Figure 2.

Organellar Mapping in Dendritic Cells

(A) Schematic representation of the fractionation profiling approach for making organellar maps. Metabolically labeled (SILAC heavy—vehicle-treated and light—vehicle- or drug-treated) MutuDCs are lysed mechanically. Post-nuclear supernatant from light labeled cells is subjected to a series of differential centrifugation steps to separate organelles partially. In parallel, post-nuclear supernatant from heavy labeled cells is pelleted at high speed to obtain a reference fraction, which is spiked into each of the light fractions. Quantitative mass spectrometry allows the accurate determination of abundance distribution profiles across the light subfractions for individual proteins. Proteins associated with the same organelle have similar profiles, and different organelles have distinct profiles. Principal component analysis is used to visualize organellar clusters.

(B) Examples of the log2 heavy/light ratios for proteins in selected organelles and protein complexes from vehicle treated MutuDCs (mean ± 95% confidence interval [CI]).

(C) Organellar maps of MutuDCs visualized by principal component analysis. The first two principal components account for >90% of the variability in the data. Marker proteins of various organelles and known protein complexes are shown with colored circles; density gradients for proteins in each cluster are also highlighted.

See also Data S2.