Using proteomics to identify host cell interaction partners for VgrG and IglJ.

Francisella tularensis is a highly virulent intracellular bacterium and the causative agent of tularemia. The disease is characterized by the suboptimal innate immune response and consequently by the impaired adaptive immunity. The virulence of this pathogen depends on proteins encoded by a genomic island termed the Francisella Pathogenicity Island (FPI). However, the precise biological roles of most of the FPI-encoded proteins remain to be clarified. In this study, we employed stable isotope labeling by amino acids in cell culture (SILAC) in combination with affinity protein purification coupled with liquid chromatography-mass spectrometry to identify potential protein-effector binding pairs for two FPI virulence effectors IglJ and VgrG. Our results may indicate that while the IglJ protein interactions primarily affect mitochondria, the VgrG interactions affect phagosome and/or autophagosome biogenesis via targeting components of the host's exocyst complex.

Cytokines and radiation-induced pulmonary injuries.

Radiation therapy is one of the most common treatment strategies for thorax malignancies. One of the considerable limitations of this therapy is its toxicity to normal tissue. The lung is the major dose-limiting organ for radiotherapy. That is because ionizing radiation produces reactive oxygen species that induce lesions, and not only is tumor tissue damaged, but overwhelming inflammatory lung damage can occur in the alveolar epithelium and capillary endothelium. This damage may result in radiation-induced pneumonitis and/or fibrosis. While describing the lung response to irradiation generally, the main focus of this review is on cytokines and their roles and functions within the individual stages. We discuss the relationship between radiation and cytokines and their direct and indirect effects on the formation and development of radiation injuries. Although this topic has been intensively studied and discussed for years, we still do not completely understand the roles of cytokines. Experimental data on cytokine involvement are fragmented across a large number of experimental studies; hence, the need for this review of the current knowledge. Cytokines are considered not only as molecular factors involved in the signaling network in pathological processes, but also for their diagnostic potential. A concentrated effort has been made to identify the significant immune system proteins showing positive correlation between serum levels and tissue damages. Elucidating the correlations between the extent and nature of radiation-induced pulmonary injuries and the levels of one or more key cytokines that initiate and control those damages may improve the efficacy of radiotherapy in cancer treatment and ultimately the well-being of patients.

Conventional-Flow Liquid Chromatography-Mass Spectrometry for Exploratory Bottom-Up Proteomic Analyses.

Due to its sensitivity and productivity, bottom-up proteomics based on liquid chromatography-mass spectrometry (LC-MS) has become the core approach in the field. The de facto standard LC-MS platform for proteomics operates at sub-µL/min flow rates, and nanospray is required for efficiently introducing peptides into a mass spectrometer. Although this is almost a "dogma", this view is being reconsidered in light of developments in highly efficient chromatographic columns, and especially with the introduction of exceptionally sensitive MS instruments. Although conventional-flow LC-MS platforms have recently penetrated targeted proteomics successfully, their possibilities in discovery-oriented proteomics have not yet been thoroughly explored. Our objective was to determine what are the extra costs and what optimization and adjustments to a conventional-flow LC-MS system must be undertaken to identify a comparable number of proteins as can be identified on a nanoLC-MS system. We demonstrate that the amount of a complex tryptic digest needed for comparable proteome coverage can be roughly 5-fold greater, providing the column dimensions are properly chosen, extra-column peak dispersion is minimized, column temperature and flow rate are set to levels appropriate for peptide separation, and the composition of mobile phases is fine-tuned. Indeed, we identified 2 835 proteins from 2 µg of HeLa cells tryptic digest separated during a 60 min gradient at 68 µL/min on a 1.0 mm × 250 mm column held at 55 °C and using an aqua-acetonitrile mobile phases containing 0.1% formic acid, 0.4% acetic acid, and 3% dimethyl sulfoxide. Our results document that conventional-flow LC-MS is an attractive alternative for bottom-up exploratory proteomics.