TMT-based Phosphatase Activity Assay

We adapted our previous phosphatase method^{2 (link)} to make use of TMT. Briefly, five dried down desalted digests were resuspended in 100 mM EPPS pH 8.5 and separated into two equivalent 50 μg aliquots. Each digest corresponded to a biological replicate. Each aliquot was labeled with a TMT10 reagent for 90 min at room temperature and then quenched with hydroxylamine. The quenched reaction was flash frozen and dried down in a vacuum centrifuge and then resuspended in CutSmart Buffer (New England Biolabs) and one labeled aliquot from each replicate was treated with 200 units of calf intestinal phosphatase (New England Biolabs) while the other aliquot from the replicate was treated with water. All aliquots were incubated at 37 °C for 3 h and then acidified with formic acid to a final concentration of 1%. All aliquots were then combined at a 1:1:1:1:1:1:1:1:1:1 ratio.^{11 (link)} The pooled sample was then subjected to C18 SPE (Sep-Pak, Waters) and then dried down in a vacuum centrifuge before resuspension in 10 mM ammonium bicarbonate and 5% acetonitrile for off-line basic pH reversed-phase (BPRP) fractionation.