ABSTRACT
The Comet database search software was initially released as an open source project in late 2012. Prior to that, Comet existed as the University of Washington's academic version of the SEQUEST database search tool. Despite its availability and widespread use over the years, some details about its implementation have not been previously disseminated or are not well understood. We address a few of these details in depth and highlight new features available in the latest release. Comet is freely available for download at http://comet-ms.sourceforge.net or it can be accessed as a component of a number of larger software projects into which it has been incorporated. Graphical Abstract á .
Subject(s)
Databases, Protein , Proteomics/methods , Software , Tandem Mass Spectrometry/methods , AlgorithmsABSTRACT
Previous work has shown conflicting roles for Tec family kinases in regulation of TLR-dependent signaling in myeloid cells. In the present study, we performed a detailed investigation of the role of the Tec kinases Btk and Tec kinases in regulating TLR signaling in several types of primary murine macrophages. We demonstrate that primary resident peritoneal macrophages deficient for Btk and Tec secrete less proinflammatory cytokines in response to TLR stimulation than do wild-type cells. In contrast, we found that bone marrow-derived and thioglycollate-elicited peritoneal macrophages deficient for Btk and Tec secrete more proinflammatory cytokines than do wild-type cells. We then compared the phosphoproteome regulated by Tec kinases and LPS in primary peritoneal and bone marrow-derived macrophages. From this analysis we determined that Tec kinases regulate different signaling programs in these cell types. In additional studies using bone marrow-derived macrophages, we found that Tec and Btk promote phosphorylation events necessary for immunoreceptor-mediated inhibition of TLR signaling. Taken together, our results are consistent with a model where Tec kinases (Btk, Tec, Bmx) are required for TLR-dependent signaling in many types of myeloid cells. However, our data also support a cell type-specific TLR inhibitory role for Btk and Tec that is mediated by immunoreceptor activation and signaling via PI3K.
Subject(s)
Macrophages/immunology , Phosphoproteins/immunology , Protein-Tyrosine Kinases/immunology , Agammaglobulinaemia Tyrosine Kinase , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/drug effects , Bone Marrow Cells/immunology , Gene Expression Profiling , Gene Expression Regulation , Lipopolysaccharides/pharmacology , Macrophages/cytology , Macrophages/drug effects , Mice , Mice, Inbred C57BL , Mice, Knockout , Organ Specificity , Peritoneal Cavity/cytology , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/immunology , Phosphoproteins/genetics , Phosphorylation , Primary Cell Culture , Protein-Tyrosine Kinases/genetics , Signal Transduction , Toll-Like Receptors/genetics , Toll-Like Receptors/immunologyABSTRACT
Proteomics research routinely involves identifying peptides and proteins via MS/MS sequence database search. Thus the database search engine is an integral tool in many proteomics research groups. Here, we introduce the Comet search engine to the existing landscape of commercial and open-source database search tools. Comet is open source, freely available, and based on one of the original sequence database search tools that has been widely used for many years.
Subject(s)
Databases, Protein , Peptides/genetics , Proteins/genetics , Software , Algorithms , Amino Acid Sequence , Humans , Proteomics , Search Engine , Tandem Mass SpectrometryABSTRACT
Human immunodeficiency virus (HIV-1) depends upon host-encoded proteins to facilitate its replication while at the same time inhibiting critical components of innate and/or intrinsic immune response pathways. To characterize the host cell response on protein levels in CD4+ lymphoblastoid SUP-T1 cells after infection with HIV-1 strain LAI, we used mass spectrometry (MS)-based global quantitation with iTRAQ (isobaric tag for relative and absolute quantification). We found 266, 60 and 22 proteins differentially expressed (DE) (P-value ≤ 0.05) at 4, 8, and 20 hours post-infection (hpi), respectively, compared to time-matched mock-infected samples. The majority of changes in protein abundance occurred at an early stage of infection well before the de novo production of viral proteins. Functional analyses of these DE proteins showed enrichment in several biological pathways including protein synthesis, cell proliferation, and T-cell activation. Importantly, these early changes before the time of robust viral production have not been described before.
