Figure 2.

Principle of the activity correlation profiling approach. ( a) A Triton X-100 extract (TE) of salt-washed yeast membranes, i.e. an extract that is enriched in ER membrane proteins, is fractionated by velocity gradient sedimentation. Half of each fraction is reconstituted into large unilamellar vesicles and assayed for M5-DLO scramblase activity as outlined in Fig. 1b to generate a profile of scramblase activity across the gradient. The remaining half of each fraction is subjected to mass spectrometric analysis using the tandem mass tag (TMT) system. Thus, each fraction is digested with trypsin, and the resulting peptides are labeled with TMT multiplex reagents (a unique TMT mass tag is used for each fraction). The digests are pooled and analyzed by tandem mass spectrometry to quantify the relative amount of identified proteins across fractions, thereby generating protein profiles. The protein profiles are quantitatively compared with the activity profile by obtaining a Pearson correlation score. (b) Schematic illustration of the readout from the activity correlation profiling experiment. The activity profile (solid blue line) can be compared to the profile of several different proteins (dashed lines) identified by tandem mass spectrometry. Only a protein whose profile is highly correlated with the activity profile (orange dashed line) is considered as a scramblase candidate. Other proteins that fractionate differently (brown and green dashed lines) are considered irrelevant to scramblase activity.