The Proteomics Core applies a number of techniques to interrogate protein content and activity in mouse bone marrow-derived macrophages. The resulting data provide an important complement to the global gene expression data sets generated by the Genomics Core. We have developed methods to quantitatively compare the protein profiles of different biological samples. To effectively reduce sample complexity so that proteins of lower abundance can be measured, we developed techniques to selectively capture and study membrane-bound, secreted, or phosphorylated proteins. Another proteomic technique that yields important information about protein-protein interactions within cells combines tandem affinity purification with mass spectrometry (see our Technology page for more details on all our techniques). We have thus far focused on membrane, secreted, and phagosomal proteins in the Toll-like Receptor (TLR) response of mouse macrophages. A number of computational tools have been developed to support and validate proteomic analysis, more details of which are provided on the Software page.
Proteomics
Proteomics techniques are used to study the protein content and characteristics of biological samples. The major
tool of proteomics approaches is mass spectrometry. Mass spectrometry is used to identify proteins and, in
conjuction with isotopic labeling, this technique can also determine relative amounts of proteins in 2 or more
samples. In order to identify as many proteins as possible, samples can be fractionated and their complexity reduced
by isolating particular cellular compartments, such as membrane or secreted proteins, or proteins with particular
modifications such as glycosylated or phosphorylated proteins. The identity of proteins that are members of large
complexes can also be identified using techniques such as protein tagging followed by affinity purification. Data
generated by mass spectrometers is analyzed using a comprehensive software package known as the Trans-Proteomic
Pipeline that was developed at the ISB.
Proteomics and the Innate Immune Response
We are using the proteomics techniques to
study changes in the proteome of immune cells in response to whole pathogens and pathogen-associated molecular
patterns (PAMPs) that are known to activate Toll-like receptors (TLR). We are studying changes in the surface and
secreted proteins of macrophages and dendritic cells that have been challenged with these microbes and their
components. In addition, we are using affinity purification techniques to characterize protein-protein interactions
of specific proteins that have been identified as important through techniques and screening methods used in the
various cores such as Forward Genetics, Genomics, Computation, and Signaling.
Changes in the cell surface composition and in proteins secreted by immune cells are important to the activity and identity of the immune cell and its interaction with other cells. These activities can affect the whole immune response; adaptive and innate. The results of this multipronged approach will give us a dataset of protein expression in response to pathogens that will complement our comprehensive set of RNA expression data and quide us towards an understanding of the consequences of this expression.