Changes in Arabidopsis Phosphorylation During Drought Response: Quantitative Phosphoproteomics via Metabolic Labeling

摘要

1. We are studying water stress response signaling in Arabidopsis via relative quantitation. Plants are grown in natural abundance or ~(15)N-enriched media. 2. Plants are incubated with 300mM mannitol (stressed) or media (control). Plants are combined and proteins are isolated by tissue homogenization, centrifugation, and precipitation. 3 Proteins are digested with trypsin, peptide carboxylates are converted to their methyl ester form, and phosphopeptides are enriched using Fe~(3+) IMAC. 4. Peptides are separated by microbore C_(18) liquid chromatography and spotted directly onto LC/MALDI plates using a modified Agilent 1100 fraction collector. 5. Spotted peptides are analyzed using an Applied Biosystems 4800 TOF-TOF. 6. Data is processed by Mascot, and differential quantitation is achieved through in-house created scripts.