Plasma Proteome Signature of Sepsis: a Functionally Connected Protein Network

Sepsis is an extreme host response to infection that leads to loss of organ function and cardiovascular integrity. Mortality from sepsis is on the rise. Despite more than three decades of research and clinical trials, specific diagnostic and therapeutic strategies for sepsis are still absent. The use of LFQ‐ and TMT‐based quantitative proteomics is reported here to study the plasma proteome in five mouse models of sepsis. A knowledge‐based interpretation of the data reveals a protein network with extensive connectivity through documented functional or physical interactions. The individual proteins in the network all have a documented role in sepsis and are known to be extracellular. The changes in protein abundance observed in the mouse models of sepsis have for the most part the same directionality (increased or decreased abundance) as reported in the literature for human sepsis. This network has been named the Plasma Proteome Signature of Sepsis (PPSS). The PPSS is a quantifiable molecular readout that can supplant the current symptom‐based approach used to diagnose sepsis. This type of molecular interpretation of sepsis, its progression, and its response to therapeutic intervention are an important step in advancing our understanding of sepsis, and for discovering and evaluating new therapeutic strategies.     

A Fast and Economical Sample Preparation Protocol for Interaction Proteomics Analysis

A simple and fast immunoprecipitation (IP) protocol is designed with the sample preparation incorporated, applicable to both low and high throughput. This new protocol combines two procedures based on magnetic beads in 96‐well plate format. Protein complexes are captured by antibodies and magnetic beads conjugated with protein A. Proteins are washed and on‐bead digested by using Single‐Pot solid‐phase sample preparation (SP3). The whole IP‐SP3 approach can be completed in one day, which is considerably faster compared to the classical approach. No major quantitative differences are found between SP3 and FASP (filter‐aided sample preparation) or a longer incubation protocol. Taken together, the IP‐SP3 protocol is a fast and economical approach easily applicable for large‐scale protein interactome analysis.     

Identification of Prolificacy‐Related Differentially Expressed Proteins from Sheep (Ovis aries) Hypothalamus by Comparative Proteomics

Reproduction, as a physiologically complex process, can significantly affect the development of the sheep industry. However, a lack of overall understanding to sheep fecundity has long blocked the progress in sheep breeding and husbandry. In the present study, the aim is to identify differentially expressed proteins (DEPs) from hypothalamus in sheep without FecB mutation in two comparison groups: polytocous (PF) versus monotocous (MF) sheep at follicular phase and polytocous (PL) versus monotocous (ML) sheep at luteal phase. Totally 5058 proteins are identified in sheep hypothalamus, where 22 in PF versus MF, and 39 proteins in PL versus ML are differentially expressed, respectively. A functional analysis is then conducted including Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis to reveal the potential roles of these DEPs. The proteins ENSOARP00000020097, ENSOARP00000006714, growth hormone (GH), histone deacetylase 4 (HDAC4), and 5′‐3′ exoribonuclease 2 (XRN2) in PF versus MF, and bcl‐2‐associated athanogene 4 (BAG4), insulin‐like growth factor‐1 receptor (IGF1R), hydroxysteroid 11‐beta dehydrogenase 1 (HSD11B1), and transthyretin (TTR) in PL versus ML appear to modulate reproduction, presumably by influencing the activities of gonadotropin‐releasing hormone (GnRH). This study provides an alternative method to identify DEPs associated with sheep prolificacy from the hypothalamus. The mass spectrometry data are available via ProteomeXchange with identifier PXD013822.     

Oncogenic Regulation of Extracellular Vesicle Proteome and Heterogeneity

Mutational and epigenetic driver events profoundly alter intercellular communication pathways in cancer. This effect includes deregulated release, molecular composition, and biological activity of extracellular vesicles (EVs), membranous cellular fragments ranging from a few microns to less than 100 nm in diameter and filled with bioactive molecular cargo (proteins, lipids, and nucleic acids). While EVs are usually classified on the basis of their physical properties and biogenetic mechanisms, recent analyses of their proteome suggest a larger than expected molecular diversity, a notion that is also supported by multicolour nano‐flow cytometry and other emerging technology platforms designed to analyze single EVs. Both protein composition and EV diversity are markedly altered by oncogenic transformation, epithelial to mesenchymal transition, and differentiation of cancer stem cells. Interestingly, only a subset of EVs released from mutant cells may carry oncogenic proteins (e.g., EGFRvIII), hence, these EVs are often referred to as “oncosomes”. Indeed, oncogenic transformation alters the repertoire of EV‐associated proteins, increases the presence of pro‐invasive cargo, and alters the composition of distinct EV populations. Molecular profiling of single EVs may reveal a more intricate effect of transforming events on the architecture of EV populations in cancer and shed new light on their biological role and diagnostic utility.     

Identification of Rabbit Oviductal Fluid Proteins Involved in Pre‐Fertilization Processes by Quantitative Proteomics

Oviductal fluid (ODF) proteins modulate and support reproductive processes in the oviduct. In the present study, proteins involved in the biological events that precede fertilization have been identified in the rabbit ODF proteome, isolated from the ampulla and isthmus of the oviduct at different time points within 8 h after intrauterine insemination. A workflow is used that integrates lectin affinity capture with stable‐isotope dimethyl labeling prior to nanoLC‐MS/MS analysis. In total, over 400 ODF proteins, including 214 lectin enriched glycoproteins, are identified and quantified. Selected data are validated by Western blot analysis. Spatiotemporal alterations in the abundance of ODF proteins in response to insemination are detected by global analysis. A subset of 63 potentially biologically relevant ODF proteins is identified, including extracellular matrix components, chaperones, oxidoreductases, and immunity proteins. Functional enrichment analysis reveals an altered peptidase regulator activity upon insemination. In addition to protein identification and abundance changes, N‐glycopeptide analysis further identifies 281 glycosites on 199 proteins. Taken together, these results show, for the first time, the evolving oviductal milieu early upon insemination. The identified proteins are likely those that modulate in vitro processes, including spermatozoa function.     

Proteomic Analysis of Horseweed (Conyza canadensis) Subjected to Caprylic Acid Stress

Caprylic acid (CAP) is anticipated to be a potential biocontrol herbicide in the control of weeds, however the molecular mechanism of how CAP affects weeds is poorly understood. Here, the physiological and biochemical (protein‐level) changes in horseweed (Conyza canadensis L.) are studied under CAP treatment, with infrared gas analyzer and label‐free quantitative proteomics methods. In total, 112 differentially‐accumulated proteins (DAPs) (>1.5 fold change, p < 0.05) are present between treated horseweed and control samples, with 46 up‐regulated and 66 down‐regulated proteins. These DAPs are involved in 28 biochemical pathways, including photosynthesis pathways. In particular, six photosynthesis proteins show significant abundance changes in the CAP‐treated horseweed. The qRT‐PCR results confirm three of the six genes involved in photosynthesis. Moreover, by measuring photosynthesis characteristics, CAP was shown to decrease photosynthetic rate, stomatal conductance, intercellular CO₂ concentration, and the transpiration rate of horseweed. These results suggest that photosystem I is one of the main biological processes involved in the response of horseweed to CAP.     

Quantitative Proteome Analysis of Brain Subregions and Spinal Cord from Experimental Autoimmune Encephalomyelitis Mice by TMT‐Based Mass Spectrometry

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS); its cause is unknown. To understand the pathogenesis of MS, researchers often use the experimental autoimmune encephalomyelitis (EAE) mouse model. Here, the aim is to build a proteome map of the biological changes that occur during MS at the major onset sites—the brain and the spinal cord. Quantitative proteome profiling is performed in five specific brain regions and the spinal cord of EAE and healthy mice with high‐resolution mass spectrometry based on tandem mass tags. On average, 7400 proteins per region are quantified, with the most differentially expressed proteins in the spinal cord (1691), hippocampus (104), frontal cortex (83), cerebellum (63), brainstem (50), and caudate nucleus (41). Moreover, region‐specific and commonly expressed proteins in each region are identified and bioinformatics analysis is performed. Pathway analysis reveals that protein clusters resemble their functions in disease pathogenesis (i.e., by inducing inflammatory responses, immune activation, and cell–cell adhesion). In conclusion, the study provides an understanding of the pathogenesis of MS in the EAE animal model. It is expected that the comprehensive proteome map of the brain and spinal cord can be used to identify biomarkers for the pathogenesis of MS.     

Plant–Microbe Symbiosis: What Has Proteomics Taught Us?

Beneficial microbes have a positive impact on the productivity and fitness of the host plant. A better understanding of the biological impacts and underlying mechanisms by which the host derives these benefits will help to address concerns around global food production and security. The recent development of omics‐based technologies has broadened our understanding of the molecular aspects of beneficial plant–microbe symbiosis. Specifically, proteomics has led to the identification and characterization of several novel symbiosis‐specific and symbiosis‐related proteins and post‐translational modifications that play a critical role in mediating symbiotic plant–microbe interactions and have helped assess the underlying molecular aspects of the symbiotic relationship. Integration of proteomic data with other “omics” data can provide valuable information to assess hypotheses regarding the underlying mechanism of symbiosis and help define the factors affecting the outcome of symbiosis. Herein, an update is provided on the current and potential applications of symbiosis‐based “omic” approaches to dissect different aspects of symbiotic plant interactions. The application of proteomics, metaproteomics, and secretomics as enabling approaches for the functional analysis of plant‐associated microbial communities is also discussed.