Project description:Bottom-up proteomics database search algorithms used for peptide identification cannot comprehensively identify posttranslational modifications (PTMs) in a single-pass because of high false discovery rates (FDRs). A new approach to database searching enables Global PTM (G-PTM) identification by exclusively looking for curated PTMs, thereby avoiding the FDR penalty experienced during conventional variable modification searches. We identified nearly 2500 unique, high-confidence modified peptides comprising 31 different PTM types in single-pass database searches.
Project description:Bottom-up proteomics database search algorithms used for peptide identification cannot comprehensively identify posttranslational modifications (PTMs) in a single-pass because of high false discovery rates (FDRs). A new approach to database searching enables Global PTM (G-PTM) identification by exclusively looking for curated PTMs, thereby avoiding the FDR penalty experienced during conventional variable modification searches. We identified nearly 2500 unique, high-confidence modified peptides comprising 31 different PTM types in single-pass database searches. Male C57BL/6J (B6) and CAST/EiJ (CAST) mice were purchased from The Jackson Laboratories (Bar Harbor, Maine) and housed in an environmentally controlled vivarium at the University of Wisconsin Biochemistry Department. Mice were provided standard rodent chow (Purina no. 5008) and water ad libitum, and maintained on a 12-hour light/dark cycle (6 AM – 6 PM). At 10 weeks of age, mice were sacrificed by CO2 asphyxiation. All animal procedures were preapproved by the University of Wisconsin Animal Care and Use Committee.
Project description:Elucidating the role of gut microbiota in physiological and pathological processes has recently emerged as a key research aim in life sciences. In this respect, metaproteomics (the study of the whole protein complement of a microbial community) can provide a unique contribution by revealing which functions are actually being expressed by specific microbial taxa. However, its wide application to gut microbiota research has been hindered by challenges in data analysis, especially related to the choice of the proper sequence databases for protein identification. Here we present a systematic investigation of variables concerning database construction and annotation, and evaluate their impact on human and mouse gut metaproteomic results. We found that both publicly available and experimental metagenomic databases lead to the identification of unique peptide assortments, suggesting parallel database searches as a mean to gain more complete information. Taxonomic and functional results were revealed to be strongly database-dependent, especially when dealing with mouse samples. As a striking example, in mouse the Firmicutes/Bacteroidetes ratio varied up to 10-fold depending on the database used. Finally, we provide recommendations regarding metagenomic sequence processing aimed at maximizing gut metaproteome characterization, and contribute to identify an optimized pipeline for metaproteomic data analysis.
Project description:We report the use of Next Generation RNA Sequencing for confirming or improving initial identification of small regulatory RNA in Staphylococcus aureus to prodive a list of the most probable RNA molecules transcribed as independent units.
Project description:We report the use of Next Generation RNA Sequencing for confirming or improving initial identification of small regulatory RNA in Staphylococcus aureus to prodive a list of the most probable RNA molecules transcribed as independent units. Extraction of RNAs in exponential phase of growth in Newman and N315 strains