Protein pellets were prepared for mass spectrometry as previously described [80] . Pellets were solubilized by adding a volume of 50 mM ammonium bicarbonate (Sigma; St. Louis, MO)/0.1% RapiGest SF (Waters Corp.; Milford, MA) solution. Samples were left to stir overnight at 4uC and then centrifuged at 16,0006g for 5 min to pellet insoluble debris. Protein concentration of the supernatant was determined using a Quickstart Bradford assay (Biorad) and verified using 1-D SDS PAGE as described [77] . For each sample replicate, 100 mg of protein was diluted in 50 ml 50 mM ammonium bicarbonate/0.1% RapiGest SF (Waters Corp., Milford, MA) and used as the starting material for the digestion procedure. Samples were reduced with DTT at a 5 mM final concentration for 30 min at 50uC and then alkylated with IAA at a 15 mM final concentration for 30 min at room temperature, in the dark. For digestion, a 200 ng/ml trypsin (Promega; Madison, WI) solution was prepared using 0.01% acetic acid. Two mg of trypsin was added to each sample at a trypsin:protein ratio of 1:50 and incubated at 37uC for 3.5 h, with gentle vortexing every 15 min. To hydrolyze the RapiGest surfactant, samples were acidified with HCl to a pH #2, final HCl concentration of 200 mM, incubated at 37uC for 45 min, and centrifuged at 16,0006g for 10 min. The supernatant was transferred to new tubes and frozen at 280uC until mass spectrometry, approximately 48 h. Impurities were removed using mixed mode RP SCX SPE cation exchange cartridges (Waters Oasis 1cc MCX cartridge).