Bull fertility is an important trait in breeding as the semen of one bull can, potentially, be used to perform thousands of inseminations. The high number of inseminations needed to obtain reliable measures from Non-Return Rates to oestrus creates difficulties in assessing fertility accurately. Improving molecular knowledge of seminal properties may provide ways to facilitate selection of bulls with good semen quality. In this study, liquid chromatography mass spectrometry (LC-MS/MS) was used to analyze the protein content from the seminal plasma of 20 bulls with Non-Return Rates between 35 and 60%, sampled across three seasons. Overall, 1343 proteins were identified and proteins with consistent correlation to fertility across multiple seasons found. From these, nine protein groups had a significant Pearson correlation (p < 0.1) with fertility in all three seasons and 34 protein groups had a similar correlation in at least two seasons. Among notable proteins showing a high and consistent correlation across seasons were Osteopontin, a lipase (LIPA) and N-acetylglucosamine-1phosphotransferase subunit gamma. Three proteins were combined in a multiple linear regression to predict fertility (r = 0.81). These sets of proteins represent potential markers, which could be used by the breeding industry to phenotype bull fertility. SIGNIFICANCE: The ability of bull spermatozoa to fertilize oocytes is crucial for breeding efficiency. However, the reliability of this trait from field measures is relatively low and the prediction of fertility given by conventional methods to evaluate sperm quality is currently not very accurate. In this work, we identify sets of proteins in bull seminal plasma from repeated samples collected at different times of the year that correlate to fertility in a consistent way. We combined these individual proteins to build a molecular signature predictive of fertility. This study provides an overview of proteins linked to fertility in seminal plasma, thereby increasing knowledge of the bull seminal plasma proteome. Protein signatures from the latter, potentially related to fertility, may be of use to predict fertility for individual bulls.

Plasmodium blood stages, responsible for human to vector transmission, termed gametocytes, are the precursor cells that develop into gametes in the mosquito. Male gametogenesis works as a bottleneck for the parasite life cycle, where, during a peculiar and rapid exflagellation, a male gametocyte produces 8 intracellular axonemes that generate by budding 8 motile gametes. Understanding the molecular mechanisms of gametogenesis is key to design strategies for controlling malaria transmission. In the rodent P. berghei, the microtubule-based motor kinesin-8B (PbKIN8B) is essential for flagellum assembly during male gametogenesis and its gene disruption impacts on completion of the parasitic life cycle. In efforts to improve our knowledge about male gametogenesis, we performed an iTRAQ-based quantitative proteomic comparison of P. berghei mutants with disrupted kinesin-8B gene (ΔPbkin8B) and wild type parasites. During the 15 min of gametogenesis, ΔPbkin8B parasites exhibited important motor protein dysregulation that suggests an essential role of PbKIN8B for the correct interaction or integration of axonemal proteins within the growing axoneme. The energy metabolism of ΔPbkin8B mutants was further affected, as well as the response to stress proteins, protein synthesis, as well as chromatin organisation and DNA processes, although endomitoses seemed to occur. SIGNIFICANCE: Malaria continues to be a global scourge, mainly in subtropical and tropical areas. The disease is caused by parasites from the Plasmodium genus. Plasmodium life cycle alternates between female Anopheles mosquitoes and vertebrate hosts through bites. Gametocytes are the parasite blood forms responsible for transmission from vertebrates to vectors. Inside the mosquito midgut, after stimulation, male and female gametocytes transform into gametes resulting in fertilization. During male gametogenesis, one gametocyte generates eight intracytoplasmic axonemes that generate, by budding, flagellated motile gametes involving a process termed exflagellation. Sexual development has a central role in ensuring malaria transmission. However, molecular data on male gametogenesis and particularly on intracytoplasmic axoneme assembly are still lacking. Since rodent malaria parasites permit the combination of in vivo and in vitro experiments and reverse genetic studies, our group investigated the molecular events in rodent P. berghei gametogenesis. The P. berghei motor ATPase kinesin-8B is proposed as an important component for male gametogenesis. We generated Pbkin8B gene-disrupted gametocytes (ΔPbkin8B) that were morphologically similar to the wild- type (WT) parasites. However, in mutants, male gametogenesis is impaired, male gametocytes are disabled in their ability to assemble axonemes and to exflagellate to release gametes, reducing fertilization drastically. Using a comparative quantitative proteomic analysis, we associated the nonfunctional axoneme of the mutants with the abnormal differential expression of proteins essential to axoneme organisation and stability. We also observed a differential dysregulation of proteins involved in protein biosynthesis and degradation, chromatin organisation and DNA processes in ΔPbkin8B parasites, although DNA condensation, mitotic spindle formation and endomitoses seem to occur. This is the first functional proteomic study of a kinesin gene-disrupted Plasmodium parasite providing new insights into Plasmodium male gametogenesis.

Lampreys, one of the most basal jawless vertebrates, are an excellent animal model for investigating vertebrate evolution, embryonic development, and the origin of adaptive immunity. Gut-derived Shewanella strain was isolated and then proved to induce adaptive immunity response in lampreys. Using Shewanella as the antigen, the effect of gut-derived Shewanella on lamprey leukocyte proteome was investigated via label-free liquid chromatography-tandem mass spectrometry for quantitative proteomics analysis. Twenty-five differentially expressed proteins in lamprey leukocytes were identified with significant differences. The differentially expressed proteins were associated with several biological processes. Among these proteins, the signal transducer and activator of transcription 3 (STAT3) was significantly upregulated in leukocytes after Shewanella immunization, indicating that lamprey STAT3 (L-STAT3) was involved in Shewanella-lamprey interactions. Expression pattern analysis revealed that L-STAT3 was mainly distributed in the cytoplasm and upregulated in other tissues after Shewanella immunization. Moreover, L-STAT3 overexpression could promote HEK-293 T and HeLa cell proliferation. However, the functions of L-STAT3 in the adaptive immune response of lamprey induced by gut-derived Shewanella remain to be explored. Overall, the identification of leukocyte proteins involved in Shewanella-lamprey interactions provides important information for understanding the variable lymphocyte receptor-based adaptive immune signal pathways in lampreys. SIGNIFICANCE: Lampreys are considered to be an excellent animal model for studying the origin and development of adaptive immune systems in vertebrates. Lampreys use variable lymphocyte receptors (VLRs) in recognizing antigens. However, the understanding of the VLR-based adaptive immune signal pathways in lampreys remains unclear. Intestinal bacteria could regulate the development of host immune systems. The attempts of inducing lamprey leukocyte differentially expressed proteins using the gut bacterial as the antigen will supply an promising avenue to explore the molecular mechanism of the intestinal bacteria interaction with it's host. Also, the identification of differentially expressed proteins involved in interactions between gut-derived Shewanella and lamprey will supply clues for understanding the variable lymphocyte receptor-based adaptive immune signal pathways in lampreys.

The phytopathogenic fungus Chrysoporthe cubensis has a great capacity to produce highly efficient enzymes for the hydrolysis of lignocellulosic biomass. The bioinfosecretome of C. cubensis was identified by computational predictions of secreted proteins combined with protein analysis using 1D-LC-MS/MS. The in silico secretome predicted 562 putative genes capable of encoding secreted proteins, including 273 CAZymes. Proteomics analysis confirmed the existence of 313 proteins, including 137 CAZymes classified as Glycosyl Hydrolases (GH), Polysaccharide Lyases (PL), Carbohydrate Esterases (CE) and Auxiliary Activities enzymes (AA), which indicates the presence of classical and oxidative cellulolytic mechanisms. The enzymes diversity in the extract shows fungal versatility to act in complex biomasses. This study provides an insight into the lignocellulose-degradation mechanisms by C. cubensis and allows the identification of the enzymes that are potentially useful in improving industrial process of bioconversion of lignocellulose. SIGNIFICANCE: Chrysoporthe cubensis is an important deadly canker pathogen of commercially cultivated Eucalyptus species. The effective depolymerisation of the recalcitrant plant cell wall performed by this fungus is closely related to its high potential of lignocellulolytic enzymes secretion. Since the degradation of biomass occurs in nature almost exclusively by enzyme secretion systems, it is reasonable to suggest that the identification of C. cubensis lignocellulolytic enzymes is relevant in contributing to new sustainable alternatives for industrial solutions. As far as we know, this work is the first accurate proteomic evaluation of the enzymes secreted by this species of fungus. The integration of the gel-based proteomic approach, the bioinformatic prediction of the secretome and the analyses of enzymatic activity are powerful tools in the evaluation of biotechnological potential of C. cubensis in producing carbohydrate-active enzymes. In addition, analysis of the C. cubensis secretome grown in wheat bran draws attention to this plant pathogen and its extracellular enzymatic machinery, especially regarding the identification of promising new enzymes for industrial applications. The results from this work allowed for explanation and reinforce previous research that revealed C. cubensis as a strong candidate to produce enzymes to hydrolyse sugarcane bagasse and similar substrates.

MARCH8 is an E3 ligase, primarily involved in immune-modulation. Recently, we reported its aberrant expression in human esophageal squamous cell carcinoma. However, exact mechanisms by which it regulates cancer have been poorly understood. We applied high-throughput quantitative proteomics approach to identify downstream protein targets of MARCH8. Silencing of endogenous MARCH8 in ESCC cells followed by LC-MS/MS analysis led to identification of 1,029 unique proteins showing altered expression post MARCH8 knockdown. Several previously reported MARCH8 target proteins viz. TFR1, syntaxin-4, e-cadherin and CD44 were found to be upregulated. Furthermore, new putative targets of MARCH8, including β2M, were identified in the present study. We demonstrated that MARCH8 interacts with and ubiquitinates CDH1 and β2M. Inhibiting proteasome activity with MG132 prevented CDH1 and β2M degradation, indicating that MARCH8 might be targeting CDH1 and β2M for proteasomal degradation. Further, loss of β2M and CDH1 expression significantly and inversely correlated with MARCH8 expression in ESCC tissues (r =  -0.737 and  - 0.651, respectively; p < 0.01). In conclusion, our present study has led to identification of new targets of MARCH8 and suggests the role of MARCH8 in regulating CDH1 and β2M turnover in esophageal cancer cells. SIGNIFICANCE: The use of quantitative proteomics carried out has led to the recognition of new targets of MARCH8. The present study gives a broad understanding of the molecular remodeling arising in the ESCC after MARCH8 knockdown. The study also solidifies the idea that role of MARCH8 is not just limited to immunomodulation as silencing of MARCH8 affects various other processes such as protein processing and localization. This study might help in understanding the regulation of MARCH8 in ESCCs and the mechanism by which MARCH8 might be facilitating cancer cells to evade immune surveillance.

Animal fertility is one of the most important characteristics for the livestock breeding industry. Conventional semen analysis provides basic information on sperm quality, but the predictive value of such analysis with regard to fertility remains questionable. Therefore, it is important to determine and predict male fertility more accurately in the clinic. To identify seminal plasma proteins involved in fertility, isobaric tags for relative and absolute quantitation (iTRAQ) and liquid chromatography with tandem mass spectrometry (quantitative proteomic analysis) were used to identify differentially abundant proteins (DAPs) in seminal plasma between high- and low-reproductive-efficiency Landrace boars. A total of 141 DAPs were identified, of which 125 upregulated and 16 downregulated proteins were subjected to bioinformatics analysis. These DAPs were found to be mainly involved in proteolysis, ATP binding, and energy metabolism. We investigated the relevance of three DAPs-ceruloplasmin, carboxypeptidase E (CPE), and serpin family A member 12 (SERPINA12)-in an in vitro fertility assay. This assay revealed that the inhibition of these proteins with antibodies can reduce or increase the fertilization rate. These results indicate possible biomarkers for the selection of high-fertility boars and provide a theoretical basis for the use of protein biomarkers in the livestock breeding industry. SIGNIFICANCE: Our study identified differentially abundant proteins in the seminal plasma of high-reproductive-efficiency and low-reproductive-efficiency Landrace boars. These proteins may be used as biomarkers to screen out high-fertility boars. The study can provide not only a new method for improving the effects of artificial insemination and reproductive efficiency of boars but also an important reference for boar breeding. Meanwhile, because pigs and humans have similar physiological parameters and organ sizes, our findings can also serve as a reference for human reproduction research.

We used data-independent acquisition (DIA) proteomics technology followed by ELISAs and automated biochemical analyses to identify and validate protein expression levels in Intrahepatic Cholestasis of Pregnancy (ICP) and healthy pregnant controls. We employed bioinformatics to identify metabolic processes associated with differentially expressed proteins.The expression levels of two proteins (S100-A9 and the L-lactate dehydrogenase A chain) were significantly higher in ICP patients than in controls; the areas under the receiver operating characteristic (ROC) curves (AUCs) were 0.774 and 0.828, respectively. The expression levels of two other proteins (apolipoprotein A-I and cholinesterase) were significantly lower in patients, with values of 0.900 and 0.842, respectively. Multiple logistic regression showed that a combination of the levels of the four proteins optimized the AUC (0.962), thus more reliably diagnosing ICP. The levels of all four proteins were positively associated with that of total bile acids. Bioinformatics analyses indicated that the four proteins principally affected neutrophil activation involved in the immune response, cell adhesion, lipoprotein metabolism, and the PPAR signaling pathway. SIGNIFICANCE: This preliminary work improves our understanding of changes in serum levels of protein in pregnant women with ICP. The four proteins may serve as novel noninvasive biomarkers for ICP.

Mikania micrantha is one of the world's most invasive plants, which causes severe damage to natural ecosystems and agroforestry systems due to its rapid stem growth. This work investigated the proteomic and transcriptomic profiles of M. micrantha in different stem tissues (pre-internode, post-internode, and internode), as well as in adventitious roots and primary roots with the final goal of elucidating differentially expressed genes and proteins responsible for the rapid growth of stem. The objective was approached by using DIA-based proteomic and RNA-Seq technologies. More than seven giga-transcriptome clean reads were sequenced, and 5196 protein species were identified. Differentially expressed genes identified in all stem tissues were significantly enriched in photosynthesis and carbon fixation, suggesting that the stem possesses a strong photosynthetic capacity in order to maintain the energy supply for this species. Analysis of differentially expressed proteins showed that proteins related to photosystem I/II and the cytochrome b6/f complex, such as D1, D2, and cp43, were also highly accumulated in the adventitious roots, corroborating the transcriptome analysis results. These results provided basic proteomic and transcriptional expression information about the M. micrantha stem and adventitious root, thereby improving our understanding of the molecular mechanism underlying rapid growth in this species. SIGNIFICANCE: This is the first study to investigate the proteomic and transcriptomic profiles of Mikania micrantha, a highly invasive plant, in different stem tissues (pre-internode, post-internode, and internode), as well as in adventitious and primary roots, using the latest DIA-based (data-independent acquisition mode) proteomic and RNA-Seq technologies. A comprehensive study was carried out, and differentially expressed genes and differentially expressed proteins identified in the pre-internode, post-internode, and internode tissues were significantly enriched during photosynthesis and carbon fixation, suggesting that the M. micrantha stem possesses a strong photosynthetic capacity that allows the plant to maintain a high energy supply. Enriched plant hormone signal transduction pathway analysis revealed an interaction between auxin and other phytohormones involved in adventitious root development. The study provided basic data on the molecular mechanism of M. micrantha vegetative propagation and the rapid growth of its stem. The novel scientific content of this study successfully builds upon the limited information currently available on the subject, therefore warranting publication.

Zooarchaeology by Mass Spectrometry (ZooMS) is rapidly becoming a staple in archaeological and cultural heritage science. Developed a decade ago, this peptide mass fingerprinting technique is expanding from a small group of researchers mainly involved in method development to a broader group of scientists using it as another tool in their toolboxes. With new researchers beginning to use the method, it is imperative that a user-friendly, standardized approach be established. A major barrier has been the often haphazard and changing nomenclature used to label peptide markers necessary for taxonomic identification. Consistent, reliable, and easy-to-understand nomenclature is key to the growth of ZooMS, particularly as the reference library continues to expand. We propose a new set of standardized guidelines for peptide markers based on their position in the type I collagen sequence from the beginning of the highly conserved, helical region. Since this region has no insertions or deletions over a wide range of taxonomic groups, the proposed nomenclature system can be used reliably and consistently across all vertebrate taxa. We propose to label ZooMS peptide markers with the gene, followed by the position of the first and last amino acid of the marker from the start of the helical region. SIGNIFICANCE STATEMENT: We propose a standardized nomenclature system for ZooMS peptide markers that provides consistent labels across multiple, broad taxonomic groups. This system unambiguously locates the marker peptides in the type I collagen sequence, avoids duplication of marker names, and facilitates the creation of large ZooMS databases which can include all vertebrates.

We describe here a modern tool for exploring documents pertaining to the world Cultural Heritage while avoiding their contamination or damage. Known under the acronym EVA, it consists of a plastic foil of Ethylene Vinyl Acetate studded with strong cation and anion resins admixed with C8 and C18 hydrophobic beads. When applied to any surface such foils can harvest any type of surface material, which is then eluted and analyzed via standard means, such as GS/MS (typically for metabolites), MS/MS (for peptide and protein analysis), X-ray (for elemental analysis). We briefly review here a number of past data, such as screening of original documents by Bulgakov, Chekov, Casanova, Kepler, while dealing in extenso with very recent data, pertaining to Orwell and Stalin and analysis of the skin of an Egyptian mummy. The technique was also successfully applied to paintings, such as the Donna Nuda at the Hermitage in St. Petersburg, attributed to Leonardo and his school. This novel methodology represents a formidable tool for exploring the past life of famous authors, scientist and literates in that it can detect traces of their pathologies and even drug consumption left by saliva and sweat traces on their original hand-written documents.

The proteomics of formalin-fixed, paraffin-embedded (FFPE) samples has advanced significantly during the last two decades, but there are many protocols and few studies comparing them directly. There is no consensus on the most effective protocol for shotgun proteomic analysis. We compared the in-solution digestion with RapiGest and Filter Aided Sample Preparation (FASP) of FFPE prostate tissues stored 7 years and mirroring fresh frozen samples, using two label-free data-independent LC-MS/MS acquisitions. RapiGest identified more proteins than FASP, with almost identical numbers of proteins from fresh and FFPE tissues and 69% overlap, good preservation of high-MW proteins, no bias regarding isoelectric point, and greater technical reproducibility. On the other hand, FASP yielded 20% fewer protein identifications in FFPE than in fresh tissue, with 64-69% overlap, depletion of proteins >70 kDa, lower efficiency in acidic and neutral range, and lower technical reproducibility. Both protocols showed highly similar subcellular compartments distribution, highly similar percentages of extracted unique peptides from FFPE and fresh tissues and high positive correlation between the absolute quantitation values of fresh and FFPE proteins. In conclusion, RapiGest extraction of FFPE tissues delivers a proteome that closely resembles the fresh frozen proteome and should be preferred over FASP in biomarker and quantification studies. SIGNIFICANCE: Here we analyzed the performance of two sample preparation methods for shotgun proteomic analysis of FFPE tissues to give a comprehensive overview of the obtained proteomes and the resemblance to its matching fresh frozen counterparts. These findings give us better understanding towards competent proteomics analysis of FFPE tissues. It is hoped that it will encourage further assessments of available protocols before establishing the most effective protocol for shotgun proteomic FFPE tissue analysis.

Avocado is a nutritious and economically important fruit, generating significant income for exporter countries. Recently, by-products of this fruit such as seeds and peels, have raised interest in different industries. However, the biochemical features of the nutraceutical value of these tissues have not been analyzed using molecular approaches during the postharvest shelf life (PSL). We carried out comparative proteomics using tandem mass tagging (TMT) and synchronous-precursor selection (SPS)-MS3. We analyzed testa, cotyledon, and embryo axes from avocado seeds at detachment from the tree (unripe), and after five (breaker) and ten days (ripe) of PSL. We identified 1968 proteins, from which 933 were specific to the testa, 167 to the embryo axis, and 23 to the cotyledon. The testa had a more dynamic proteome than the other tissues, resembling similar stress responses to those observed in peel tissues, such as down-accumulation of translational machinery, cell wall catabolism and synthesis of secondary metabolites. In contrast, the up-accumulation of the biosynthesis of l-glutamine, L-isoleucine, and l-serine was observed in all tissues. Our study provides the basic biochemical and physiological features of avocado seed during PSL and demonstrates that avocado seed tissues could potentially be used as a costless source of high-value compounds. SIGNIFICANCE: Avocado seed as a fruit by-product is a source of different valuable molecules, including those with nutraceutical properties. During PSL, several biochemical and physiological modifications occur in this dispersal unit, which also includes the alteration of several key metabolites' content. However, the proteome profile associated with different metabolic pathways that regulate the inner content of seed metabolites has not been previously studied. Our tissue-specific proteomics TMT-SPS-MS3-based provides the first evidence of molecular and physiological changes in avocado tissues during PSL delivering fundamental knowledge of this organ. In this vein, the modulation of secondary metabolites, amino acid, and sugar metabolism of avocado tissues during PLS can encourage these by-products exploitation in multiple industries.

Proteins can be post-translationally modified and this can be important in the regulation of cellular processes and function. However, little is known about whether protein phosphorylation plays a role in regulating wool fibre properties. In this study, we used a chemical labelling method combined with a high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis to compare the phosphopeptides present in the wool of three Tan sheep with highly crimped wool and three Tan sheep with straighter wool. Thirty-six phosphopeptides that had differences in relative abundance between these two types of wool were identified. These peptides were derived from 28 to 33 different proteins, including two keratins (Ks) and 7 to 12 keratin-associated proteins (KAPs), with these proteins being common structural components of the wool fibre. The crimped wool had a higher relative abundance of phosphorylated K38, K72 and KAP13-x, whereas the straighter wool had a higher relative abundance of phosphorylated KAP2-1, KAP6-1, KAP4-x, KAP10-x and KAP13-y. These results confirm the phosphorylation of wool Ks and KAPs, and suggest that differential phosphorylation of Ks and KAPs may affect wool fibre crimping in Tan sheep. SIGNIFICANCE: Protein phosphorylation can alter the structural conformation and interaction of a protein, and hence affect the cellular processes that the protein undertakes. In this study, we compared the suite of phosphorylated proteins in crimped and straight wool from Chinese Tan sheep and found that some keratins and keratin-associated proteins were phosphorylated. Crimped wool had more keratin phosphorylation, while straight wool had more keratin-associated protein phosphorylation, with this suggesting that wool fibre crimping may be a regulated by phosphorylation of some wool proteins. This suggests that wool traits may be under epigenetic control and that post-translation modifications need to be considered in breeding for different wool types.

The database search method is a widely accepted method to assign a peptide to the tandem mass spectra. In this study, a new flexible method- FPTMS is introduced to interpret the tandem mass spectra with the known peptide sequences in a protein database. Here the frequency of occurrence of fragment ion peaks extracted from the extensive spectral library is used to predict the theoretical tandem mass spectra of the peptides. The dot product scoring and windowed-xcorr scoring methods were implemented to score the experimental spectrum against the theoretical peptide spectra. Windowed-xcorr is introduced to tackle the mass errors and the cleavage position of the fragmentation process. The new method with windowed-xcorr shows an improved identification rate compared to the existing search engines Crux-Tide and X!Tandem at 1% False Discovery Rate (FDR) for the dataset considered in this study. SIGNIFICANCE: Identifying or sequencing of the peptide from tandem mass spectra is an important application in mass spectrometry-based proteomics. Collision-induced dissociation (CID) fragmentation spectra have been widely used to develop a peptide identification algorithm using database search strategy. CID fragmentation behavior is a complex process and found to have dependency on the sequences of peptide, charge state, and residue content. The inclusion of more features of peptide fragmentation behavior and adaptable scoring algorithm improves the efficiency of the peptide identification algorithm.

Plants must rapidly adapt to changes in nutrient conditions. Especially adaptations to changing nitrogen environments are very complex involving also major adjustments on the protein level. Here, we used a size-exclusion chromatography-coupled to mass spectrometry approach to study the dynamics of protein-protein interactions induced by transition from full nutrition to nitrogen starvation. Comparison of interaction networks established for each nutrient condition revealed a large overlap of proteins which were part of the protein-protein interaction network, but that same set of proteins underwent different interactions at each treatment. Network topology parameter betweenness centrality (BC) was found to best reflect the relevance of individual proteins in the information flow within each network. Changes in BC for individual proteins may therefore indicate their involvement in the cellular adjustments to the new condition. Based on this analysis, a set of proteins was identified showing high nitrogen-dependent changes in their BC values: The receptor kinase AT5G49770, co-receptor QSK1, and proton-ATPase AHA2. Mutants of those proteins showed a nitrate-dependent root growth phenotype. Individual interactions within the reconstructed network were tested using FRET-FLIM technology. Taken together, we present a systematic strategy comparing dynamic changes in protein-protein interaction networks based on their network parameters to identify regulatory nodes. SIGNIFICANCE: Protein-protein interactions are known to be important in cellular signaling events, but the dynamic changes in interaction networks induced by external stimuli are still rarely studied. We systematically analyzed how changes in the nutrient environment induced a rewiring of protein-protein interactions in roots. We observed small changes in overall protein abundances, but instead a rewiring of pairwise protein-protein interactions. Betweenness centrality was found to be the optimal network topology parameter to identify protein candidates with high relevance to the information flow in the (dynamic) network. Predicted interactions of those relevant nodes were confirmed in FLIM/FRET experiments and in phenotypic analysis. The network approach described here may be a useful application in dynamic network analysis more generally.

FAIMS interface is gaining popularity because of the impressive 100-fold signal to noise enhancement in addition to the recent coupling to the Orbitrap technology, the most important analyzer developed in the last 20 years. The selection of group of ions and effective removal of single-charged ones at particular compensation voltages increases around 50% the proteome coverage at expenses of lower peptides coverage. However, specific setting for phosphoproteome analysis is yet poorly described. Here we have found the maximum transmission for several tryptic phosphopeptides isolated from a single complex mixture and we have set an experimental method based on five compensation voltages partially different to the ones described previously, demonstrating the relevance of voltages higher than 47 V, with an increase of around 20% of unique phosphopeptides. Using this experimental setup two complex phosphoproteomes isolates (SH-SY5Y cell line and plasma) were analyzed and found increments of 50% on phosphopeptides identification with the proposed method with respect to a previous one, for the cell line extract. Meanwhile for plasma 109 of the detected phosphopeptides are found for first time in this body fluid, presumably due to the release of intracellular proteins. With this FAIMS setup, 60% of the proteins identified are classified as very low abundant proteins.

OBJECTIVE: Endometrial cancer is the most common gynecologic cancer and yet much is still unknown about this disease. Our goal was to identify unique biomarkers of disease by performing a comprehensive proteomic analysis of early stage, low-grade endometrial cancer through analysis of serum collected from patients pre- and post-definitive surgery. METHODS: We used mass spectrometry (MS)-based proteomics to identify serum proteins from these patients. Serum samples from women undergoing hysterectomy with bilateral salpingo-oophorectomy for benign reasons served as control samples for the correlative studies. We then correlated our findings with The Cancer Genome Atlas (TCGA) database for additional confirmation. RESULTS: The Ingenuity Pathway Analysis of proteins that were differentially expressed in endometrial cancer showed increased cell survival and decreased organismal death, the most common hallmarks of cancer. We identified over expression of FAM83D (family with sequence similarity 83, member D) in the serum of patients with early stage low-grade endometrial cancer and verified the same in the endometrial cancer cell lines and patient tumors. We also confirmed our hypothesis that FAM83D may serve as a biomarker for endometrial cancer in a cohort of patients with endometrial cancer from The Cancer Genome Atlas (TCGA) project. CONCLUSION: Comprehensive proteomic analysis is a feasible strategy for potential biomarker identification. Using this technique, FAM83D was identified as a candidate biomarker in early endometrial cancer in our patient samples and was not present in benign control samples. FAM83D has been associated with poor clinical outcomes in several human malignancies. SIGNIFICANCE: Our manuscript describes an alternative approach to comprehensive protein analysis in a model pre and post tumor removal for a sample of patients with early endometrial cancer. The model is innovative and the findings of over expression FAM83D in this population of early cancer may be useful in the study of a disease where there are few biomarkers or targetable therapies.

Complications from pathologic myopia (PM) are a major cause of visual impairment and blindness. However, an efficient clinical therapeutic strategy for PM is still lacking. The aim of this study was to quantitatively compare the proteomic profiles of aqueous humor between PM and non-PM cataract patients. Twenty aqueous humor samples from each group were analyzed with label-free quantitative proteomic analysis to identify the differentially expressed proteins for function enrichment analyses and protein-protein interaction network construction. Hub protein was validated with ELISA using an independent cohort consisting of 20 samples from each group and its receiver operating characteristic (ROC) curve analysis was conducted. A total of 583 proteins were identified and 101 proteins were found to be differentially expressed, including 63 up-regulated proteins and 38 down-regulated proteins. The bioinformatics analysis suggested that PM is closely associated with immunity and inflammation interactions, and remodeling of extracellular matrix. Apolipoprotein A-I (ApoA1) was enriched as the hub protein of the network with the highest score, degree and centrality. ROC analysis showed that ApoA1 could distinguish PM from controls with an area under the curve of 0.963 (p < 0.001). The findings could provide potential clues for further study on the molecular mechanisms and developing new treatments for PM, especially related to immunity and inflammation interactions. ApoA1 may be a potential key protein and therapeutic target in human PM. SIGNIFICANCE: It is important and urgent to discover the mechanisms of pathologic myopia (PM) to inhibit its progression. This study applied the quantitative proteomic analysis to study aqueous humor from patients with or without PM, aiming to discover dysregulated proteins related to PM. Our results suggested that those dysregulated proteins are closely associated with immunity and inflammation interactions, and remodeling of extracellular matrix. The findings from this study could provide potential clues for further research on the molecular mechanisms and developing new treatments for PM, especially related to immunity and inflammation. ApoA1 may be a potential key protein and therapeutic target in human PM.

Previously, we developed a novel separation technique, namely, supported molecular matrix electrophoresis (SMME), which separates mucins on a PVDF membrane that impregnated with a hydrophilic polymer (such as polyvinyl alcohol), so it has the characteristics that are compatible with glycan analysis of the separated bands. Here, we describe the first instance of the application of SMME to mouse sera fractionation and demonstrate their differences from the pooled human sera fractionation by SMME. Furthermore, we have developed a fixation method for the lectin blotting of SMME-separated glycoproteins by immersing the SMME membranes into acetone solvent followed by heating. It showed that the amount of protein samples required for SMME were reduced more than 4-fold than that of the process of SDS-PAGE. We applied these techniques for the detection of glycosylation patterns of serum proteins from Fut8+/+ and Fut8-/- mice, further analyzed N-linked and O-linked glycans from the separated γ-bands by mass spectrometry, and demonstrated that there are α2,8-sialylated O-glycans contained in mouse sera glycoproteins. SMME can provide simple, rapid sera fractionation, glycan profiling differences between the bands of two samples and a new insight into the underlying mechanism that responsible for related diseases. SIGNIFICANCE: We describe that the first application of SMME can separate mouse serum proteins into six bands and identify the major protein components of each fraction in mouse serum separated by SMME. Furthermore, we successfully developed a fixation method for lectin blotting of SMME-separated glycoproteins and applied to the detection of glycosylation patterns of serum glycoproteins from Fut8+/+ and Fut8-/- mice, also, the method is promising for detecting glycan profiling differences between two samples in both research and clinical settings.

In this study, we performed an integrated physiological and chloroplast proteome analysis of wheat seedling leaves under salt and osmotic stresses by label-free based quantitative proteomic approach. Both salt and osmotic stresses significantly increased the levels of abscisic acid and methyl jasmonate and led to damages of chloroplast ultrastructure. Main parameters of chlorophyll fluorescence and gas exchange showed a significant decline under both stresses. Quantitative proteomic analysis identified 194 and 169 chloroplast-localized differentially accumulated proteins (DAPs) responsive to salt and osmotic stresses, respectively. The abundance of main DAPs involved in light-dependent reaction were increased under salt stress, but decreased in response to osmotic stress. On the contrary, salt stress induced a significant upregulation of the DAPs associated with Calvin cycle, transcription and translation, amino acid metabolism, carbon and nitrogen metabolism, and some of them exhibited a downregulation under osmotic stress. In particular, both treatments significantly upregulated the DAPs involved in plastoglobule development, protein folding and proteolysis, hormone and vitamin synthesis. Finally, we proposed a putative synergistic responsive network of wheat chloroplast proteome under salt and osmotic stresses, aiming to provide new insights into the underlying response and defense mechanisms of wheat chloroplast proteome in response to abiotic stresses. SIGNIFICANCE: Salt and osmotic stresses are the two most common abiotic stresses that severely affect crop growth and productivity. As the main site of photosynthesis of plant cells, the chloroplast also plays important role in plant tolerance to abiotic stress. However, the response of chloroplast proteome to salt and osmotic is still poorly understood by using the traditional two-dimensional electrophoresis (2-DE) method due to a poor resolution of chloroplast protein separation and low throughput identification of differentially accumulated proteins (DAPs). In this study, we employed label-free based quantitative proteomic approach to perform an integrated physiological and large-scale chloroplast proteome analysis of wheat seedling leaves under salt and osmotic stresses, which laid a solid foundation for future studies into the response and defense mechanisms of wheat chloroplast in response to abiotic stresses.

Using high-throughput BioPlex assays, we determined that six fractions from the venom of Conus nux inhibit the adhesion of various recombinant PfEMP-1 protein domains (PF08_0106 CIDR1α3.1, PF11_0521 DBL2β3, and PFL0030c DBL3X and DBL5e) to their corresponding receptors (CD36, ICAM-1, and CSA, respectively). The protein domain-receptor interactions permit P. falciparum-infected erythrocytes (IE) to evade elimination in the spleen by adhering to the microvasculature in various organs including the placenta. The sequences for the main components of the fractions, determined by tandem mass spectrometry, yielded four T-superfamily conotoxins, one (CC-Loop-CC) with I-IV, II-III connectivity and three (CC-Loop-CXaaC) with a I-III, II-IV connectivity. The 3D structure for one of the latter, NuxVA = GCCPAPLTCHCVIY, revealed a novel scaffold defined by double turns forming a hairpin-like structure stabilized by the two disulfide bonds. Two other main fraction components were a miniM conotoxin, and a O2-superfamily conotoxin with cysteine framework VI/VII. This study is the first one of its kind suggesting the use of conotoxins for developing pharmacological tools for anti-adhesion adjunct therapy against malaria. Similarly, mitigation of emerging diseases like AIDS and COVID-19, can also benefit from conotoxins as inhibitors of protein-protein interactions as treatment. BIOLOGICAL SIGNIFICANCE: Among the 850+ species of cone snail species there are hundreds of thousands of diverse venom exopeptides that have been selected throughout several million years of evolution to capture prey and deter predators. They do so by targeting several surface proteins present in target excitable cells. This immense biomolecular library of conopeptides can be explored for potential use as therapeutic leads against persistent and emerging diseases affecting non-excitable systems. We aim to expand the pharmacological reach of conotoxins/conopeptides by revealing their in vitro capacity to disrupt protein-protein and protein-polysaccharide interactions that directly contribute to pathology of Plasmodium falciparum malaria. This is significant for severe forms of malaria, which might be deadly even after treated with current parasite-killing drugs because of persistent cytoadhesion of P. falciparum infected erythrocytes even when parasites within red blood cells are dead. Anti-adhesion adjunct drugs would de-sequester or prevent additional sequestration of infected erythrocytes and may significantly improve survival of malaria patients. These results provide a lead for further investigations into conotoxins and other venom peptides as potential candidates for anti-adhesion or blockade-therapies. This study is the first of its kind and it suggests that conotoxins can be developed as pharmacological tools for anti-adhesion adjunct therapy against malaria. Similarly, mitigation of emerging diseases like AIDS and COVID-19, can also benefit from conotoxins as potential inhibitors of protein-protein interactions as treatment.

Protobothrops mucrosquamatus, also known as the brown spotted pit viper or Taiwanese habu, is a medically significant venomous snake in Taiwan, especially in the northern area. To more fully understand the proteome profile of P. mucrosquamatus, we characterized its venom composition using a bottom-up proteomic approach. Whole venom components were fractionated by RP-HPLC and then analyzed by SDS-PAGE. Each protein band in gels was excised and subjected to protein identification by LC-MS/MS. A subsequent proteomic analysis revealed the presence of 61 distinct proteins belonging to 19 families in P. mucrosquamatus venom. Snake venom metalloproteinase (SVMP; 29.4%), C-type lectin (CLEC; 21.1%), snake venom serine protease (SVSP; 17.6%) and phospholipase A2 (PLA2; 15.9%) were the most abundant protein families, whereas several low-abundance proteins, categorized into eight protein families, were demonstrated in P. mucrosquamatus venom for the first time. Because PLA2 is known to make a major contribution to venom lethality, we evaluated whether the known PLA2 inhibitor, varespladib, was capable of preventing the toxic effects of P. mucrosquamatus venom. This small-molecule drug demonstrated the ability to inhibit PLA2 activity in vitro (IC50 = 101.3 nM). It also blunted lethality in vivo, prolonging survival following venom injection in a mouse model, but it showed limited potency against venom-induced local hemorrhage in this model. Our findings provide essential biological and pathophysiological insights into the composition of P. mucrosquamatus venom and suggest PLA2 inhibition as an adjunctive or alternative therapeutic strategy in the clinical management of P. mucrosquamatus envenoming in emergency medicine. SIGNIFICANCE: P. mucrosquamatus envenomation is a significant medical concern in Taiwan, especially in the northern region. Although antivenom is commonly used for rescuing P. mucrosquamatus envenoming, severe clinical events still occur, with more than 20% of cases requiring surgical intervention. Small-molecule therapy offers several advantages as a potential adjunctive, or even alternative, to antivenom treatment, such as heat stability, low antigenicity and ease of administration, among others. A deeper understanding of the venom proteome of P. mucrosquamatus would aid in the discovery of small-molecule drugs that could be repurposed to target specific venom proteins. Here, we applied a bottom-up proteomic approach to characterize the protein profile of P. mucrosquamatus venom. Varespladib, a small-molecule drug used to treat inflammatory disease, was repurposed to inhibit the toxicity of P. mucrosquamatus venom, and was shown to reduce the lethal effects of P. mucrosquamatus envenomation in a rodent model. Varespladib might be used as a first-aid therapeutic against P. mucrosquamatus envenoming in the pre-referral period and/or as an adjunctive agent administered together with anti-P. mucrosquamatus antivenom.