Shedding light on black boxes in protein identification

Performing a well thought-out proteomics data analysis can be a daunting task, especially for newcomers to the field. Even researchers experienced in the proteomics field can find it challenging to follow existing publication guidelines for MS-based protein identification and characterization in detail. One of the primary goals of bioinformatics is to enable any researcher to interpret the vast amounts of data generated in modern biology, by providing user-friendly and robust end-user applications, clear documentation, and corresponding teaching materials. In that spirit, we here present an extensive tutorial for peptide and protein identification, available at http://compomics.com/bioinformatics-for-proteomics. The material is completely based on freely available and open-source tools, and has already been used and refined at numerous international courses over the past 3 years. During this time, it has demonstrated its ability to allow even complete beginners to intuitively conduct advanced bioinformatics workflows, interpret the results, and understand their context. This tutorial is thus aimed at fully empowering users, by removing black boxes in the proteomics informatics pipeline.acceptedVersio

SpectroGene: A Tool for Proteogenomic Annotations Using Top-Down Spectra

In the past decade, proteogenomics has emerged as a valuable technique that contributes to the state-of-the-art in genome annotation; however, previous proteogenomic studies were limited to bottom-up mass spectrometry and did not take advantage of top-down approaches. We show that top-down proteogenomics allows one to address the problems that remained beyond the reach of traditional bottom-up proteogenomics. In particular, we show that top-down proteogenomics leads to the discovery of previously unannotated genes even in extensively studied bacterial genomes and present SpectroGene, a software tool for genome annotation using top-down tandem mass spectra. We further show that top-down proteogenomics searches (against the six-frame translation of a genome) identify nearly all proteoforms found in traditional top-down proteomics searches (against the annotated proteome). SpectroGene is freely available at http://github.com/fenderglass/SpectroGene

Mzion enables deep and precise identification of peptides in data-dependent acquisition proteomics

Sensitive and reliable identification of proteins and peptides pertains the basis of proteomics. We introduce Mzion, a new database search tool for data-dependent acquisition (DDA) proteomics. Our tool utilizes an intensity tally strategy and achieves generally a higher performance in terms of depth and precision across 20 datasets, ranging from large-scale to single-cell proteomics. Compared to several other search engines, Mzion matches on average 20% more peptide spectra at tryptic enzymatic specificity and 80% more at no enzymatic specificity from six large-scale, global datasets. Mzion also identifies more phosphopeptide spectra that can be explained by fewer proteins, demonstrated by six large-scale, local datasets corresponding to the global data. Our findings highlight the potential of Mzion for improving proteomic analysis and advancing our understanding of protein biology

Thioloxidoreductase HP0231 of Helicobacter pylori impacts HopQ-dependent CagA translocation

Thioloxidoreductase HP0231 of Helicobacter pylori plays essential roles in gastric colonization and related gastric pathology. Comparative proteomics and analysis of complexes between HP0231 and its protein substrates suggested that several Hop proteins are its targets. HP0231 is a dimeric oxidoreductase that functions in an oxidizing Dsb (disulfide bonds) pathway of H. pylori. H. pylori HopQ possesses six cysteine residues, which generate three consecutive disulfide bridges. Comparison of the redox state of HopQ in wild-type cells to that in hp0231-mutated cells clearly indicated that HopQ is a substrate of HP0231. HopQ binds CEACAM1, 3, 5 and 6 (carcinoembryonic antigen-related cell adhesion molecules). This interaction enables T4SS-mediated translocation of CagA into host cells and induces host signaling. Site directed mutagenesis of HopQ (changing cysteine residues into serine) and analysis of the functioning of HopQ variants showed that HP0231 influences the delivery of CagA into host cells, in part through its impact on HopQ redox state. Introduction of a C382S mutation into HopQ significantly affects its reaction with CEACAM receptors, which disturbs T4SS functioning and CagA delivery. An additional effect of HP0231 on other adhesins and their redox state, resulting in their functional impairment, cannot be excluded

Identification of a target protein of Hydra actinoporin-like toxin-1 (HALT-1)using GST affinity purification and SILAC-based quantitative proteomics

Hydra actinoporin-like toxin-1 (HALT-1) is a 20.8 kDa pore-forming toxin isolated from Hydra magnipapillata. HALT-1 shares structural similarity with actinoporins, a family that is well known for its haemolytic and cytolytic activity. However, the precise pore-forming mechanism of HALT-1 remains an open question since little is known about the specific target binding for HALT-1. For this reason, a comprehensive proteomic analysis was performed using affinity purification and SILAC-based mass spectrometry to identify potential protein-protein interactions between mammalian HeLa cell surface proteins and HALT-1. A total of 4 mammalian proteins was identified, of which only folate receptor alpha was further verified by ELISA. Our preliminary results highlight an alternative-binding mode of HALT-1 to the human plasma membrane. This is the first evidence showing that HALT-1, an actinoporin-like protein,binds to a membrane protein, the folate receptor alpha. This study would advance our understanding of the molecular basis of toxicity of pore-forming toxins and provide new insights in the production of more potent inhibitors for the toxin-membrane receptor interactions

Plasmodium falciparum CRK4 directs continuous rounds of DNA replication during schizogony.

: Plasmodium parasites, the causative agents of malaria, have evolved a unique cell division cycle in the clinically relevant asexual blood stage of infection(1). DNA replication commences approximately halfway through the intracellular development following invasion and parasite growth. The schizont stage is associated with multiple rounds of DNA replication and nuclear division without cytokinesis, resulting in a multinucleated cell. Nuclei divide asynchronously through schizogony, with only the final round of DNA replication and segregation being synchronous and coordinated with daughter cell assembly(2,3). However, the control mechanisms for this divergent mode of replication are unknown. Here, we show that the Plasmodium-specific kinase PfCRK4 is a key cell-cycle regulator that orchestrates multiple rounds of DNA replication throughout schizogony in Plasmodium falciparum. PfCRK4 depletion led to a complete block in nuclear division and profoundly inhibited DNA replication. Quantitative phosphoproteomic profiling identified a set of PfCRK4-regulated phosphoproteins with greatest functional similarity to CDK2 substrates, particularly proteins involved in the origin of replication firing. PfCRK4 was required for initial and subsequent rounds of DNA replication during schizogony and, in addition, was essential for development in the mosquito vector. Our results identified an essential S-phase promoting factor of the unconventional P. falciparum cell cycle. PfCRK4 is required for both a prolonged period of the intraerythrocytic stage of Plasmodium infection, as well as for transmission, revealing a broad window for PfCRK4-targeted chemotherapeutics.<br/

Quantitative comparative analysis of human erythrocyte surface proteins between individuals from two genetically distinct populations

Abstract: Red blood cells (RBCs) play a critical role in oxygen transport, and are the focus of important diseases including malaria and the haemoglobinopathies. Proteins at the RBC surface can determine susceptibility to disease, however previous studies classifying the RBC proteome have not used specific strategies directed at enriching cell surface proteins. Furthermore, there has been no systematic analysis of variation in abundance of RBC surface proteins between genetically disparate human populations. These questions are important to inform not only basic RBC biology but additionally to identify novel candidate receptors for malarial parasites. Here, we use ‘plasma membrane profiling’ and tandem mass tag-based mass spectrometry to enrich and quantify primary RBC cell surface proteins from two sets of nine donors from the UK or Senegal. We define a RBC surface proteome and identify potential Plasmodium receptors based on either diminished protein abundance, or increased variation in RBCs from West African individuals

Proteome-wide systems analysis of a cellulosic biofuel-producing microbe

We apply mass spectrometry-based ReDi proteomics to quantify the Clostridium phytofermentans proteome during fermentation of cellulosic substrates. ReDi proteomics gives accurate, low-cost quantification of an extra and intracellular microbial proteome. When combined with physiological measurements, these methods form a general systems biology strategy to evaluate the efficiency of cellulosic bioconversion and to identify enzyme targets to engineer for improving this process.C. phytofermentans expressed more than 100 carbohydrate-active enzymes, of which distinct subsets were upregulated on cellulose and hemicellulose. Numerous extracellular enzymes cleave insoluble plant polysaccharides into oligosaccharides, which are transported into the cell to be further degraded by intracellular carbohydratases. Sugars are catabolized by EMP glycolysis incorporating alternative glycolytic enzymes to maximize the ATP yield of anaerobic metabolism.During cellulosic fermentation, cells adhered to the substrate and altered metabolic processes such as upregulation of tryptophan and nicotinamide synthesis proteins and repression of proteins for fatty acid metabolism and cell motility. These diverse metabolic changes highlight how a systems approach can identify novel ways to optimize cellulosic fermentation