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.

• MeSH
• Adaptive Immunity physiology   MeSH
• Bacterial Proteins metabolism   MeSH
• Francisella tularensis metabolism   MeSH
• Genomic Islands * MeSH
• Mass Spectrometry MeSH
• Immunity, Innate physiology   MeSH
• Proteomics MeSH
• Gene Expression Regulation, Bacterial * MeSH
• Tularemia microbiology   MeSH
• Virulence MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH

Mass spectrometry (MS) is a powerful and sensitive method often used for the identification of phosphoproteins. However, in phosphoproteomics, there is an identified need to compensate for the low abundance, insufficient ionization, and suppression effects of non-phosphorylated peptides. These may hamper the subsequent liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis, resulting in incomplete phosphoproteome characterization, even when using high-resolution instruments. To overcome these drawbacks, we present here an effective microgradient chromatographic technique that yields specific fractions of enriched phosphopeptides compatible with LC-MS/MS analysis. The purpose of our study was to increase the number of identified phosphopeptides, and thus, the coverage of the sample phosphoproteome using the reproducible and straightforward fractionation method. This protocol includes a phosphopeptide enrichment step followed by the optimized microgradient fractionation of enriched phosphopeptides and final LC-MS/MS analysis of the obtained fractions. The simple fractionation system consists of a gas-tight microsyringe delivering the optimized gradient mobile phase to reversed-phase microcolumn. Our data indicate that combining the phosphopeptide enrichment with the microgradient separation is a promising technique for in-depth phosphoproteomic analysis due to moderate input material requirements and more than 3-fold enhanced protein identification.

• Keywords
• acetonitrile, enrichment, fractionation, gradient, mass spectrometry, phosphopeptides, titanium dioxide,
• MeSH
• Acetonitriles chemistry   MeSH
• Chemical Fractionation methods   MeSH
• Chromatography, Liquid methods   MeSH
• Phosphopeptides chemistry   MeSH
• Phosphoproteins metabolism   MeSH
• Hydrogen-Ion Concentration MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Proteome MeSH
• Proteomics MeSH
• Tandem Mass Spectrometry methods   MeSH
• Titanium chemistry   MeSH
• Pressure MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• acetonitrile MeSH Browser
• Acetonitriles MeSH
• Phosphopeptides MeSH
• Phosphoproteins MeSH
• Proteome MeSH
• Titanium MeSH
• titanium dioxide MeSH Browser

Transferrins are a superfamily of iron-binding proteins and are recognized as multifunctional proteins. In the present study, transcriptomic and proteomic methods were used to identify transferrins in the reproductive organs and sperm of out-of-spawning and spermiating sterlet (Acipenser ruthenus) males. The results showed that seven transferrin transcripts were identified in the transcriptome of sterlet, and these transcripts were qualified as two different transferrin genes, serotransferrin and melanotransferrin, with several isoforms present for serotransferrin. The relative abundance of serotransferrin isoforms was higher in the kidneys and Wolffian ducts in the spermiating males compared to out-of-spawning males. In addition, transferrin was immunodetected in sterlet seminal plasma, but not in sterlet spermatozoa extract. Mass spectrometry identification of transferrin in seminal plasma but not in spermatozoa corroborates immunodetection. The identification of transferrin in the reproductive organs and seminal plasma of sterlet in this study provides the potential function of transferrin during sturgeon male reproduction.

• Keywords
• Wolffian duct, kidney, sperm, testes, transcriptome,
• Publication type
• Journal Article MeSH

This work reports highly selective phosphopeptide enrichment using amorphous TiO2 nanotubes (TiO2NTs) and the same material decorated with superparamagnetic Fe3O4 nanoparticles (TiO2NTs@Fe3O4NPs). TiO2NTs and TiO2NTs@Fe3O4NPs materials were applied for phosphopeptide enrichment both from a simple peptide mixture (tryptic digest of bovine serum albumin and α-casein) and from a complex peptide mixture (tryptic digest of Jurkat T cell lysate). The obtained enrichment efficiency and selectivity for phosphopeptides of TiO2NTs and TiO2NTs@Fe3O4NPs were increased to 28.7 and 25.3%, respectively, as compared to those of the well-established TiO2 microspheres. The enrichment protocol was extended for a second elution step facilitating the identification of additional phosphopeptides. It further turned out that both types of amorphous TiO2 nanotubes provide qualitatively new physicochemical features that are clearly advantageous for highly selective phosphopeptide enrichment. This has been confirmed experimentally resulting in substantial reduction of non-phosphorylated peptides in the enriched samples. In addition, TiO2NTs@Fe3O4NPs combine high selectivity and ease of handling due to the superparamagnetic character of the material. The presented materials and performances are further promising for applications toward a whole range of other types of biomolecules to be treated in a similar fashion.

• Publication type
• Journal Article MeSH

BACKGROUND: The availability of tick in vitro cell culture systems has facilitated many aspects of tick research, including proteomics. However, certain cell lines have shown a tissue-specific response to infection. Thus, a more thorough characterization of tick cell lines is necessary. Proteomic comparative studies of various tick cell lines will contribute to more efficient application of tick cell lines as model systems for investigation of host-vector-pathogen interactions. RESULTS: Three cell lines obtained from a hard tick, Ixodes ricinus, and two from I. scapularis were investigated. A cell mass spectrometry approach (MALDI-TOF MS) was applied, as well as classical proteomic workflows. Using PCA, tick cell line MS profiles were grouped into three clusters comprising IRE/CTVM19 and ISE18, IRE11 and IRE/CTVM20, and ISE6 cell lines. Two other approaches confirmed the results of PCA: in-solution digestion followed by nanoLC-ESI-Q-TOF MS/MS and 2D electrophoresis. The comparison of MS spectra of the cell lines and I. ricinus tick organs revealed 29 shared peaks. Of these, five were specific for ovaries, three each for gut and salivary glands, and one for Malpighian tubules. For the first time, characteristic peaks in MS profiles of tick cell lines were assigned to proteins identified in acidic extracts of corresponding cell lines. CONCLUSIONS: Several organ-specific MS signals were revealed in the profiles of tick cell lines.

• Keywords
• Biotyping, MALDI-TOF MS, Tick, Tick cell line, Tick organs,
• MeSH
• Electrophoresis, Gel, Two-Dimensional MeSH
• Cell Line * cytology   metabolism   MeSH
• Insect Proteins metabolism   MeSH
• Ixodes cytology   MeSH
• Proteomics MeSH
• Salivary Glands cytology   MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization * MeSH
• Tandem Mass Spectrometry MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH
• Names of Substances
• Insect Proteins MeSH

Although the role of high-risk human papillomaviruses (hrHPVs) as etiological agents in cancer development has been intensively studied during the last decades, there is still the necessity of understanding the impact of the HPV E6 and E7 oncogenes on host cells, ultimately leading to malignant transformation. Here, we used newly established immortalized human keratinocytes with a well-defined HPV16 E6E7 expression cassette to get a more complete and less biased overview of global changes induced by HPV16 by employing transcriptome sequencing (RNA-Seq) and stable isotope labeling by amino acids in cell culture (SILAC). This is the first study combining transcriptome and proteome data to characterize the impact of HPV oncogenes in human keratinocytes in comparison with their virus-negative counterparts. To enhance the informative value and accuracy of the RNA-Seq data, four different bioinformatic workflows were used. We identified potential novel upstream regulators (e.g., CNOT7, SPDEF, MITF, and PAX5) controlling distinct clusters of genes within the HPV-host cell network as well as distinct factors (e.g., CPPED1, LCP1, and TAGLN) with essential functions in cancer. Validated results in this study were compared to data sets from The Cancer Genome Atlas (TCGA), demonstrating that several identified factors were also differentially expressed in cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) and HPV-positive head and neck squamous cell carcinomas (HNSCs). This highly integrative approach allows the identification of novel HPV-induced cellular changes that are also reflected in cancer patients, providing a promising omics data set for future studies in both basic and translational research.IMPORTANCE Human papillomavirus (HPV)-associated cancers still remain a big health problem, especially in developing countries, despite the availability of prophylactic vaccines. Although HPV oncogenes have been intensively investigated for decades, a study applying recent advances in RNA-Seq and quantitative proteomic approaches to a precancerous model system with well-defined HPV oncogene expression alongside HPV-negative parental cells has been missing until now. Here, combined omics analyses reveal global changes caused by the viral oncogenes in a less biased way and allow the identification of novel factors and key cellular networks potentially promoting malignant transformation. In addition, this system also provides a basis for mechanistic research on novel key factors regulated by HPV oncogenes, especially those that are confirmed in vivo in cervical cancer as well as in head and neck cancer patient samples from TCGA data sets.

• Keywords
• HPV, RNA-Seq, SILAC, TCGA, cervical cancer, head and neck cancer, integrated analysis,
• MeSH
• Adenocarcinoma genetics   virology   MeSH
• Squamous Cell Carcinoma of Head and Neck genetics   virology   MeSH
• Gene Regulatory Networks * MeSH
• Carcinogenesis genetics   MeSH
• Keratinocytes virology   MeSH
• Humans MeSH
• Human papillomavirus 16 genetics   MeSH
• Cell Transformation, Neoplastic MeSH
• Uterine Cervical Neoplasms genetics   virology   MeSH
• Oncogene Proteins, Viral genetics   MeSH
• Proteome genetics   MeSH
• Proteomics MeSH
• Carcinoma, Squamous Cell genetics   virology   MeSH
• Gene Expression Profiling MeSH
• Transcriptome * MeSH
• Computational Biology MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Check Tag
• Humans MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Oncogene Proteins, Viral MeSH
• Proteome MeSH