Novel integrated computational AMP discovery approaches highlight diversity in the helminth AMP repertoire.

Antimicrobial Peptides (AMPs) are immune effectors that are key components of the invertebrate innate immune system providing protection against pathogenic microbes. Parasitic helminths (phylum Nematoda and phylum Platyhelminthes) share complex interactions with their hosts and closely associated microbiota that are likely regulated by a diverse portfolio of antimicrobial immune effectors including AMPs. Knowledge of helminth AMPs has largely been derived from nematodes, whereas the flatworm AMP repertoire has not been described.

Generation of a mouse SWATH-MS spectral library to quantify 10148 proteins involved in cell reprogramming.

Murine models are amongst the most widely used systems to study biology and pathology. Targeted quantitative proteomic analysis is a relatively new tool to interrogate such systems. Recently the need for relative quantification on hundreds to thousands of samples has driven the development of Data Independent Acquisition methods.

Mitochondrial fusion is required for spermatogonial differentiation and meiosis.

Differentiating cells tailor their metabolism to fulfill their specialized functions. We examined whether mitochondrial fusion is important for metabolic tailoring during spermatogenesis. Acutely after depletion of mitofusins and , spermatogenesis arrests due to failure to accomplish a metabolic shift during meiosis.

Diagnosis of epithelial ovarian cancer using a combined protein biomarker panel.

Background: An early detection tool for EOC was constructed from analysis of biomarker expression data from serum collected during the UKCTOCS.

Methods: This study included 49 EOC cases (19 Type I and 30 Type II) and 31 controls, representing 482 serial samples spanning seven years pre-diagnosis. A logit model was trained by analysis of dysregulation of expression data of four putative biomarkers, (CA125, phosphatidylcholine-sterol acyltransferase, vitamin K-dependent protein Z and C-reactive protein); by scoring the specificity associated with dysregulation from the baseline expression for each individual.

A combined biomarker panel shows improved sensitivity for the early detection of ovarian cancer allowing the identification of the most aggressive type II tumours.

Background: There is an urgent need for biomarkers for the early detection of ovarian cancer (OC). The purpose of this study was to assess whether changes in serum levels of lecithin-cholesterol acyltransferase (LCAT), sex hormone-binding globulin (SHBG), glucose-regulated protein, 78 kDa (GRP78), calprotectin and insulin-like growth factor-binding protein 2 (IGFBP2) are observed before clinical presentation and to assess the performance of these markers alone and in combination with CA125 for early detection.

Methods: This nested case-control study used samples from the United Kingdom Collaborative Trial of Ovarian Cancer Screening trial.

Novel risk models for early detection and screening of ovarian cancer.

Purpose: Ovarian cancer (OC) is the most lethal gynaecological cancer. Early detection is required to improve patient survival. Risk estimation models were constructed for Type I (Model I) and Type II (Model II) OC from analysis of Protein Z, Fibronectin, C-reactive protein and CA125 levels in prospectively collected samples from the United Kingdom Collaborative Trial of Ovarian Cancer Screening (UKCTOCS).

Protein Z: A putative novel biomarker for early detection of ovarian cancer.

Ovarian cancer (OC) has the highest mortality of all gynaecological cancers. Early diagnosis offers an approach to achieving better outcomes. We conducted a blinded-evaluation of prospectively collected preclinical serum from participants in the multimodal group of the United Kingdom Collaborative Trial of Ovarian Cancer Screening.

Cell-specific proteomic analysis in Caenorhabditis elegans.

Proteomic analysis of rare cells in heterogeneous environments presents difficult challenges. Systematic methods are needed to enrich, identify, and quantify proteins expressed in specific cells in complex biological systems including multicellular plants and animals. Here, we have engineered a Caenorhabditis elegans phenylalanyl-tRNA synthetase capable of tagging proteins with the reactive noncanonical amino acid p-azido-L-phenylalanine.

Semiquantitative analysis of clinical heat stress in Clostridium difficile strain 630 using a GeLC/MS workflow with emPAI quantitation.

Clostridium difficile is considered to be the most frequent cause of infectious bacterial diarrhoea in hospitals worldwide yet its adaptive ability remains relatively uncharacterised. Here, we used GeLC/MS and the exponentially modified protein abundance index (emPAI) calculation to determine proteomic changes in response to a clinically relevant heat stress. Reproducibility between both biological and technical replicates was good, and a 37°C proteome of 224 proteins was complemented by a 41°C proteome of 202 proteins at a 1% false discovery rate.

F-box protein FBXL16 binds PP2A-B55α and regulates differentiation of embryonic stem cells along the FLK1+ lineage.

The programmed formation of specific tissues from embryonic stem cells is a major goal of regenerative medicine. To identify points of intervention in cardiac tissue formation, we performed an siRNA screen in murine embryonic stem cells to identify ubiquitin system genes that repress cardiovascular tissue formation. Our screen uncovered an F-box protein, Fbxl16, as a repressor of one of the earliest steps in the cardiogenic lineage: FLK1+ progenitor formation.

Comprehensive proteomic profiling of outer membrane vesicles from Campylobacter jejuni.

Unlabelled: Gram-negative bacteria constitutively release outer membrane vesicles (OMVs) during cell growth that play significant roles in bacterial survival, virulence and pathogenesis. In this study, comprehensive proteomic analysis of OMVs from a human gastrointestinal pathogen Campylobacter jejuni NCTC11168 was performed using high-resolution mass spectrometry. The OMVs of C.

Identification of secreted bacterial proteins by noncanonical amino acid tagging.

Pathogenic microbes have evolved complex secretion systems to deliver virulence factors into host cells. Identification of these factors is critical for understanding the infection process. We report a powerful and versatile approach to the selective labeling and identification of secreted pathogen proteins.

Perturbations to the ubiquitin conjugate proteome in yeast δubx mutants identify Ubx2 as a regulator of membrane lipid composition.

Yeast Cdc48 (p97/VCP in human cells) is a hexameric AAA ATPase that is thought to use ATP hydrolysis to power the segregation of ubiquitin-conjugated proteins from tightly bound partners. Current models posit that Cdc48 is linked to its substrates through adaptor proteins, including a family of seven proteins (13 in human) that contain a Cdc48-binding UBX domain. However, few substrates for specific UBX proteins are known, and hence the generality of this hypothesis remains untested.

Cand1 promotes assembly of new SCF complexes through dynamic exchange of F box proteins.

The modular SCF (Skp1, cullin, and F box) ubiquitin ligases feature a large family of F box protein substrate receptors that enable recognition of diverse targets. However, how the repertoire of SCF complexes is sustained remains unclear. Real-time measurements of formation and disassembly indicate that SCF(Fbxw7) is extraordinarily stable, but, in the Nedd8-deconjugated state, the cullin-binding protein Cand1 augments its dissociation by one-million-fold.

Comprehensive profiling of N-linked glycosylation sites in HeLa cells using hydrazide enrichment.

The adenocarcinoma cell line HeLa serves as a model system for cancer research in general and cervical cancer in particular. In this study, hydrazide enrichment in combination with state-of-the art nanoLC-MS/MS analysis was used to profile N-linked glycosites in HeLa cells. N-Linked glycoproteins were selectively enriched in HeLa cells by the hydrazide capture method, which isolates all glycoproteins independent of their glycans.

Designer reagents for mass spectrometry-based proteomics: clickable cross-linkers for elucidation of protein structures and interactions.

We present novel homobifunctional amine-reactive clickable cross-linkers (CXLs) for investigation of three-dimensional protein structures and protein-protein interactions (PPIs). CXLs afford consolidated advantages not previously available in a simple cross-linker, including (1) their small size and cationic nature at physiological pH, resulting in good water solubility and cell-permeability, (2) an alkyne group for bio-orthogonal conjugation to affinity tags via the click reaction for enrichment of cross-linked peptides, (3) a nucleophilic displacement reaction involving the 1,2,3-triazole ring formed in the click reaction, yielding a lock-mass reporter ion for only clicked peptides, and (4) higher charge states of cross-linked peptides in the gas-phase for augmented electron transfer dissociation (ETD) yields. Ubiquitin, a lysine-abundant protein, is used as a model system to demonstrate structural studies using CXLs.

Protein interaction profiling of the p97 adaptor UBXD1 points to a role for the complex in modulating ERGIC-53 trafficking.

UBXD1 is a member of the poorly understood subfamily of p97 adaptors that do not harbor a ubiquitin association domain or bind ubiquitin-modified proteins. Of clinical importance, p97 mutants found in familial neurodegenerative conditions Inclusion Body Myopathy Paget's disease of the bone and/or Frontotemporal Dementia and Amyotrophic Lateral Sclerosis are defective at interacting with UBXD1, indicating that functions regulated by a p97-UBXD1 complex are altered in these diseases. We have performed liquid chromatography-mass spectrometric analysis of UBXD1-interacting proteins to identify pathways in which UBXD1 functions.

Click chemistry facilitates formation of reporter ions and simplified synthesis of amine-reactive multiplexed isobaric tags for protein quantification.

We report the development of novel reagents for cell-level protein quantification, referred to as Caltech isobaric tags (CITs), which offer several advantages in comparison with other isobaric tags (e.g., iTRAQ and TMT).

LogViewer: a software tool to visualize quality control parameters to optimize proteomics experiments using Orbitrap and LTQ-FT mass spectrometers.

Visualization tools that allow both optimization of instrument's parameters for data acquisition and specific quality control (QC) for a given sample prior to time-consuming database searches have been scarce until recently and are currently still not freely available. To address this need, we have developed the visualization tool LogViewer, which uses diagnostic data from the RAW files of the Thermo Orbitrap and linear trap quadrupole-Fourier transform (LTQ-FT) mass spectrometers to monitor relevant metrics. To summarize and visualize the performance on our test samples, log files from RawXtract are imported and displayed.

Novel proteomic tools reveal essential roles of SRP and importance of proper membrane protein biogenesis.

The signal recognition particle (SRP), which mediates cotranslational protein targeting to cellular membranes, is universally conserved and essential for bacterial and mammalian cells. However, the current understanding of the role of SRP in cell physiology and pathology is still poor, and the reasons behind its essential role in cell survival remain unclear. Here, we systematically analyzed the consequences of SRP loss in E.

Quantitative proteomic analysis of the heat stress response in Clostridium difficile strain 630.

Clostridium difficile is a serious nosocomial pathogen whose prevalence worldwide is increasing. Postgenomic technologies can now be deployed to develop understanding of the evolution and diversity of this important human pathogen, yet little is known about the adaptive ability of C. difficile.

Proteomics in the microbial sciences.

Mass spectrometry based proteomics is now widely used in the microbial sciences. In conjunction with transcriptomics it has greatly enhanced the field of microbial biology and has provide microbiologists with unparalleled insights into cellular processes and functions. Proteomics allows the dynamic nature of the entire protein network to be mapped providing a deeper understanding of microbial systems, their evolution and role in disease states.

Broad activation of the ubiquitin-proteasome system by Parkin is critical for mitophagy.

Parkin, an E3 ubiquitin ligase implicated in Parkinson's disease, promotes degradation of dysfunctional mitochondria by autophagy. Using proteomic and cellular approaches, we show that upon translocation to mitochondria, Parkin activates the ubiquitin-proteasome system (UPS) for widespread degradation of outer membrane proteins. This is evidenced by an increase in K48-linked polyubiquitin on mitochondria, recruitment of the 26S proteasome and rapid degradation of multiple outer membrane proteins.

The steady-state repertoire of human SCF ubiquitin ligase complexes does not require ongoing Nedd8 conjugation.

The human genome encodes 69 different F-box proteins (FBPs), each of which can potentially assemble with Skp1-Cul1-RING to serve as the substrate specificity subunit of an SCF ubiquitin ligase complex. SCF activity is switched on by conjugation of the ubiquitin-like protein Nedd8 to Cul1. Cycles of Nedd8 conjugation and deconjugation acting in conjunction with the Cul1-sequestering factor Cand1 are thought to control dynamic cycles of SCF assembly and disassembly, which would enable a dynamic equilibrium between the Cul1-RING catalytic core of SCF and the cellular repertoire of FBPs.

Proteomic analysis of the insoluble subproteome of Clostridium difficile strain 630.

Clostridium difficile, a Gram-positive spore-forming anaerobe, causes infections in humans ranging from mild diarrhoeal to potentially life-threatening pseudomembranous colitis. The availability of genomic information for a range of C. difficile strains affords researchers the opportunity to better understand not only the evolution of these organisms but also their basic physiology and biochemistry.