Fig. 2.

Diagrammatic representation of workflow for protein quantitation. Cells from each harvested time point were lysed, and extracted proteins were processed to produce reduced, alkylated tryptic peptides separately. Peptides were chemically labeled with the indicated tandem-mass tags and were combined at a 1:1 ratio following quenching of the labeling reaction. The combined peptides were fractionated by high-pH reverse phase chromatography into 24 fractions which were prepared for mass spectrometry and acquired on a Fusion mass spectrometer using the MultiNotch MS3 method (47).