High performance liquid chromatography grade acetonitrile

Samples (2 μL containing 1 μg protein) for SRM analysis were subjected to injection using a manual injection loop with an analytical Eksigent HALO fused-core C18 2.7 μm, 0.3 × 100 mm connected to the Applied Biosystems 5500 iontrap fit with a turbo V electrospray source fitted for micro-flow. The samples were subjected to a gradient of 26 minutes with an Agilent 1100 micro-flow HPLC. The samples were subjected to the following gradient at a flow rate of 8 μL/minute: 0–1 minute at 5% B; 3–18 minutes a gradient to 46% B; 18–21 minutes at 95% B; and equilibrated from 21–23 minutes at 5% B, (Buffer A: Water, 0.1% formic acid; Buffer B: high-performance liquid chromatography grade acetonitrile (Fisher Scientific, Pittsburg, PA, USA), 0.1% formic acid). Transitions monitored are listed below. These were established using the instrument optimization mode with direct injection of synthetic phosphorylated and unphosphorylated peptides representing their tryptic digest of DAM1 protein. The entrance potential (EP) was 10 V with a collision energy (CE) of 28.0 V and collision cell exit potential (CXP) of 15.0. The data were analyzed using MultiQuant™ (ABI Sciex, Framingham, MA, ). The peak area ratio of phosphorylated/unphosphorylated DAM1 peptide was used to determine the percent of phosphorylation peptide in the reaction. Samples were run in triplicate and randomized.