iPOND-SILAC mass spectrometry

Light lysine and arginine labeled mESCs were incubated with 10 μM EdU for 10 min and treated with 4 mM HU for 3 hours. Heavy lysine and arginine labeled mESCs were incubated with 10 μM EdU for 10 min. iPOND mass spectrometry was performed essentially as described. At least two peptides were required for protein identification. Quantitation is reported as the log₂ of the normalized heavy/light ratios. SILAC data were analyzed using MaxQuant. The resulting output tables of two independent experiments were merged and used as the input for calculating the average fold change to identify significantly up-regulated proteins in unperturbed forks and stalled forks based on the ratio of heavy and light peptides (H/L ratio) in the SILAC experiment in MaxQuant software (9).