Body fluid proteome has been intensively studied as a primary source for disease biomarker discovery. Using advanced proteomics technologies, early research success has resulted in increasingly accumulated proteins detected in different body fluids, among which many are promising biomarkers. However, despite a handful of small-scale and specific data resources, current research is clearly lacking effort compiling published body fluid proteins into a centralized and sustainable repository that can provide users with systematic analytic tools. In this study, we developed a new database of human body fluid proteome (HBFP) that focuses on experimentally validated proteome in 17 types of human body fluids. The current database archives 11 827 unique proteins reported by 164 scientific publications, with a maximal false discovery rate of 0.01 on both the peptide and protein levels since 2001, and enables users to query, analyze and download protein entries with respect to each body fluid. Three unique features of this new system include the following: (i) the protein annotation page includes detailed abundance information based on relative qualitative measures of peptides reported in the original references, (ii) a new score is calculated on each reported protein to indicate the discovery confidence and (iii) HBFP catalogs 7354 proteins with at least two non-nested uniquely mapping peptides of nine amino acids according to the Human Proteome Project Data Interpretation Guidelines, while the remaining 4473 proteins have more than two unique peptides without given sequence information. As an important resource for human protein secretome, we anticipate that this new HBFP database can be a powerful tool that facilitates research in clinical proteomics and biomarker discovery. Database URL: https://bmbl.bmi.osumc.edu/HBFP/.

Cell surface proteins are responsible for many critical functions. Systematical profiling of these proteins would provide a unique molecular fingerprint to classify cells and provide important information to guide immunotherapy. Cell surface biotinylation method is one of the effective methods for cell surface proteome profiling. However, classical workflows suffer the disadvantage of poor sensitivity. In this work, we presented an optimized protocol which enabled identification of more cell surface proteins from a smaller number of cells. When this protocol was combined with a tip based fractionation scheme, 4510 proteins, including 2055 annotated cell surface-associated proteins, were identified with only 20 microgram protein digest, showing the superior sensitivity of the approach. To enable process 10 times fewer cells, a pipet tip based protocol was developed, which led to the identification of about 600 cell surface-associated proteins. Finally, the new protocol was applied to compare the cell surface proteomes of two breast cancer cell lines, BT474 and MCF7. It was found that many cell surface-associated proteins were differentially expressed. The new protocols were demonstrated to be easy to perform, time-saving, and yielding good selectivity and high sensitivity. We expect this protocol would have broad applications in the future. SIGNIFICANCE: Cell surface proteins confer specific cellular functions and are easily accessible. They are often used as drug targets and potential biomarkers for prognostic or diagnostic purposes. Thus, efficient methods for profiling cell surface proteins are highly demanded. Cell surface biotinylation method is one of the effective methods for cell surface proteome profiling. However, classical workflows suffer the disadvantage of poor sensitivity. In this work, we presented an optimized protocol which enabled identification of more cell surface proteins from a smaller number of starting cells. The new protocol is easier to perform, time-saving and has less protein loss. By using a special pipet tip, sensitive and in-depth cell surface proteome analysis could be achieved. In combination with label-free quantitative MS, the new protocol can be applied to the differential analysis of the cell surface proteomes between different cell lines to find genetically- or drug-induced changes. We expect this protocol would have broad application in cell surface protein studies, including the discovery of diagnostic marker proteins and potential therapeutic targets.

Fully understanding the target spaces of drugs is essential for investigating the mechanism of drug action and side effects, as well as for drug discovery and repurposing. In this study, we present an energetics-based approach, termed pH-dependent protein precipitation (pHDPP), to probe the ligand-induced protein stability shift for proteome-wide drug target identification. We demonstrate that pHDPP works for a diverse array of ligands, including a folate derivative, an ATP analog, a CDK inhibitor and an immunosuppressant, enabling highly specific identification of target proteins from total cell lysates. This approach is compared to thermal and solvent-induced denaturation approaches with a pan-kinase inhibitor as the model drug, demonstrating its high sensitivity and high complementarity to other approaches. Dihydroartemisinin (DHA), a dominant derivative of artemisinin to treat malaria, is known to have an extraordinary effect on the treatment of various cancers. However, the anti-tumor mechanisms remain unknown. pHDPP was applied to reveal the target space of DHA and 45 potential target proteins were identified. Pathway analysis indicated that these target proteins were mainly involved in metabolism and apoptosis pathways. Two cancer-related target proteins, ALDH7A1 and HMGB1, were validated by structural simulation and AI-based target prediction methods. And they were further validated to have strong affinity to DHA by using cellular thermal shift assay (CETSA). In summary, pHDPP is a powerful tool to construct the target protein space to reveal the mechanism of drug action and would have broad application in drug discovery studies.

Although the use of detergents in thermal proteome profiling (TPP) has become a common practice to identify membrane protein targets in complex biological samples, surprisingly, there is no proteome-wide investigation into the impacts of detergent introduction on the target identification performance of TPP. In this study, we assessed the target identification performance of TPP in the presence of a commonly used non-ionic detergent or a zwitterionic detergent using a pan-kinase inhibitor staurosporine, our results showed that the addition of either of these detergents significantly impaired the identification performance of TPP at the optimal temperature for soluble target protein identification. Further investigation showed that detergents destabilized the proteome and increased protein precipitation. By lowering the applied temperature point, the target identification performance of TPP with detergents is significantly improved and is comparable to that in the absence of detergents. Our findings provide valuable insight into how to select the appropriate temperature range when detergents are used in TPP. In addition, our results also suggest that the combination of detergent and heat may serve as a novel precipitation-inducing force that can be applied for target protein identification.

Ascorbate peroxidase (APEX)-based proximity labeling coupled with mass spectrometry has a great potential for spatiotemporal identification of proteins proximal to a protein complex of interest. Using this approach is feasible to define the proteome neighborhood of important protein complexes in a popular photosynthetic model cyanobacterium Synechocystis sp. PCC6803 (hereafter named as Synechocystis). To this end, we developed a robust workflow for APEX2-based proximity labeling in Synechocystis and used the workflow to identify proteins proximal to the photosystem II (PS II) oxygen evolution complex (OEC) through fusion APEX2 with a luminal OEC subunit, PsbO. In total, 38 integral membrane proteins (IMPs) and 93 luminal proteins were identified as proximal to the OEC. A significant portion of these proteins are involved in PS II assembly, maturation, and repair, while the majority of the rest were not previously implicated with PS II. The IMPs include subunits of PS II and cytochrome b6/f, but not of photosystem I (except for PsaL) and ATP synthases, suggesting that the latter two complexes are spatially separated from the OEC with a distance longer than the APEX2 labeling radius. Besides, the topologies of six IMPs were successfully predicted because their lumen-facing regions exclusively contain potential APEX2 labeling sites. The luminal proteins include 66 proteins with a predicted signal peptide and 57 proteins localized also in periplasm, providing important targets to study the regulation and selectivity of protein translocation. Together, we not only developed a robust workflow for the application of APEX2-based proximity labeling in Synechocystis and showcased the feasibility to define the neighborhood proteome of an important protein complex with a short radius but also discovered a set of the proteins that potentially interact with and regulate PS II structure and function.

To understand the molecular mechanism underlying salt stress response in radish, iTRAQ-based proteomic analysis was conducted to investigate the differences in protein species abundance under different salt treatments. In total, 851, 706, and 685 differential abundance protein species (DAPS) were identified between CK vs. Na100, CK vs. Na200, and Na100 vs. Na200, respectively. Functional annotation analysis revealed that salt stress elicited complex proteomic alterations in radish roots involved in carbohydrate and energy metabolism, protein metabolism, signal transduction, transcription regulation, stress and defense and transport. Additionally, the expression levels of nine genes encoding DAPS were further verified using RT-qPCR. The integrative analysis of transcriptomic and proteomic data in conjunction with miRNAs was further performed to strengthen the understanding of radish response to salinity. The genes responsible for signal transduction, ROS scavenging and transport activities as well as several key miRNAs including miR171, miR395, and miR398 played crucial roles in salt stress response in radish. Based on these findings, a schematic genetic regulatory network of salt stress response was proposed. This study provided valuable insights into the molecular mechanism underlying salt stress response in radish roots and would facilitate developing effective strategies toward genetically engineered salt-tolerant radish and other root vegetable crops.

Osteoporosis is a common, complex disease of bone with a strong heritable component, characterized by low bone mineral density, microarchitectural deterioration of bone tissue and an increased risk of fracture. Due to limited drug selection for osteoporosis and increasing morbidity, mortality of osteoporotic fractures, osteoporosis has become a major health burden in aging societies. Current researches for identifying specific loci or genes involved in osteoporosis contribute to a greater understanding of the pathogenesis of osteoporosis and the development of better diagnosis, prevention and treatment strategies. However, little is known about how most causal genes work and interact to influence osteoporosis. Therefore, it is greatly significant to collect and analyze the studies involved in osteoporosis-related genes. Unfortunately, the information about all these osteoporosis-related genes is scattered in a large amount of extensive literature. Currently, there is no specialized database for easily accessing relevant information about osteoporosis-related genes and miRNAs.

Citrate Synthase (Cs) gene mutation (locus ahL4) has been found to play an important role in progressive hearing loss of A/J mice. HEI-OC1 cells have been widely used as an in vitro system to study cellular and molecular mechanisms related to hearing lose. We previously reported the increased apoptosis and the accumulation of reactive oxygen species in shRNACs-1429 cells, a Cs low-expressed cell model from HEI-OCI. The details of the mechanism of ROS production and apoptosis mediated by the abnormal expression of Cs needed to research furtherly.

Although protein phosphorylation is an important post-translational modification affecting protein function and metabolism, dynamic changes in this process during ontogenesis remain unexplored in woody angiosperms.

The ocean harbors a variety of bacteria that contain huge protease resources and offer a great potential for industrial and biotechnological applications. Here, we isolated a protease-secreting bacterium Vibrio pomeroyi strain 12613 from Atlantic seawater and purified a protease VP9 from strain 12613. VP9 was identified as a metalloprotease of the M4 family. VP9 could hydrolyze casein and gelatin but not elastin and collagen. With gelatin as the substrate, VP9 showed the highest activity at 40°C and pH 6.0-8.0. It was stable at temperatures of 50°C and less and in the range of pH 5.0-11.0. VP9 also had good tolerance to NaCl, non-ionic detergents, and organic solvent methanol. Unlike other M4 metalloproteases, VP9 has distinct collagen-swelling ability, and its collagen-swelling effect was concentration dependent. The relative expansion volume of collagen increased by approximately eightfold after treatment with 10 μM VP9 at 37°C for 12 h. The collagen-swelling mechanism of VP9 on bovine-insoluble type I collagen was further studied. Atomic force microscopy observation and biochemical analyses showed that VP9 can degrade proteoglycans in collagen fibers, resulting in the release of collagen fibrils from collagen fibers and the swelling of the latter. In addition, VP9 can degrade glycoproteins, a non-collagenous constituent interacting with collagen in the skin. The characteristics of VP9, such as sufficient specificity toward proteoglycans and glycoproteins but no activity toward collagen, suggest its promising potential in the unhairing and fiber-opening processing in leather industry.

Sperm cryopreservation is challenging, often resulting in irreversible damage to spermatozoa, as indicated by decreased motility, viability, and/or acrosomal integrity. Developing cryopreservation protocols for gametes of endangered species compounds the complexity of technique optimization; samples are difficult to obtain and numbers are limited. Cryopreservation of sperm collected from the critically endangered addra gazelle (Nanger dama ruficollis), a member of the Bovidae family, resulted in significant loss of motility, which was prevented by pretreatment with cholesterol-loaded cyclodextrin (CLC). This study investigated the proteome of sperm (fresh and cryopreserved), processed in the absence and presence of 0.5 mg/ml CLC in the addra gazelle. The proteome of Bos taurus, the closest domestic relative, was used as a reference. Mass spectrometry analysis of the addra gazelle sperm proteome revealed 287 proteins. The concentrations of 85 proteins differed between fresh and frozen/thawed samples; nearly all were decreased. Most were associated with metabolic processes, specifically glycolysis, which may explain the decrease in post-thaw motility observed in this species. CLC pretreatment partially prevented the loss of various proteins involved in metabolism including CAPZB (gene = CAPZB), HS90A (gene = HSP90AA1), and PGAM2 (gene = PGAM2). To our knowledge, this is the first study to evaluate the proteome of any wild bovids' sperm, and the first to compare protein levels in sperm pretreated with CLC.

The giant panda (Ailuropoda melanoleuca) is a vulnerable mammal herbivore living wild in central China. Viral infections have become a potential threat to the health of these endangered animals, but limited information related to these infections is available.

Alternative splicing (AS) plays important roles in gene expression and proteome diversity. Single nucleotide polymorphism (SNP) and insertion/deletion (InDel) are abundant polymorphisms and co-dominant inheritance markers, which have been widely used in germplasm identification, genetic mapping and marker-assisted selection in plants. So far, however, little information is available on utilization of AS events and development of SNP and InDel markers from transcriptome in radish.

The fleshy taproot of radish is an important storage organ determining its yield and quality. Taproot thickening is a complex developmental process in radish. However, the molecular mechanisms governing this process remain unclear at the proteome level. In this study, a comparative proteomic analysis was performed to analyze the proteome changes at three developmental stages of taproot thickening using iTRAQ approach. In total, 1862 differentially expressed proteins (DEPs) were identified from 6342 high-confidence proteins, among which 256 up-regulated proteins displayed overlapped accumulation in S1 (pre-cortex splitting stage) vs. S2 (cortex splitting stage) and S1 vs. S3 (expanding stage) pairs, whereas 122 up-regulated proteins displayed overlapped accumulation in S1 vs. S3 and S2 vs. S3 pairs. Gene Ontology (GO) and pathway enrichment analysis showed that these DEPs were mainly involved in several processes such as "starch and sucrose metabolism", "plant hormone signal transduction", and "biosynthesis of secondary metabolites". A high concordance existed between iTRAQ and RT-qPCR at the mRNA expression levels. Furthermore, association analysis showed that 187, 181, and 96 DEPs were matched with their corresponding differentially expressed genes (DEGs) in S1 vs. S2, S1 vs. S3, and S2 vs. S3 comparison, respectively. Notably, several functional proteins including cell division cycle 5-like protein (CDC5), expansin B1 (EXPB1), and xyloglucan endotransglucosylase/hydrolase protein 24 (XTH24) were responsible for cell division and expansion during radish taproot thickening process. These results could facilitate a better understanding of the molecular mechanism underlying taproot thickening, and provide valuable information for the identification of critical genes/proteins responsible for taproot thickening in root vegetable crops.

Cytotoxic T cells (CTLs) bind to peptides presented by MHC I (pMHC) through T cell receptors of various affinities. Low-affinity CTLs are important for the control of intracellular pathogens and cancers; however, the mechanisms by which these lower affinity CTLs are activated and maintained are not well understood. We recently discovered that fully activated CTLs stimulated by strong-affinity peptides in the presence of IL-12 are able to secrete exosomes that, in turn, stimulate bystander CTLs without requiring the presence of antigen. We hypothesized that exosomes secreted by high-affinity CTLs could strengthen the activation of low-affinity CTLs. Naive OT-I CD8+ cells were stimulated with altered N4 peptides of different affinities in the presence or absence of Exo. The presence of Exo preferentially increased cell proliferation and enhanced the production of IFNγ in CTLs stimulated by low-affinity peptides. The expression of granzyme B (GZB) was augmented in all affinities, with higher GZB production in low-affinity stimulated CTLs than in high-affinity stimulated ones. Exosomes promoted the rapid activation of low-affinity CTLs, which remained responsive to exosomes for a prolonged duration. Unexpectedly, exosomes could be induced quickly (24 h) following CTL activation and at a higher quantity per cell than later (72 h). While exosome protein profiles vary significantly between early exosomes and their later-derived counterparts, both appear to have similar downstream functions. These results reveal a potential mechanism for fully activated CTLs in activating lower-affinity CTLs that may have important implications in boosting the function of low-affinity CTLs in immunotherapy for cancers and chronic viral infections.

Viral infection is a complex pathogenesis and the underlying molecular mechanisms remain poorly understood. In this study, an integrated multiple resources analysis was performed and showed that the cellular ncRNAs and proteins targeted by viruses were primarily "hubs" and "bottlenecks" in the human ncRNA/protein-protein interaction. The common proteins targeted by both viral ncRNAs and proteins tended to skew toward higher degrees and betweenness compared with other proteins, showed significant enrichment in the cell death process. Specifically, >800 pairs of human cellular ncRNAs and viral ncRNAs that exhibited a high degree of functional homology were identified, representing potential ncRNA-mediated co-regulation patterns of viral invasion. Additionally, clustering analysis further revealed several distinct viral clusters with obvious functional divergence. Overall, this is the first attempt to systematically explore the invasive mechanism via global ncRNA-associated virus-host crosstalk. Our results provide useful information in comprehensively understanding the viral invasive mechanism.

The collagenases of Vibrio species, many of which are pathogens, have been regarded as an important virulence factor. However, there is little information on the structure and collagenolytic mechanism of Vibrio collagenase. Here, we report the crystal structure of the collagenase module (CM) of Vibrio collagenase VhaC and the conformation of VhaC in solution. Structural and biochemical analyses and molecular dynamics studies reveal that triple-helical collagen is initially recognized by the activator domain, followed by subsequent cleavage by the peptidase domain along with the closing movement of CM. This is different from the peptidolytic mode or the proposed collagenolysis of Clostridium collagenase. We propose a model for the integrated collagenolytic mechanism of VhaC, integrating the functions of VhaC accessory domains and its collagen degradation pattern. This study provides insight into the mechanism of bacterial collagenolysis and helps in structure-based drug design targeting of the Vibrio collagenase.

Cell metabolism plays a pivotal role in tumor progression, and targeting cancer metabolism might effectively kill cancer cells. We aimed to investigate the role of hexokinases in prostate cancer (PCa) and identify a crucial target for PCa treatment.

Intervertebral disc degeneration (IVDD) is a major cause of low back pain. Although the mechanism of degeneration remains unclear, aging has been recognized as a key risk factor for IVDD. Most studies seeking to identify IVDD-associated molecular alterations in the context of human age-related IVDD have focused only on a limited number of proteins. Differential proteomic analysis is an ideal method for comprehensively screening altered protein profiles and identifying the potential pathways related to pathological processes such as disc degeneration.

Dry-cured meat products are worldwide food with high-salt content, and filamentous fungi are beneficial to the maturation process. However, some salt-tolerant strains of Aspergillus and Penicillium produce ochratoxin A (OTA) on these products and thus threaten food safety. In our study, proteomic analysis was performed to reveal the mechanism of adaptability to high-salt environment by Aspergillus ochraceus. Twenty g/L and 70 g/L NaCl substrates were used to provide medium- and high-NaCl content environments, respectively. The NaCl addition could induce fungal growth, but only 20 g/L NaCl addition could induce spore production while 70 g/L repressed it. Proteomics analysis identified 2646 proteins in A. ochraceus fc-1, of which 237 and 251 were differentially expressed with 20 g/L and 70 g/L NaCl addition, respectively. Potential factors affecting fungal growth and development were identified by GO and KEGG analyses of biological process, cellular component, and molecular function terms. The results revealed that ergosterol synthesis pathway was significantly upregulated with 20 g/L and 70 g/L NaCl addition. However, fungal growth and development including OTA production were complex processes associated with many factors including nutrient uptake, cell membrane integrity, cell cycle, energy metabolism, intracellular redox homeostasis, protein synthesis and processing, autophagy, and secondary metabolism. Reactive oxygen species may be an important window to understand the mechanism that medium-salt content was conducive to intracellular signal transduction while high-salt content caused oxidative stress. The findings would help to improve the processes and storage conditions of dry-cured meat products.