The proteomics core is using glyco-capture and ultra centrifugation to isolate proteins associated with the plasma
membrane of bone marrow derived macrophages (BMDM) and dendritic cells (BMDC). To enrich for glyco-proteins on the cell
surface we label the plasma membrane glyco-proteins with biocytin hydrazide. We can then enrich for glyco-peptides using
streptavidin affinity chromatography. To examine quantitative differences in the plasma membrane after the cells are
infected with different pathogens we will grow the BMDM and BMDCs in SILAC media. We are also examining how all membrane
constituents change in response to infection by obtaining a crude membrane pellet from our cells, post infection. We
separate and fractionate our membrane proteins by SDS-PAGE. Each fraction is then analyzed in the LTQ orbitrap mass
spectrometer. We can obtain label-free quantitation of our samples using software developed at the Institute for Systems
Biology called Corra. See the preliminary datasets for membrane associated proteins from each technique.
To study the secretome of bone marrow derived macrophages and dendritic cells we are using ultra-filtration to
concentrate the media. The proteins in the concentrated media are separated using SDS-PAGE. The gel lanes are
sectioned into 6 fractions. These fractions are digested and then analyzed by an LTQ orbitrap mass spectrometer. We
will also use Corra to quantitate the changes in the secretome during infection with different pathogens. See the
preliminary datasheets for proteins detected in the media of LPS stimulated BMDM.
Preliminary Data