Institute for Systems Biology

About: Institute for Systems Biology is a nonprofit organization based out in Seattle, Washington, United States. It is known for research contribution in the topics: Population & Proteomics. The organization has 1277 authors who have published 2777 publications receiving 353165 citations.

Proteome-wide cellular protein concentrations of the human pathogen Leptospira interrogans

Abstract: Mass-spectrometry-based methods for relative proteome quantification have broadly affected life science research. However, important research directions, particularly those involving mathematical modelling and simulation of biological processes, also critically depend on absolutely quantitative data--that is, knowledge of the concentration of the expressed proteins as a function of cellular state. Until now, absolute protein concentration measurements of a considerable fraction of the proteome (73%) have only been derived from genetically altered Saccharomyces cerevisiae cells, a technique that is not directly portable from yeast to other species. Here we present a mass-spectrometry-based strategy to determine the absolute quantity, that is, the average number of protein copies per cell in a cell population, for a large fraction of the proteome in genetically unperturbed cells. Applying the technology to the human pathogen Leptospira interrogans, a spirochete responsible for leptospirosis, we generated an absolute protein abundance scale for 83% of the mass-spectrometry-detectable proteome, from cells at different states. Taking advantage of the unique cellular dimensions of L. interrogans, we used cryo-electron tomography morphological measurements to verify, at the single-cell level, the average absolute abundance values of selected proteins determined by mass spectrometry on a population of cells. Because the strategy is relatively fast and applicable to any cell type, we expect that it will become a cornerstone of quantitative biology and systems biology.

The Toll-Like Receptor 5 Stimulus Bacterial Flagellin Induces Maturation and Chemokine Production in Human Dendritic Cells

Abstract: Toll-like receptors (TLRs) are pattern recognition receptors that serve an important function in detecting pathogens and initiating inflammatory responses. Upon encounter with foreign Ag, dendritic cells (DCs) go through a maturation process characterized by an increase in surface expression of MHC class II and costimulatory molecules, which leads to initiation of an effective immune response in naive T cells. The innate immune response to bacterial flagellin is mediated by TLR5, which is expressed on human DCs. Therefore, we sought to investigate whether flagellin could induce DC maturation. Immature DCs were cultured in the absence or presence of flagellin and monitored for expression of cell surface maturation markers. Stimulation with flagellin induced increased surface expression of CD83, CD80, CD86, MHC class II, and the lymph node-homing chemokine receptor CCR7. Flagellin stimulated the expression of chemokines active on neutrophils (IL-8/CXC chemokine ligand (CXCL)8, GRO-alpha/CXCL1, GRO-beta/CXCL2, GRO-gamma/CXCL3), monocytes (monocyte chemoattractant protein-1/CC chemokine ligand (CCL)2), and immature DCs (macrophage-inflammatory protein-1 alpha/CCL3, macrophage-inflammatory protein-1 beta/CCL4), but not chemokines active on effector T cells (IFN-inducible protein-10 kDa/CXCL10, monokine induced by IFN-gamma/CXCL9, IFN-inducible T cell alpha chemoattractant/CXCL11). However, stimulating DCs with both flagellin and IFN-inducible protein-10 kDa, monokine induced by IFN-gamma, and IFN-inducible T cell alpha chemoattractant expression, whereas stimulation with IFN-beta or flagellin alone failed to induce these chemokines. In functional assays, flagellin-matured DCs displayed enhanced T cell stimulatory activity with a concomitant decrease in endocytic activity. Finally, DCs isolated from mouse spleens or bone marrows were shown to not express TLR5 and were not responsive to flagellin stimulation. These results demonstrate that flagellin can directly stimulate human but not murine DC maturation, providing an additional mechanism by which motile bacteria can initiate an acquired immune response.

Quantitative proteome analysis by solid-phase isotope tagging and mass spectrometry.

Abstract: The adaptation of sequences of chemical reactions to a solid-phase format has been essential to the automation, reproducibility, and efficiency of a number of biotechnological processes including peptide and oligonucleotide synthesis and sequencing. Here we describe a method for the site-specific, stable isotopic labeling of cysteinyl peptides in complex peptide mixtures through a solid-phase capture and release process, and the concomitant isolation of the labeled peptides. The recovered peptides were analyzed by microcapillary liquid chromatography and tandem mass spectrometry (microLC-MS/MS) to determine their sequences and relative quantities. The method was used to detect galactose-induced changes in protein abundance in the yeast Saccharomyces cerevisiae. A side-by-side comparison with the isotope-coded affinity tag (ICAT) method demonstrated that the solid-phase method for stable isotope tagging of peptides is comparatively simpler, more efficient, and more sensitive.