proteins quantified by our approach far exceeds what is traditionally used in plasma protein tests.

A novel network-based approach for predicting missing proteins (MPs) is proposed here. This approach, PROTREC (short for PROtein RECovery), dominates existing network-based methods - such as Functional Class Scoring (FCS), Hypergeometric Enrichment (HE), and Gene Set Enrichment Analysis (GSEA) - across a variety of proteomics datasets derived from different proteomics data acquisition paradigms: Higher PROTREC scores are much more closely correlated with higher recovery rates of MPs across sample replicates. The PROTREC score, unlike methods reporting p-values, can be directly interpreted as the probability that an unreported protein in a proteomic screen is actually present in the sample being screened. SIGNIFICANCE: Mass spectrometry (MS) has developed rapidly in recent years; however, an obvious proportion of proteins is still undetected, leading to missing protein problems. A few existing protein recovery methods are based on biological networks, but the performance is not satisfactory. We propose a new protein recovery method, PROTREC, a Bayesian-inspired approach based on biological networks, which shows exceptional performance across multiple validation strategies. It does not rely on peptide information, so it avoids the ambiguity issue that most protein assembly methods face.

Viral infections facilitate bacterial trafficking to the lower respiratory tract resulting in bacterial-viral co-infections. Bacterial dissemination to the lower respiratory tract is enhanced by influenza A virus induced epithelial cell damage and dysregulation of immune responses. Epithelial cells act as a line of defense and detect pathogens by a high variety of pattern recognition receptors. The post-translational modification ubiquitin is involved in almost every cellular process. Moreover, ubiquitination contributes to the regulation of host immune responses, influenza A virus uncoating and transport within host cells. We applied proteomics with a special focus on ubiquitination to assess the impact of single bacterial and viral as well as bacterial-viral co-infections on bronchial epithelial cells. We used Tandem Ubiquitin Binding Entities to enrich polyubiquitinated proteins and assess changes in the ubiquitinome. Infecting 16HBE cells with Streptococcus pyogenes led to an increased abundance of proteins related to mitochondrial translation and energy metabolism in proteome and ubiquitinome. In contrast, influenza A virus infection mainly altered the ubiquitinome. Co-infections had no additional impact on protein abundances or affected pathways. Changes in protein abundance and enriched pathways were assigned to imprints of both infecting pathogens. SIGNIFICANCE: Viral and bacterial co-infections of the lower respiratory tract are a burden for health systems worldwide. Therefore, it is necessary to elucidate the complex interplay between the host and the infecting pathogens. Thus, we analyzed the proteome and the ubiquitinome of co-infected bronchial epithelial cells to elaborate a potential synergism of the two infecting organisms. The results presented in this work can be used as a starting point for further analyses.

Candida albicans is the most common human fungal pathogen in immunocompromised individuals. With the emergence of clinical fungal resistance, there is an urgent need to develop novel antifungal agents. AMP-17, a novel antimicrobial peptide from Musca domestica, has an antifungal effect against C. albicans, but its mechanism of antifungal action remains unclear. In the current study, we performed a proteomics analysis in C. albicans using TMT technique under the treatment of AMP-17. A total of 3931 proteins were identified, of which 3600 included quantitative information. With a 1.5-fold change threshold and a t-test p-value < 0.05 as standard, 423 differentially expressed proteins (DEPs) were up-regulated and 180 DEPs were down-regulated in the AMP-17/control. Notably, GO enrichment revealed that DEPs associated with the cell wall, RNA and oxidative stress were significantly up-regulated, while DEPs involved in ergosterol metabolism and membrane were significantly down-regulated in the AMP-17/control. KEGG pathway enrichment revealed that DEPs involved seven significant metabolic pathways, mainly involved oxidative phosphorylation, RNA degradation, propanoate metabolism and fatty acid metabolism. These results show that AMP-17 induces a complex organism response in C. albicans, indicating that AMP-17 may inhibit growth by affecting multiple targets in C. albicans cells. SIGNIFICANCE: Antimicrobial peptides (AMPs) are an important part of the innate immune system of organisms and having broad range of activity against fungi, bacteria and viruses. These AMPs are considered as probable candidate for forthcoming drugs, due to their broad range of activity, lesser toxicity and decreased resistance development by target cells. AMP-17, a novel antimicrobial peptide from M. domestica, has significant antifungal activity against C. albicans. It has been confirmed that AMP-17 can play an antifungal effect by destroying the cell wall and cell membrane of C. albicans in previous studies, but its mechanism of action at the protein level is currently unclear. In the current study, using the TMT-based quantitative proteomics method, 603 differentially expressed proteins were identified in the cells of C. albicans treated with AMP-17 for 12 h, and these DEPs were closely related to cell wall, cell membrane, RNA degradation and oxidative stress. The results provide new insights into the potential mechanism of action of AMP- 17 against C. albicans. Meanwhile, it provides certain technical support and theoretical basis for the research and development of novel peptide drugs.

Listeria monocytogenes presents a dimorphism associated to the SecA2 activity with cells having a normal rod shape or a dysmorphic elongated filamentous form. Besides variation of the cell and colony morphotype, this cell differentiation has profound ecophysiological and physiopathological implications with collateral effects on virulence and pathogenicity, biotope colonisation, bacterial adhesion and biofilm formation. This suggests the SecA2-only protein export could influence the listerial cell surface, which was investigated first by characterising its properties in L. monocytogenes wt and ΔsecA2. The degree of hydrophilicity and Lewis acid-base properties appeared significantly affected upon SecA2 inactivation. As modification of electrostatic properties would owe to modification in the composition of cell-surface proteins, the proteosurfaceome was further investigated by shotgun label-free proteomic analysis with a comparative relative quantitative approach. Following secretomic analysis, the protein secretion routes of the identified proteins were mapped considering the cognate transport and post-translocational maturation systems, as well as protein categories and subcellular localisation. Differential protein abundance profiles coupled to network analysis revealed the SecA2 dependence of 48 proteins, including some related to cell envelope biogenesis, translation and protein export, which could account for modifications of adhesion and surface properties of L. monocytogenes upon SecA2 inactivation. This investigation unravelled the profound influence of SecA2 activity on the cell surface properties and proteosurfaceome of L. monocytogenes, which provides advanced insights about its ecophysiopathology. SIGNIFICANCE: L. monocytogenes is a foodborne zoonotic pathogen and etiological agent of human listeriosis. This species presents a cellular dimorphism associated to the SecA2 activity that has profound physiopathological and ecophysiological implications with collateral effects on bacterial virulence and colonisation. To explore the influence of the SecA2-only protein export on the listerial cell, the surface properties of L. monocytogenes expressing or depleted of SecA2 was characterised by microelectrophoresis, microbial affinity to solvents and contact angles analyses. As modifications of hydrophilicity and Lewis acid-base electrostatic properties would owe to modification in the composition of cell-surface proteins, the proteinaceous subset of the surfaceome, i.e. the proteosurfaceome, was investigated further by shotgun label-free proteomic analysis. This subproteome appeared quite impacted upon SecA2 inactivation with the identification of proteins accounting for modifications in the cell surface properties. The profound influence of SecA2 activity on the cell surface of L. monocytogenes was unravelled, which provides advanced insights about its ecophysiopathology.

Some carboxydotrophs like Rhodospirillum rubrum are able to grow with CO as their sole source of energy using a Carbone monoxide dehydrogenase (CODH) and an Energy conserving hydrogenase (ECH) to perform anaerobically the so called water-gas shift reaction (WGSR) (CO + H2O → CO2 + H2). Several studies have focused at the biochemical and biophysical level on this enzymatic system and a few OMICS studies on CO metabolism. Knowing that CO is toxic in particular due to its binding to heme iron atoms, and is even considered as a potential antibacterial agent, we decided to use a proteomic approach in order to analyze R. rubrum adaptation in term of metabolism and management of the toxic effect. In particular, this study allowed highlighting a set of proteins likely implicated in ECH maturation, and important perturbations in term of cofactor biosynthesis, especially metallic cofactors. This shows that even this CO tolerant microorganism cannot avoid completely CO toxic effects associated with its interaction with metallic ions. SIGNIFICANCE: This proteomic study highlights the fact that even in a microorganism able to handle carbon monoxide and in some way detoxifying it via the intrinsic action of the carbon monoxide dehydrogenase (CODH), CO has important effects on metal homeostasis, metal cofactors and metalloproteins. These effects are direct or indirect via transcription regulation, and amplified by the high interdependency of cofactors biosynthesis.

Global analysis of protein phosphorylation by mass spectrometry proteomic techniques has emerged in the last decades as a powerful tool in biological and biomedical research. However, there are several factors that make the global study of the phosphoproteome more challenging than measuring non-modified proteins. The low stoichiometry of the phosphorylated species and the need to retrieve residue specific information require particular attention on sample preparation, data acquisition and processing to ensure reproducibility, qualitative and quantitative robustness and ample phosphoproteome coverage in phosphoproteomic workflows. Aiming to investigate the effect of different variables in the performance of proteome wide phosphoprotein analysis protocols, ProteoRed-ISCIII and EuPA launched the Proteomics Multicentric Experiment 11 (PME11). A reference sample consisting of a yeast protein extract spiked in with different amounts of a phosphomix standard (Sigma/Merck) was distributed to 31 laboratories around the globe. Thirty-six datasets from 23 laboratories were analyzed. Our results indicate the suitability of the PME11 reference sample to benchmark and optimize phosphoproteomics strategies, weighing the influence of different factors, as well as to rank intra and inter laboratory performance.

SAPHO syndrome is an inflammatory disease invading the skin and bones, whose diagnosis has been difficult due to its low incidence and diversified manifestation. We investigated the serum proteomic profile of SAPHO patients to identify key proteins associated with SAPHO syndrome, trying to find clinical biomarkers or functional molecules for this rare disease. Blood samples from 8 SAPHO patients and 8 healthy controls were detected and analyzed using data independent acquisition (DIA) method to identify differentially expressed proteins (DEPs) specific to SAPHO. A total of 57 differentially expressed proteins were identified (p < 0.05, fold change >1.2), in which 27 proteins were upregulated and 30 downregulated. DEPs may participate in GO terms such as "lipid particle" and "Notch signaling pathway", as well as KEGG pathways including "complement and coagulation cascades" and "mTOR signaling pathway". The overexpression of inhibitors of the complement system (CFH and C4BP), were verified in a larger cohort (16 SAPHO patients, 8 AS patients and 24 healthy controls) with ELISA, and the combined diagnostic ability of CFH and C4BP was predicted by ROC curve with an AUC of 0.91, which may be molecular candidates for further study on diagnosis and pathology of this rare disease. SIGNIFICANCE: Our research provided the first insight into plasma proteomic profile for SAPHO patients，offering potential biomarkers for disease diagnosis. We found that inhibitors of complement system such as CFH and C4BP were up-regulated in SAPHO syndrome, which may play important roles in the pathogenesis of SAPHO syndrome.

Dissolved oxygen is one of the determinants in the healthy farming of Pelteobagrus vachelli. This study, we conducted quantitative proteomics on the juvenile P. vachelli livers using iTRAQ. P. vachelli were treated by 3.75 ± 0.25 mg O2/L (hypoxia group) and 7.25 ± 0.25 mg O2/L (control group) for 90 days. The results revealed that under hypoxic conditions, P. vachelli grew slower than control group. Proteomic profiling enabled us to identify 2618 proteins, of which 176 were significantly differentially abundant proteins (DAPs). Verification of protein regulation based on qRT-PCR indicated that the proteomics data were reliable. The top 20 significantly DAPs (10 up-regulated, 10 down-regulated) were primarily involved in energy metabolism, apoptosis inhibition, and heavy metal detoxification. KEGG pathway enrichment analysis revealed significant enrichment of 'protein digestion and absorption', 'glycolysis/gluconeogenesis', and 'phagosome'. Combining the proteomics results of short-term hypoxia (treated with 0.70 ± 0.10 mg O2 /L for 4 h), we screened 36 common DAPs. The analysis of the 36 common DAPs indicated that P. vachelli responded to the hypoxia by regulating energy supply, inhibiting apoptosis, and disturbing defensive system. Our results lay a theoretical foundation for the cultivation of hypoxia-tolerant species and eco-breeding of P. vachelli. SIGNIFICANCE OF THE STUDY: The hypoxia tolerance of Pelteobagrus vachelli is poor, which will seriously lead to its death in high-density culture. This study analysed the liver proteome of P. vachelli under long-term hypoxia stress (treated for 90 days at 3.75 ± 0.25 mg O2/L), and then combined the proteome results of short-term hypoxia stress (treated for 4 h at 0.70 ± 0.10 mg O2/L). The results showed P. vachelli responded to the hypoxia by regulating energy supply, inhibiting apoptosis and disturbing defensive system. The study contributes to the breeding of new hypoxia-tolerant species of P. vachelli and lays the theoretical foundation for eco-breeding.

Extracellular vesicles (EVs) are involved in a wide range of pathological processes and recognized as potential and novel biomarkers for oral squamous cell carcinoma (OSCC). Here, we describe the plasma EV proteome of rats with 4-nitroquinoline-1-oxide (4NQO)-induced OSCC or moderate dysplasia (MD), which can progress to OSCC, by tandem mass tag (TMT)-labeled mass spectrometry. The proteomic profiles suggest the differential expression of various proteins in MD and OSCC, some well-recognized pathological changes (e.g., translation, ATP metabolism, and mesenchymal transition), and some novel pathological changes (e.g., podosome, focal adhesion, and S100 binding). We re-examined the presence of traditional exosomal markers and the reported novel pan-EV markers. In summary, these results suggest potential EV biomarkers and underlying pathological changes in early OSCC as well as the presence of oral-derived EVs in plasma and the need for pan-EV markers. SIGNIFICANCE: This research suggests potential EV biomarkers and underlying pathological changes in early OSCC as well as the presence of oral-derived EVs in plasma and the need for pan-EV markers.

The functions of proteins at the onset of puberty in goats remain largely unexplored. To identify the proteins regulating puberty in goats, we analysed protein abundance and pathways in the hypothalamus of female goats. We applied tandem mass tag (TMT) labelling, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and parallel reaction monitoring (PRM) to examine hypothalamus of pubertal (cases; n = 3) and prepubertal (controls; n = 3) goats. We identified 5119 proteins, including 69 differentially abundant proteins (DAPs), of which 35 were upregulated and 34 were downregulated. Fourteen DAPs were randomly selected to verify these results using PRM, and the results were consistent with the TMT quantitative results. DAPs were enriched in MAPK signalling pathway, Ras signalling pathway, Autophagy-animal, Endocytosis, and PI3K/Akt/mTOR signalling pathway categories. These pathways are related to embryogenesis, cell proliferation, cell differentiation, and promoting the release of gonadotropin-releasing hormone (GnRH) in the hypothalamus. In particular, PDGFRβ and MAP3K7 occupied important locations in the protein-protein interaction network. The results demonstrate that DAPs and their related signalling pathways are crucial in regulating puberty in goats. However, further research is needed to explore the functions of DAPs and their pathways to provide new insights into the mechanism of puberty onset. SIGNIFICANCE: In domestic animals, reaching the age of puberty is an event that contributes significantly to lifetime reproductive potential. And the hypothalamus functions directly in the complex systemic changes that control puberty. Our study was the first TMT proteomics analysis on hypothalamus tissues of pubertal goats, which revealed the changes of protein and pathways that are related to the onset of puberty. We identified 69 DAPs, which were enriched in the MAPK signaling pathway, the Ras signaling pathway, and the IGF-1/PI3K/Akt/mTOR pathway, suggesting that these processes were probably involved in the onset of puberty.

During the last decade, the evidences on the relationship between neurodevelopmental disorders and the microbial communities of the intestinal tract have considerably grown. Particularly, the role of gut microbiota (GM) ecology and predicted functions in Autism Spectrum Disorders (ASD) has been especially investigated by 16S rRNA targeted and shotgun metagenomics, trying to assess disease signature and their correlation with cognitive impairment or gastrointestinal (GI) manifestations of the disease. Herein we present a metaproteomic approach to point out the microbial gene expression profiles, their functional annotations, and the taxonomic distribution of gut microbial communities in ASD children. We pursued a LC-MS/MS based investigation, to compare the GM profiles of patients with those of their respective relatives and aged-matched controls, providing a quantitative evaluation of bacterial metaproteins by SWATH analysis. All data were managed by a multiple step bioinformatic pipeline, including network analysis. In particular, comparing ASD subjects with CTRLs, up-regulation was found for some metaproteins associated with Clostridia and with carbohydrate metabolism (glyceraldehyde-3-phosphate and glutamate dehydrogenases), while down-regulation was observed for others associated with Bacteroidia (SusC and SusD family together with the TonB dependent receptor). Moreover, network analysis highlighted specific microbial correlations among ASD subgroups characterized by different functioning levels and GI symptoms. SIGNIFICANCE: To the best of our knowledge, this study represents the first metaproteomic investigation on the gut microbiota of ASD children compared with relatives and age-matched CTRLs. Remarkably, the applied SWATH methodology allowed the attribution of differentially regulated functions to specific microbial taxa, offering a novel and complementary point of view with respect to previous studies.

Preparation of stable isotope-labeled internal standard peptides is crucial for mass spectrometry (MS)-based targeted proteomics. Herein, we developed versatile and multiplexed absolute protein quantification method using MS. A previously developed method based on the cell-free peptide synthesis system, termed MS-based quantification by isotope-labeled cell-free products (MS-QBiC), was improved for multiple peptide synthesis in one-pot reaction. We pluralized the quantification tags used for the quantification of synthesized peptides and thus, made it possible to use cell-free synthesized isotope-labeled peptides as mixtures for the absolute quantification. The improved multiplexed MS-QBiC method was proved to be applied to clarify ribosomal proteins stoichiometry in the ribosomal subunit, one of the largest cellular complexes. The study demonstrates that the developed method enables the preparation of several dozens and even several hundreds of internal standard peptides within a few days for quantification of multiple proteins with only a single-run of MS analysis. SIGNIFICANCE: The developed method can be applied for the preparation of internal standard peptides without limiting the number of peptides to be synthesized, which may result in more practical screening of quantitatively reliable peptides, one of the fundamental steps in the reliable absolute quantification using MS. Furthermore, the method is highly versatile for proteome analysis of any organisms or species without any cDNA or SIL peptide libraries. The quantification can be finished in a few days including design and preparation of appropriate SIL peptides using small-scale batch cell-free reactions, which has a potential to be a part of the standard methodology in a field of quantitative proteomics.

The sea cucumber Apostichopus japonicus is an important aquaculture species in China because of its high nutritional and medicinal values. Gender, as a factor affecting the physiology of organisms, is always considered when improving the breeding efficiency of economically important animals. In the present study, protein expression profiles of the gonads and tube feet of male and female A. japonicus were investigated using a comparative proteomics approach. A set of 7499 proteins were identified, which covered a broad range of functions based on function annotations. A significant difference in protein expression profiles was observed between the gonads and tube feet of A. japonicus; gonads showed more apparent gender differences than tube feet. Moreover, the findings revealed that male A. japonicus had more specific functions and most of these functions were associated with energy consumption. Further analyses suggested that the regulation of ERK activity and the capacity of tyrosine production and virus immunity might be more powerful in male and female A. japonicus, respectively. Some candidate proteins were also recognized as potential targets for gender identification of A. japonicus. Overall, our study provides new insights into the understanding of molecular mechanisms underlying gender-based physiological differences in A. japonicus. SIGNIFICANCE: The current study aimed to reveal gender differences in the physiological characteristics of gonads and tube feet of the sea cucumber A. japonicus. To the best of our knowledge, this is the first proteomics study to analyze the differences in the protein expression profiles of external organs between male and female A. japonicus. The analysis revealed gender differences in the protein expression profiles of both gonads and tube feet of A. japonicus, and the gender differences in gonads were quite apparent. Moreover, according to the recognition of differentially expressed proteins and the enrichment analyses based on Kyoto Encyclopedia of Genes and Genomes, a draft view of how the physiological functions of A. japonicus were affected by gender was obtained. Male A. japonicus could have more specific functions related to energy consumption than females. The regulation of ERK activity and virus immunity might be more robust in male and female A. japonicus, respectively. Some candidate proteins were also recognized as potential targets for gender identification of A. japonicus. The findings presented here will improve the understanding of researchers about the molecular mechanisms underlying gender-based differences in A. japonicus and contribute to the meticulous breeding of A. japonicus.

Glycosylation of ApoC-III modulates its function in TG metabolism, with some variants being associated with a more atherogenic lipid profile. These associations have been studied in whole plasma but rarely in individual lipoprotein fractions. In this study, we aimed to measure the relative content of ApoC-III glycoforms in each lipoprotein fraction as a potential biomarker for TG metabolism and cardiovascular risk. Lipoprotein fractions were separated by differential ultracentrifugation of plasma samples from healthy subjects. Relative concentrations of seven ApoC-III variants were measured by MSIA. ApoC-III1, ApoC-III0b and ApoC-III2 were the most abundant glycoforms. There was high interindividual variability in the distribution of glycoforms across the study population but a uniform proportion in all lipoprotein fractions of each given subject. Two ApoC-III variants, ApoC-III0b and ApoC-III1d, negatively correlated with plasma and VLDL triglycerides irrespectively of VLDL size and were associated with increased LDL size when transported in LDL particles. ApoC-III0b also showed a negative correlation with lipoprotein-insulin resistance score. We have been able to measure seven ApoC-III glycoforms in each lipoprotein fraction, setting the basis for future studies exploring their role on cardiovascular risk. Some glycoforms suggest a less proatherogenic role on TG and lipoprotein metabolism. SIGNIFICANCE: Apo CIII has an important role on plasma TG metabolism through different mechanisms and it is also involved in type 1 and type 2 Diabetes Mellitus. Different glycosylated forms of Apo CIII exist and they show different roles. For this reason, this protein has gained interest in the las years and the relationship between ApoC-III glycoforms and lipids, lipoproteins and metabolic disorders has been increasingly studied in the last years. Apo CIII glycoforms have been previously analysed in plasma, and the function of the main four glycoforms has been assessed in a variety of cohorts; but in the present study, ApoC-III glycoforms are measured in each lipoprotein fraction, which may be of clinical interest.

Tandem mass spectrometry has been the principal method in shotgun proteomics for peptide and protein identification. However, incorrect identifications reported by proteome search engines are still unknown, and further validation methods are needed. We have proposed a validation method pValid before, but its scope of application is limited because two features used in pValid are related to open database search and sub-optimal peptide candidates for tandem mass spectra, and the performance on complex datasets still has room for improvement. In this study, we developed a more comprehensive validation method, pValid 2, to break these limitations by removing the two features and bringing in a new feature related to the retention time predicted by a deep learning-based method pPredRT. pValid 2 yielded an average false positive rate of 0.03% and an average false negative rate of 1.37% on three testing datasets, better than those of pValid, and flagged 8.47% to 11.31% more incorrect identifications than pValid on two complex datasets. Moreover, pValid 2 flagged almost all decoy identifications in validating the open-search datasets. In addition, the function of validating identifications given by MaxQuant and MS-GF+ was implemented in pValid 2, and the validation results showed that pValid 2 performed dramatically better than three metabolic labeling validation methods. Further considering its cost-effectiveness as a pure computational approach, pValid 2 has the potential to be a widely used validation tool for peptide identifications of any proteome search engines in shotgun proteomics. SIGNIFICANCE: Identification results given by shotgun proteomics are vital to life science research. The correctness of identifications deeply affects the precision of the subsequent studies about protein structures and functions, protein-protein interactions, pathogenic mechanism, and targeted drugs. Thus, validating the correctness of identifications is crucial and urgent. In 2019, we developed an identification credibility validation method named pValid, whose false positive rate (FPR) is 0.03% and false negative rate (FNR) is 1.79%, comparable to those of the gold standard, i.e., the Synthetic-peptide validation method. However, pValid can only be used for validating the results from pFind, and its validation performance on a few complex datasets still has room for improvement. So, in this submission, we proposed pValid 2, a more comprehensive computational validation method that can validate identifications from any proteome search engines with increased discriminating power.

Vibrio cholerae can cause pandemic cholera in humans. The bacterium resides in aquatic environments worldwide. Identification of risk factors of V. cholerae in aquatic products is imperative for assuming food safety. In this study, we determined virulence-associated genes, cross-resistance between antibiotics and heavy metals, and genome fingerprinting profiles of non O1/O139 V. cholerae isolates (n = 20) recovered from 16 species of consumable aquatic animals. Secretomes and proteomes of V. cholerae with distinct genotypes and phenotypes were obtained by using two-dimensional gel electrophoresis (2D-GE) and/or liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques. Comparative secretomic analysis revealed 4 common and 45 differential extracellular proteins among 20 V. cholerae strains, including 13 virulence- and 8 resistance-associated proteins. A total of 21,972 intracellular proteins were identified, and comparative proteomic analysis revealed 215 common and 913 differential intracellular proteins, including 22 virulence- and 8 resistance-associated proteins. Additionally, different secretomes and proteomes were observed between V. cholerae isolates of fish and shellfish origins. A number of novel proteins with unknown function and strain-specific proteins were also discovered in the V. cholerae isolates. SIGNIFICANCE: V. cholerae can cause pandemic cholera in humans. The bacterium is distributed in aquatic environments worldwide. Identification of risk factors of V. cholerae in aquatic products is imperative for assuming food safety. Non-O1/O139 V. cholerae has been reported to cause sporadic cholera-like diarrhea and bacteremia diseases, which indicates virulence factors rather than the major cholera toxin (CT) exist. This study for the first time investigated proteomes and secretomes of non-O1/O139 V. cholerae originating from aquatic animals. This resulted in the identification of a number of virulence and coresistance-related factors, as well as novel proteins and strain-specific proteins in V. cholerae isolates recovered from 16 species of consumable aquatic animals. These results fill gaps for better understanding of pathogenesis and resistance of V. cholerae, and also support the increasing need for novel diagnosis and vaccine targets against the leading waterborne pathogen worldwide.

Reactive oxygen species (ROS) play a crucial role as signaling molecules in plant responses to pathogen infection. It is highly reactive with cellular components such as DNA, lipids and proteins, thereby leading to serious oxidative damages. Cysteine residues are sensitive targets of ROS in a post-translational modification known as sulfenylation. However, during plant-pathogen interaction, it is still unclear which specific proteins can be oxidized by ROS and undergo sulfenic modification to regulate the interaction process. Here, we observed a biphasic production of ROS in Nicotiana benthamiana after inoculation with Botrytis cinerea. RT-qPCR results showed that the biphasic increase in ROS production was closely related to the expression of NbRbohA, NbRbohB and NbRbohC. Furthermore, a ROS-dependent sulfenome analysis was performed and finally 183 differentially sulfenylated proteins were identified. Their post-translational sulfenylation modification in response to B. cinerea infection was further confirmed by western blot and mass spectrometry analysis. Virus-induced gene silencing of those genes encoding sulfenylated proteins resulted in reduced resistance to B. cinerea. Taken together, our data demonstrate that B. cinerea infection induces ROS burst in N. benthamiana, which triggers protein sulfenylation to ensure the transduction of ROS signals and further function in plant-pathogen interaction. SIGNIFICANCE: Reactive oxygen species (ROS) induced by Botrytis cinerea infection trigger changes in cellular redox status through protein sulfenylation to be involved in plant-pathogen interaction.

A striking feature of skin organization is that the extracellular matrix (ECM) occupies a larger volume than the cells. Skin ECM also directly contributes to aging and most cutaneous diseases. In recent years, specific ECM enrichment protocols combined with in silico approaches allowed the proteomic description of the matrisome of various organs and tumor samples. Nevertheless, the skin matrisome remains under-studied and protocols allowing the efficient recovery of the diverse ECM found in skin are still to be described. Here, we compared four protocols allowing the enrichment of ECM proteins from adult mouse back skin and found that all protocols led to a significant enrichment (up to 65%) of matrisome proteins when compared to total skin lysates. The protocols based on decellularization and solubility profiling gave the best results in terms of numbers of proteins identified and confirmed that skin matrisome proteins exhibit very diverse solubility and abundance profiles. We also report the first description of the skin matrisome of healthy adult mice that includes 236 proteins comprising 95 core matrisome proteins and 141 associated matrisome proteins. These results provide a reliable basis for future characterizations of skin ECM proteins and their dysregulations in disease-specific contexts. SIGNIFICANCE: Extracellular matrix proteins are key players in skin physiopathology and have been involved in several diseases such as genetic disorders, wound healing defects, scleroderma and skin carcinoma. However, skin ECM proteins are numerous, diverse and challenging to analyze by mass spectrometry due to the multiplicity of their post-translational modifications and to the heterogeneity of their solubility profiles. Here, we performed the thorough evaluation of four ECM enrichment protocols compatible with the proteomic analysis of mouse back skin and provide the first description of the adult mouse skin matrisome in homeostasis conditions. Our work will greatly facilitate the future characterization of skin ECM alterations in preclinical mouse models and will inspire new optimizations to analyze the skin matrisome of other species and of human clinical samples.

The gram-negative bacterium Vibrio (Listonella) anguillarum (VA) is the causative agent of vibriosis, a terminal hemorrhagic septicemia affecting the aquacultural industry across the globe. In the current study we used label-free quantitative proteomics to investigate how VA adapts to conditions that mimic defined aspects of vibriosis-related stress such as exposure to oxidative stress (H2O2), exposure to humoral factors of innate immunity through incubation with Atlantic salmon serum, and iron deprivation upon supplementation of 2,2'-dipyridyl (DIP) to the growth medium. We also investigated how regulation of virulence factors may be governed by the VA growth phase and availability of nutrients. All experimental conditions explored revealed stress-specific proteomic adaption of VA and only nine proteins were found to be commonly regulated in all conditions. A general observation made for all stress-related conditions was regulation of multiple metabolic pathways. Notably, iron deprivation and exposure to Atlantic salmon serum evoked upregulation of iron acquisition mechanisms. The findings made in the present study represent a source of potential virulence determinants that can be of use in the search for means to understand vibriosis. SIGNIFICANCE: Vibriosis in fish and shellfish caused by V. anguillarum (VA) is responsible for large economic losses in the aquaculture sector across the globe. However, not much is known about the defense mechanism of this pathogen to percept and adapt to the imposed stresses during infection. Analyzing the response of VA to multiple host-related physiochemical stresses, the quantitative proteomic analysis of the present study indicates modulation of several virulence determinants and key defense networks of this pathogen. Our findings provide a theoretical basis to enhance our understanding of VA pathogenesis and can be employed to improve current intervention strategies to control vibriosis in aquaculture.

The honeybee species A. mellifera and A. cerana have evolved substantial differences in olfactory-driven behaviors and in peripheral olfactory systems. Knowledge of the central nervous system regulating these olfaction differences is limited, however. We compared the phosphoproteome of the antennal lobes (ALs, the primary olfactory neuropil) of A. mellifera and A. cerana, and identified a total of 2812 phosphopeptides carrying 2971 phosphosites from 1265 phosphoproteins. Of these, 76% of the phosphoproteins were shared by both species, which were mainly presynapse and cytoskeleton components, and were involved in signal transduction and neurotransmitter secretion. This finding indicates the fundamental role of protein phosphorylation in regulating signal transduction in the ALs. The mTOR signaling pathway, the phagosome pathway, and the autophagy pathway, which are important in protein metabolism, were enriched, suggesting glomeruli plasticity and olfactory processing are intensively modulated by phosphorylation via these pathways. Compared with A. mellifera, 107 phosphoproteins associated with protein metabolism and transport were uniquely expressed in A. cerana, indicating the protein synthesis-dependent synaptic plasticity is enhanced in A. cerana to facilitate the processing of more complex floral odor clues in mountain foraging areas. This finding is further supported by the significantly upregulated key phosphoproteins of the mTOR signaling pathway in A. cerana ALs. These results provide insights into the phosphoproteomic basis of neuroplasticity that is coupled with the divergent evolution of bees in different environments. SIGNIFICANCE: To adapt to their own ecological niche, the two major honeybee species, A. mellifera and A. cerana, have developed significant difference in olfactory-driven behaviors, but our understanding of the underlying regulation of the central nervous system is still limitate. Here we performed the first comprehensive phosphoproteomic comparison of antennal lobes (Als) between A. mellifera and A. cerena. A large proportion of the identified phosphosites and phosphoproteins were shared between the two species to serve as a core network in the regulation of signal transduction and glomeruli plasticity of ALs. However, compared with A. mellifera, 107 phosphoproteins associated with protein metabolism and transport were uniquely identified in A. cerana ALs, and also several key phosphoproteins in mTOR signaling pathway were found upregulated in A. cerana. These findings indicate protein phosphorylation enhanced the protein synthesis-dependent synaptic plasticity in A. cerana to facilitate the processing of more complex floral odor clues in mountain foraging areas. Our data provide a valuable insight into phosphoproteome-driven cerebral regulation of honeybee olfactory behaviors, which is potentially useful for further neurobiological investigation in both honeybees and other insects.

Phenol and ammonia in wastewater pose a serious threat to ecosystems and human health. However, the currently limited studies on single bacterium simultaneously removing phenol and nitrogen pollution have not fully elucidated the relevant metabolic mechanisms. The differences in proteomic profile after supplementing with phenol and ammonia for 6 and 24 h, respectively, were evaluated to explore the metabolic characteristics and adaptive mechanism of Cupriavidus oxalaticus T2 during the simultaneous removal process of phenol and nitrogen. Results revealed that a new potential phenol para-degradation pathway appeared in T2. Phenol induced changes in nitrogen metabolism, resulting in increased denitrification and decreased synthesis of glutamate from ammonia at 6 h. In addition, phenol exposure enhanced the expression of cytochrome oxidases with high oxygen affinity and increased ATP synthesis. The increase in chemotaxis and flagellar assembly was conducive to the uptake and utilization of phenol. The synthesis of lipoic acid and biotin was also promoted to resist phenol toxicity. Moreover, phenol triggered cellular stress response, thereby leading to the upregulation of anti-stress proteins, such as universal stress protein, iron‑sulfur cluster protein, and chaperones. This study contributes to revealing the metabolic characteristics and adaptive mechanism of T2 during simultaneous nitrogen and phenol removal. SIGNIFICANCE: Phenol and ammonia often co-exist in wastewater, causing serious environmental problems. The information on the metabolic mechanism of simultaneously removing these two pollutants by bacteria is insufficient at present. Moreover, phenol is toxic to microbial and causes cells damage. Therefore, exploring the response mechanism of bacteria to phenol stress is conducive to understand their tolerance mechanism to aromatic compounds. In this study, the metabolic characteristics and adaptive mechanism of C. oxalaticus T2 during the simultaneous removal of phenol and nitrogen process were evaluated by comparing the proteome profiles at different stages. The results revealed the degradation pathways of phenol and nitrogen by strain T2. A variety of phenol response mechanisms were determined, including enhanced energy production, improved cell motility, increased the synthesis of lipoic acid and biotin, and combined action of multiple anti-stress proteins. This study is potentially useful to future phenol and nitrogen co-pollution bioremediation strategies and provides insight into the phenolic compound resistance mechanism in bacteria.

Glutens are potential proteins with multifunctional therapeutic effects. Their covalence network structures with and without protease inhibitors are expected to enhance or to serve further properties and further technological points such as increased bioactive surfaces, gelatinization, gelation and pasting properties. The depletion of the allergic peptide sequences of gluten proteins comprising sometimes protease inhibitors are valid via the enzymatic ingestion using proteolytic enzymes that might enhance these functional and technological processes by producing active peptides having osmoregulation and regular glass transitions, surface activity for coating and encapsulation properties. In addition to further therapeutic functions such as immunoregulatory, antithrombin and opioidal activities, particularly in eradicating most of the free radicals, suppressing diabetes Mellitus II complications and inhibiting angiotensin converting enzyme cardiovascular growth diseases.