Fig. 1

The proteomics workflow utilized for defining the germinating spore phosphoproteome. a The sequence of events during spore revival of wild-type B. subtilis (PY79) as captured by phase-contrast microscopy. Shown are phase-contrast images acquired at the indicated time points. Scale bar represents 1 μm. b Purified spores of PY79 strain were incubated in a revival medium containing L-Ala and AGFK, and were harvested at the indicated time points. Left panels show corresponding phase-contrast images of the germinating spores prior to further processing. Next, spores were lysed, and proteins were extracted and subjected to trypsin digestion. Digested peptides were enriched for phosphorylated peptides by TiO₂ chromatography (five consecutive rounds). All samples were analyzed on LTQ-Orbitrap XL mass spectrometer. Label-free quantitation (LFQ) was applied to determine phosphoproteome dynamics (see Methods)